Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases
Identifieur interne : 000178 ( Pmc/Corpus ); précédent : 000177; suivant : 000179Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases
Auteurs : Adam J. Book ; Ragothaman M. Yennamalli ; Taichi E. Takasuka ; Cameron R. Currie ; George N. Phillips ; Brian G. FoxSource :
- Biotechnology for Biofuels [ 1754-6834 ] ; 2014.
Abstract
Understanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families.
Comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site.
The structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.
The online version of this article (doi:10.1186/1754-6834-7-109) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/1754-6834-7-109
PubMed: 25161697
PubMed Central: 4144037
Links to Exploration step
PMC:4144037Le document en format XML
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<author><name sortKey="Book, Adam J" sort="Book, Adam J" uniqKey="Book A" first="Adam J" last="Book">Adam J. Book</name>
<affiliation><nlm:aff id="Aff49">Department of Energy, Great Lakes Bioenergy Research Center, Madison, 1552 University Avenue, Madison, WI 53726 USA</nlm:aff>
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<affiliation><nlm:aff id="Aff50">Department of Bacteriology, University of Wisconsin-Madison, Microbial Sciences Building, 1550 Linden Dr., Madison, WI 53706 USA</nlm:aff>
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<author><name sortKey="Yennamalli, Ragothaman M" sort="Yennamalli, Ragothaman M" uniqKey="Yennamalli R" first="Ragothaman M" last="Yennamalli">Ragothaman M. Yennamalli</name>
<affiliation><nlm:aff id="Aff51">Department of Biochemistry, University of Wisconsin-Madison, Biochemistry Addition, 433 Babcock Dr., Madison, WI 53706 USA</nlm:aff>
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<affiliation><nlm:aff id="Aff52">Biosciences at Rice, Rice University, George R. Brown Hall, Houston, TX 77005 USA</nlm:aff>
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<author><name sortKey="Takasuka, Taichi E" sort="Takasuka, Taichi E" uniqKey="Takasuka T" first="Taichi E" last="Takasuka">Taichi E. Takasuka</name>
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<affiliation><nlm:aff id="Aff51">Department of Biochemistry, University of Wisconsin-Madison, Biochemistry Addition, 433 Babcock Dr., Madison, WI 53706 USA</nlm:aff>
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<author><name sortKey="Currie, Cameron R" sort="Currie, Cameron R" uniqKey="Currie C" first="Cameron R" last="Currie">Cameron R. Currie</name>
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<author><name sortKey="Phillips, George N" sort="Phillips, George N" uniqKey="Phillips G" first="George N" last="Phillips">George N. Phillips</name>
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<affiliation><nlm:aff id="Aff52">Biosciences at Rice, Rice University, George R. Brown Hall, Houston, TX 77005 USA</nlm:aff>
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<author><name sortKey="Fox, Brian G" sort="Fox, Brian G" uniqKey="Fox B" first="Brian G" last="Fox">Brian G. Fox</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases</title>
<author><name sortKey="Book, Adam J" sort="Book, Adam J" uniqKey="Book A" first="Adam J" last="Book">Adam J. Book</name>
<affiliation><nlm:aff id="Aff49">Department of Energy, Great Lakes Bioenergy Research Center, Madison, 1552 University Avenue, Madison, WI 53726 USA</nlm:aff>
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<affiliation><nlm:aff id="Aff50">Department of Bacteriology, University of Wisconsin-Madison, Microbial Sciences Building, 1550 Linden Dr., Madison, WI 53706 USA</nlm:aff>
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<author><name sortKey="Yennamalli, Ragothaman M" sort="Yennamalli, Ragothaman M" uniqKey="Yennamalli R" first="Ragothaman M" last="Yennamalli">Ragothaman M. Yennamalli</name>
<affiliation><nlm:aff id="Aff51">Department of Biochemistry, University of Wisconsin-Madison, Biochemistry Addition, 433 Babcock Dr., Madison, WI 53706 USA</nlm:aff>
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<affiliation><nlm:aff id="Aff52">Biosciences at Rice, Rice University, George R. Brown Hall, Houston, TX 77005 USA</nlm:aff>
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<author><name sortKey="Takasuka, Taichi E" sort="Takasuka, Taichi E" uniqKey="Takasuka T" first="Taichi E" last="Takasuka">Taichi E. Takasuka</name>
<affiliation><nlm:aff id="Aff49">Department of Energy, Great Lakes Bioenergy Research Center, Madison, 1552 University Avenue, Madison, WI 53726 USA</nlm:aff>
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<affiliation><nlm:aff id="Aff51">Department of Biochemistry, University of Wisconsin-Madison, Biochemistry Addition, 433 Babcock Dr., Madison, WI 53706 USA</nlm:aff>
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<author><name sortKey="Currie, Cameron R" sort="Currie, Cameron R" uniqKey="Currie C" first="Cameron R" last="Currie">Cameron R. Currie</name>
<affiliation><nlm:aff id="Aff49">Department of Energy, Great Lakes Bioenergy Research Center, Madison, 1552 University Avenue, Madison, WI 53726 USA</nlm:aff>
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<affiliation><nlm:aff id="Aff50">Department of Bacteriology, University of Wisconsin-Madison, Microbial Sciences Building, 1550 Linden Dr., Madison, WI 53706 USA</nlm:aff>
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<author><name sortKey="Phillips, George N" sort="Phillips, George N" uniqKey="Phillips G" first="George N" last="Phillips">George N. Phillips</name>
<affiliation><nlm:aff id="Aff49">Department of Energy, Great Lakes Bioenergy Research Center, Madison, 1552 University Avenue, Madison, WI 53726 USA</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff51">Department of Biochemistry, University of Wisconsin-Madison, Biochemistry Addition, 433 Babcock Dr., Madison, WI 53706 USA</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff52">Biosciences at Rice, Rice University, George R. Brown Hall, Houston, TX 77005 USA</nlm:aff>
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<author><name sortKey="Fox, Brian G" sort="Fox, Brian G" uniqKey="Fox B" first="Brian G" last="Fox">Brian G. Fox</name>
<affiliation><nlm:aff id="Aff49">Department of Energy, Great Lakes Bioenergy Research Center, Madison, 1552 University Avenue, Madison, WI 53726 USA</nlm:aff>
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<affiliation><nlm:aff id="Aff51">Department of Biochemistry, University of Wisconsin-Madison, Biochemistry Addition, 433 Babcock Dr., Madison, WI 53706 USA</nlm:aff>
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<series><title level="j">Biotechnology for Biofuels</title>
<idno type="eISSN">1754-6834</idno>
<imprint><date when="2014">2014</date>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>Understanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families.</p>
</sec>
<sec><title>Results</title>
<p>Comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site.</p>
</sec>
<sec><title>Conclusions</title>
<p>The structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.</p>
</sec>
<sec><title>Electronic supplementary material</title>
<p>The online version of this article (doi:10.1186/1754-6834-7-109) contains supplementary material, which is available to authorized users.</p>
</sec>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Himmel, Me" uniqKey="Himmel M">ME Himmel</name>
</author>
<author><name sortKey="Ding, Sy" uniqKey="Ding S">SY Ding</name>
</author>
<author><name sortKey="Johnson, Dk" uniqKey="Johnson D">DK Johnson</name>
</author>
<author><name sortKey="Adney, Ws" uniqKey="Adney W">WS Adney</name>
</author>
<author><name sortKey="Nimlos, Mr" uniqKey="Nimlos M">MR Nimlos</name>
</author>
<author><name sortKey="Brady, Jw" uniqKey="Brady J">JW Brady</name>
</author>
<author><name sortKey="Foust, Td" uniqKey="Foust T">TD Foust</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Faaij, Apc" uniqKey="Faaij A">APC Faaij</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wilson, Db" uniqKey="Wilson D">DB Wilson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lynd, Lr" uniqKey="Lynd L">LR Lynd</name>
</author>
<author><name sortKey="Weimer, Pj" uniqKey="Weimer P">PJ Weimer</name>
</author>
<author><name sortKey="Van Zyl, Wh" uniqKey="Van Zyl W">WH van Zyl</name>
</author>
<author><name sortKey="Pretorius, Is" uniqKey="Pretorius I">IS Pretorius</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Culpepper, Ma" uniqKey="Culpepper M">MA Culpepper</name>
</author>
<author><name sortKey="Rosenzweig, Ac" uniqKey="Rosenzweig A">AC Rosenzweig</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Forsberg, Z" uniqKey="Forsberg Z">Z Forsberg</name>
</author>
<author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
<author><name sortKey="Westereng, B" uniqKey="Westereng B">B Westereng</name>
</author>
<author><name sortKey="Bunaes, Ac" uniqKey="Bunaes A">AC Bunaes</name>
</author>
<author><name sortKey="Stenstrom, Y" uniqKey="Stenstrom Y">Y Stenstrom</name>
</author>
<author><name sortKey="Mackenzie, A" uniqKey="Mackenzie A">A MacKenzie</name>
</author>
<author><name sortKey="Sorlie, M" uniqKey="Sorlie M">M Sorlie</name>
</author>
<author><name sortKey="Horn, Sj" uniqKey="Horn S">SJ Horn</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Harris, Pv" uniqKey="Harris P">PV Harris</name>
</author>
<author><name sortKey="Welner, D" uniqKey="Welner D">D Welner</name>
</author>
<author><name sortKey="Mcfarland, Kc" uniqKey="Mcfarland K">KC McFarland</name>
</author>
<author><name sortKey="Re, E" uniqKey="Re E">E Re</name>
</author>
<author><name sortKey="Navarro Poulsen, Jc" uniqKey="Navarro Poulsen J">JC Navarro Poulsen</name>
</author>
<author><name sortKey="Brown, K" uniqKey="Brown K">K Brown</name>
</author>
<author><name sortKey="Salbo, R" uniqKey="Salbo R">R Salbo</name>
</author>
<author><name sortKey="Ding, H" uniqKey="Ding H">H Ding</name>
</author>
<author><name sortKey="Vlasenko, E" uniqKey="Vlasenko E">E Vlasenko</name>
</author>
<author><name sortKey="Merino, S" uniqKey="Merino S">S Merino</name>
</author>
<author><name sortKey="Xu, F" uniqKey="Xu F">F Xu</name>
</author>
<author><name sortKey="Cherry, J" uniqKey="Cherry J">J Cherry</name>
</author>
<author><name sortKey="Larsen, S" uniqKey="Larsen S">S Larsen</name>
</author>
<author><name sortKey="Lo Leggio, L" uniqKey="Lo Leggio L">L Lo Leggio</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hemsworth, Gr" uniqKey="Hemsworth G">GR Hemsworth</name>
</author>
<author><name sortKey="Taylor, Ej" uniqKey="Taylor E">EJ Taylor</name>
</author>
<author><name sortKey="Kim, Rq" uniqKey="Kim R">RQ Kim</name>
</author>
<author><name sortKey="Gregory, Rc" uniqKey="Gregory R">RC Gregory</name>
</author>
<author><name sortKey="Lewis, Sj" uniqKey="Lewis S">SJ Lewis</name>
</author>
<author><name sortKey="Turkenburg, Jp" uniqKey="Turkenburg J">JP Turkenburg</name>
</author>
<author><name sortKey="Parkin, A" uniqKey="Parkin A">A Parkin</name>
</author>
<author><name sortKey="Davies, Gj" uniqKey="Davies G">GJ Davies</name>
</author>
<author><name sortKey="Walton, Ph" uniqKey="Walton P">PH Walton</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Schnellmann, J" uniqKey="Schnellmann J">J Schnellmann</name>
</author>
<author><name sortKey="Zeltins, A" uniqKey="Zeltins A">A Zeltins</name>
</author>
<author><name sortKey="Blaak, H" uniqKey="Blaak H">H Blaak</name>
</author>
<author><name sortKey="Schrempf, H" uniqKey="Schrempf H">H Schrempf</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Suzuki, K" uniqKey="Suzuki K">K Suzuki</name>
</author>
<author><name sortKey="Suzuki, M" uniqKey="Suzuki M">M Suzuki</name>
</author>
<author><name sortKey="Taiyoji, M" uniqKey="Taiyoji M">M Taiyoji</name>
</author>
<author><name sortKey="Nikaidou, N" uniqKey="Nikaidou N">N Nikaidou</name>
</author>
<author><name sortKey="Watanabe, T" uniqKey="Watanabe T">T Watanabe</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
<author><name sortKey="Westereng, B" uniqKey="Westereng B">B Westereng</name>
</author>
<author><name sortKey="Horn, Sj" uniqKey="Horn S">SJ Horn</name>
</author>
<author><name sortKey="Liu, Z" uniqKey="Liu Z">Z Liu</name>
</author>
<author><name sortKey="Zhai, H" uniqKey="Zhai H">H Zhai</name>
</author>
<author><name sortKey="Sorlie, M" uniqKey="Sorlie M">M Sorlie</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Levasseur, A" uniqKey="Levasseur A">A Levasseur</name>
</author>
<author><name sortKey="Drula, E" uniqKey="Drula E">E Drula</name>
</author>
<author><name sortKey="Lombard, V" uniqKey="Lombard V">V Lombard</name>
</author>
<author><name sortKey="Coutinho, Pm" uniqKey="Coutinho P">PM Coutinho</name>
</author>
<author><name sortKey="Henrissat, B" uniqKey="Henrissat B">B Henrissat</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Quinlan, Rj" uniqKey="Quinlan R">RJ Quinlan</name>
</author>
<author><name sortKey="Sweeney, Md" uniqKey="Sweeney M">MD Sweeney</name>
</author>
<author><name sortKey="Lo Leggio, L" uniqKey="Lo Leggio L">L Lo Leggio</name>
</author>
<author><name sortKey="Otten, H" uniqKey="Otten H">H Otten</name>
</author>
<author><name sortKey="Poulsen, Jc" uniqKey="Poulsen J">JC Poulsen</name>
</author>
<author><name sortKey="Johansen, Ks" uniqKey="Johansen K">KS Johansen</name>
</author>
<author><name sortKey="Krogh, Kb" uniqKey="Krogh K">KB Krogh</name>
</author>
<author><name sortKey="Jorgensen, Ci" uniqKey="Jorgensen C">CI Jorgensen</name>
</author>
<author><name sortKey="Tovborg, M" uniqKey="Tovborg M">M Tovborg</name>
</author>
<author><name sortKey="Anthonsen, A" uniqKey="Anthonsen A">A Anthonsen</name>
</author>
<author><name sortKey="Tryfona, T" uniqKey="Tryfona T">T Tryfona</name>
</author>
<author><name sortKey="Walter, Cp" uniqKey="Walter C">CP Walter</name>
</author>
<author><name sortKey="Dupree, P" uniqKey="Dupree P">P Dupree</name>
</author>
<author><name sortKey="Xu, F" uniqKey="Xu F">F Xu</name>
</author>
<author><name sortKey="Davies, Gj" uniqKey="Davies G">GJ Davies</name>
</author>
<author><name sortKey="Walton, Ph" uniqKey="Walton P">PH Walton</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Westereng, B" uniqKey="Westereng B">B Westereng</name>
</author>
<author><name sortKey="Ishida, T" uniqKey="Ishida T">T Ishida</name>
</author>
<author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
<author><name sortKey="Wu, M" uniqKey="Wu M">M Wu</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
<author><name sortKey="Igarashi, K" uniqKey="Igarashi K">K Igarashi</name>
</author>
<author><name sortKey="Samejima, M" uniqKey="Samejima M">M Samejima</name>
</author>
<author><name sortKey="Stahlberg, J" uniqKey="Stahlberg J">J Stahlberg</name>
</author>
<author><name sortKey="Horn, Sj" uniqKey="Horn S">SJ Horn</name>
</author>
<author><name sortKey="Sandgren, M" uniqKey="Sandgren M">M Sandgren</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Moser, F" uniqKey="Moser F">F Moser</name>
</author>
<author><name sortKey="Irwin, D" uniqKey="Irwin D">D Irwin</name>
</author>
<author><name sortKey="Chen, S" uniqKey="Chen S">S Chen</name>
</author>
<author><name sortKey="Wilson, Db" uniqKey="Wilson D">DB Wilson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Forsberg, Z" uniqKey="Forsberg Z">Z Forsberg</name>
</author>
<author><name sortKey="Rohr, Ak" uniqKey="Rohr A">AK Rohr</name>
</author>
<author><name sortKey="Mekasha, S" uniqKey="Mekasha S">S Mekasha</name>
</author>
<author><name sortKey="Andersson, Kk" uniqKey="Andersson K">KK Andersson</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
<author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
<author><name sortKey="Sorlie, M" uniqKey="Sorlie M">M Sorlie</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Aachmann, Fl" uniqKey="Aachmann F">FL Aachmann</name>
</author>
<author><name sortKey="Sorlie, M" uniqKey="Sorlie M">M Sorlie</name>
</author>
<author><name sortKey="Skjak Braek, G" uniqKey="Skjak Braek G">G Skjak-Braek</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
<author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Li, X" uniqKey="Li X">X Li</name>
</author>
<author><name sortKey="Beeson, Wt" uniqKey="Beeson W">WT Beeson</name>
</author>
<author><name sortKey="Phillips, Cm" uniqKey="Phillips C">CM Phillips</name>
</author>
<author><name sortKey="Marletta, Ma" uniqKey="Marletta M">MA Marletta</name>
</author>
<author><name sortKey="Cate, Jh" uniqKey="Cate J">JH Cate</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Phillips, Cm" uniqKey="Phillips C">CM Phillips</name>
</author>
<author><name sortKey="Beeson, Wt" uniqKey="Beeson W">WT Beeson</name>
</author>
<author><name sortKey="Cate, Jh" uniqKey="Cate J">JH Cate</name>
</author>
<author><name sortKey="Marletta, Ma" uniqKey="Marletta M">MA Marletta</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kim, S" uniqKey="Kim S">S Kim</name>
</author>
<author><name sortKey="Stahlberg, J" uniqKey="Stahlberg J">J Stahlberg</name>
</author>
<author><name sortKey="Sandgren, M" uniqKey="Sandgren M">M Sandgren</name>
</author>
<author><name sortKey="Paton, Rs" uniqKey="Paton R">RS Paton</name>
</author>
<author><name sortKey="Beckham, Gt" uniqKey="Beckham G">GT Beckham</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Vu, Vv" uniqKey="Vu V">VV Vu</name>
</author>
<author><name sortKey="Beeson, Wt" uniqKey="Beeson W">WT Beeson</name>
</author>
<author><name sortKey="Phillips, Cm" uniqKey="Phillips C">CM Phillips</name>
</author>
<author><name sortKey="Cate, Jhd" uniqKey="Cate J">JHD Cate</name>
</author>
<author><name sortKey="Marletta, Ma" uniqKey="Marletta M">MA Marletta</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Isaksen, T" uniqKey="Isaksen T">T Isaksen</name>
</author>
<author><name sortKey="Westereng, B" uniqKey="Westereng B">B Westereng</name>
</author>
<author><name sortKey="Aachmann, Fl" uniqKey="Aachmann F">FL Aachmann</name>
</author>
<author><name sortKey="Agger, Jw" uniqKey="Agger J">JW Agger</name>
</author>
<author><name sortKey="Kracher, D" uniqKey="Kracher D">D Kracher</name>
</author>
<author><name sortKey="Kittl, R" uniqKey="Kittl R">R Kittl</name>
</author>
<author><name sortKey="Ludwig, R" uniqKey="Ludwig R">R Ludwig</name>
</author>
<author><name sortKey="Haltrich, D" uniqKey="Haltrich D">D Haltrich</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
<author><name sortKey="Horn, Sj" uniqKey="Horn S">SJ Horn</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hori, C" uniqKey="Hori C">C Hori</name>
</author>
<author><name sortKey="Gaskell, J" uniqKey="Gaskell J">J Gaskell</name>
</author>
<author><name sortKey="Igarashi, K" uniqKey="Igarashi K">K Igarashi</name>
</author>
<author><name sortKey="Samejima, M" uniqKey="Samejima M">M Samejima</name>
</author>
<author><name sortKey="Hibbett, D" uniqKey="Hibbett D">D Hibbett</name>
</author>
<author><name sortKey="Henrissat, B" uniqKey="Henrissat B">B Henrissat</name>
</author>
<author><name sortKey="Cullen, D" uniqKey="Cullen D">D Cullen</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Karkehabadi, S" uniqKey="Karkehabadi S">S Karkehabadi</name>
</author>
<author><name sortKey="Hansson, H" uniqKey="Hansson H">H Hansson</name>
</author>
<author><name sortKey="Kim, S" uniqKey="Kim S">S Kim</name>
</author>
<author><name sortKey="Piens, K" uniqKey="Piens K">K Piens</name>
</author>
<author><name sortKey="Mitchinson, C" uniqKey="Mitchinson C">C Mitchinson</name>
</author>
<author><name sortKey="Sandgren, M" uniqKey="Sandgren M">M Sandgren</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
<author><name sortKey="Houston, Dr" uniqKey="Houston D">DR Houston</name>
</author>
<author><name sortKey="Riemen, Ah" uniqKey="Riemen A">AH Riemen</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
<author><name sortKey="Van Aalten, Dm" uniqKey="Van Aalten D">DM van Aalten</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wong, E" uniqKey="Wong E">E Wong</name>
</author>
<author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
<author><name sortKey="Ghosh, A" uniqKey="Ghosh A">A Ghosh</name>
</author>
<author><name sortKey="Hurtado Guerrero, R" uniqKey="Hurtado Guerrero R">R Hurtado-Guerrero</name>
</author>
<author><name sortKey="Konarev, Pv" uniqKey="Konarev P">PV Konarev</name>
</author>
<author><name sortKey="Ibrahim, Af" uniqKey="Ibrahim A">AF Ibrahim</name>
</author>
<author><name sortKey="Svergun, Di" uniqKey="Svergun D">DI Svergun</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
<author><name sortKey="Chatterjee, Ns" uniqKey="Chatterjee N">NS Chatterjee</name>
</author>
<author><name sortKey="Van Aalten, Dm" uniqKey="Van Aalten D">DM van Aalten</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Vaaje Kolstad, G" uniqKey="Vaaje Kolstad G">G Vaaje-Kolstad</name>
</author>
<author><name sortKey="Bohle, La" uniqKey="Bohle L">LA Bohle</name>
</author>
<author><name sortKey="Gaseidnes, S" uniqKey="Gaseidnes S">S Gaseidnes</name>
</author>
<author><name sortKey="Dalhus, B" uniqKey="Dalhus B">B Dalhus</name>
</author>
<author><name sortKey="Bjoras, M" uniqKey="Bjoras M">M Bjoras</name>
</author>
<author><name sortKey="Mathiesen, G" uniqKey="Mathiesen G">G Mathiesen</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wu, M" uniqKey="Wu M">M Wu</name>
</author>
<author><name sortKey="Beckham, Gt" uniqKey="Beckham G">GT Beckham</name>
</author>
<author><name sortKey="Larsson, Am" uniqKey="Larsson A">AM Larsson</name>
</author>
<author><name sortKey="Ishida, T" uniqKey="Ishida T">T Ishida</name>
</author>
<author><name sortKey="Kim, S" uniqKey="Kim S">S Kim</name>
</author>
<author><name sortKey="Payne, Cm" uniqKey="Payne C">CM Payne</name>
</author>
<author><name sortKey="Himmel, Me" uniqKey="Himmel M">ME Himmel</name>
</author>
<author><name sortKey="Crowley, Mf" uniqKey="Crowley M">MF Crowley</name>
</author>
<author><name sortKey="Horn, Sj" uniqKey="Horn S">SJ Horn</name>
</author>
<author><name sortKey="Westereng, B" uniqKey="Westereng B">B Westereng</name>
</author>
<author><name sortKey="Igarashi, K" uniqKey="Igarashi K">K Igarashi</name>
</author>
<author><name sortKey="Samejima, M" uniqKey="Samejima M">M Samejima</name>
</author>
<author><name sortKey="St Hlberg, J" uniqKey="St Hlberg J">J Ståhlberg</name>
</author>
<author><name sortKey="Eijsink, Vg" uniqKey="Eijsink V">VG Eijsink</name>
</author>
<author><name sortKey="Sandgren, M" uniqKey="Sandgren M">M Sandgren</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Blake, Aw" uniqKey="Blake A">AW Blake</name>
</author>
<author><name sortKey="Mccartney, L" uniqKey="Mccartney L">L McCartney</name>
</author>
<author><name sortKey="Flint, Je" uniqKey="Flint J">JE Flint</name>
</author>
<author><name sortKey="Bolam, Dn" uniqKey="Bolam D">DN Bolam</name>
</author>
<author><name sortKey="Boraston, Ab" uniqKey="Boraston A">AB Boraston</name>
</author>
<author><name sortKey="Gilbert, Hj" uniqKey="Gilbert H">HJ Gilbert</name>
</author>
<author><name sortKey="Knox, Jp" uniqKey="Knox J">JP Knox</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hogg, D" uniqKey="Hogg D">D Hogg</name>
</author>
<author><name sortKey="Pell, G" uniqKey="Pell G">G Pell</name>
</author>
<author><name sortKey="Dupree, P" uniqKey="Dupree P">P Dupree</name>
</author>
<author><name sortKey="Goubet, F" uniqKey="Goubet F">F Goubet</name>
</author>
<author><name sortKey="Martin Orue, Sm" uniqKey="Martin Orue S">SM Martin-Orue</name>
</author>
<author><name sortKey="Armand, S" uniqKey="Armand S">S Armand</name>
</author>
<author><name sortKey="Gilbert, Hj" uniqKey="Gilbert H">HJ Gilbert</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wang, N" uniqKey="Wang N">N Wang</name>
</author>
<author><name sortKey="Zhang, Y" uniqKey="Zhang Y">Y Zhang</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q Wang</name>
</author>
<author><name sortKey="Liu, J" uniqKey="Liu J">J Liu</name>
</author>
<author><name sortKey="Wang, H" uniqKey="Wang H">H Wang</name>
</author>
<author><name sortKey="Xue, Y" uniqKey="Xue Y">Y Xue</name>
</author>
<author><name sortKey="Ma, Y" uniqKey="Ma Y">Y Ma</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lin, Es" uniqKey="Lin E">ES Lin</name>
</author>
<author><name sortKey="Wilson, Db" uniqKey="Wilson D">DB Wilson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhang, S" uniqKey="Zhang S">S Zhang</name>
</author>
<author><name sortKey="Lao, G" uniqKey="Lao G">G Lao</name>
</author>
<author><name sortKey="Wilson, Db" uniqKey="Wilson D">DB Wilson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Huang, L" uniqKey="Huang L">L Huang</name>
</author>
<author><name sortKey="Garbulewska, E" uniqKey="Garbulewska E">E Garbulewska</name>
</author>
<author><name sortKey="Sato, K" uniqKey="Sato K">K Sato</name>
</author>
<author><name sortKey="Kato, Y" uniqKey="Kato Y">Y Kato</name>
</author>
<author><name sortKey="Nogawa, M" uniqKey="Nogawa M">M Nogawa</name>
</author>
<author><name sortKey="Taguchi, G" uniqKey="Taguchi G">G Taguchi</name>
</author>
<author><name sortKey="Shimosaka, M" uniqKey="Shimosaka M">M Shimosaka</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Ronquist, F" uniqKey="Ronquist F">F Ronquist</name>
</author>
<author><name sortKey="Teslenko, M" uniqKey="Teslenko M">M Teslenko</name>
</author>
<author><name sortKey="Van Der Mark, P" uniqKey="Van Der Mark P">P van der Mark</name>
</author>
<author><name sortKey="Ayres, Dl" uniqKey="Ayres D">DL Ayres</name>
</author>
<author><name sortKey="Darling, A" uniqKey="Darling A">A Darling</name>
</author>
<author><name sortKey="Hohna, S" uniqKey="Hohna S">S Hohna</name>
</author>
<author><name sortKey="Larget, B" uniqKey="Larget B">B Larget</name>
</author>
<author><name sortKey="Liu, L" uniqKey="Liu L">L Liu</name>
</author>
<author><name sortKey="Suchard, Ma" uniqKey="Suchard M">MA Suchard</name>
</author>
<author><name sortKey="Huelsenbeck, Jp" uniqKey="Huelsenbeck J">JP Huelsenbeck</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Bey, M" uniqKey="Bey M">M Bey</name>
</author>
<author><name sortKey="Zhou, S" uniqKey="Zhou S">S Zhou</name>
</author>
<author><name sortKey="Poidevin, L" uniqKey="Poidevin L">L Poidevin</name>
</author>
<author><name sortKey="Henrissat, B" uniqKey="Henrissat B">B Henrissat</name>
</author>
<author><name sortKey="Coutinho, Pm" uniqKey="Coutinho P">PM Coutinho</name>
</author>
<author><name sortKey="Berrin, Jg" uniqKey="Berrin J">JG Berrin</name>
</author>
<author><name sortKey="Sigoillot, Jc" uniqKey="Sigoillot J">JC Sigoillot</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kittl, R" uniqKey="Kittl R">R Kittl</name>
</author>
<author><name sortKey="Kracher, D" uniqKey="Kracher D">D Kracher</name>
</author>
<author><name sortKey="Burgstaller, D" uniqKey="Burgstaller D">D Burgstaller</name>
</author>
<author><name sortKey="Haltrich, D" uniqKey="Haltrich D">D Haltrich</name>
</author>
<author><name sortKey="Ludwig, R" uniqKey="Ludwig R">R Ludwig</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Langston, Ja" uniqKey="Langston J">JA Langston</name>
</author>
<author><name sortKey="Shaghasi, T" uniqKey="Shaghasi T">T Shaghasi</name>
</author>
<author><name sortKey="Abbate, E" uniqKey="Abbate E">E Abbate</name>
</author>
<author><name sortKey="Xu, F" uniqKey="Xu F">F Xu</name>
</author>
<author><name sortKey="Vlasenko, E" uniqKey="Vlasenko E">E Vlasenko</name>
</author>
<author><name sortKey="Sweeney, Md" uniqKey="Sweeney M">MD Sweeney</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Beeson, Wt" uniqKey="Beeson W">WT Beeson</name>
</author>
<author><name sortKey="Phillips, Cm" uniqKey="Phillips C">CM Phillips</name>
</author>
<author><name sortKey="Cate, Jh" uniqKey="Cate J">JH Cate</name>
</author>
<author><name sortKey="Marletta, Ma" uniqKey="Marletta M">MA Marletta</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Takasuka, Te" uniqKey="Takasuka T">TE Takasuka</name>
</author>
<author><name sortKey="Book, Aj" uniqKey="Book A">AJ Book</name>
</author>
<author><name sortKey="Lewin, Gr" uniqKey="Lewin G">GR Lewin</name>
</author>
<author><name sortKey="Currie, Cr" uniqKey="Currie C">CR Currie</name>
</author>
<author><name sortKey="Fox, Bg" uniqKey="Fox B">BG Fox</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Roy, A" uniqKey="Roy A">A Roy</name>
</author>
<author><name sortKey="Kucukural, A" uniqKey="Kucukural A">A Kucukural</name>
</author>
<author><name sortKey="Zhang, Y" uniqKey="Zhang Y">Y Zhang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Bailey, Tl" uniqKey="Bailey T">TL Bailey</name>
</author>
<author><name sortKey="Williams, N" uniqKey="Williams N">N Williams</name>
</author>
<author><name sortKey="Misleh, C" uniqKey="Misleh C">C Misleh</name>
</author>
<author><name sortKey="Li, Ww" uniqKey="Li W">WW Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hurst, Ld" uniqKey="Hurst L">LD Hurst</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Bianchetti, Cm" uniqKey="Bianchetti C">CM Bianchetti</name>
</author>
<author><name sortKey="Harmann, Ch" uniqKey="Harmann C">CH Harmann</name>
</author>
<author><name sortKey="Takasuka, Te" uniqKey="Takasuka T">TE Takasuka</name>
</author>
<author><name sortKey="Hura, Gl" uniqKey="Hura G">GL Hura</name>
</author>
<author><name sortKey="Dyer, K" uniqKey="Dyer K">K Dyer</name>
</author>
<author><name sortKey="Fox, Bg" uniqKey="Fox B">BG Fox</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Cantarel, Bl" uniqKey="Cantarel B">BL Cantarel</name>
</author>
<author><name sortKey="Coutinho, Pm" uniqKey="Coutinho P">PM Coutinho</name>
</author>
<author><name sortKey="Rancurel, C" uniqKey="Rancurel C">C Rancurel</name>
</author>
<author><name sortKey="Bernard, T" uniqKey="Bernard T">T Bernard</name>
</author>
<author><name sortKey="Lombard, V" uniqKey="Lombard V">V Lombard</name>
</author>
<author><name sortKey="Henrissat, B" uniqKey="Henrissat B">B Henrissat</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Altschul, Sf" uniqKey="Altschul S">SF Altschul</name>
</author>
<author><name sortKey="Madden, Tl" uniqKey="Madden T">TL Madden</name>
</author>
<author><name sortKey="Schaffer, Aa" uniqKey="Schaffer A">AA Schaffer</name>
</author>
<author><name sortKey="Zhang, J" uniqKey="Zhang J">J Zhang</name>
</author>
<author><name sortKey="Zhang, Z" uniqKey="Zhang Z">Z Zhang</name>
</author>
<author><name sortKey="Miller, W" uniqKey="Miller W">W Miller</name>
</author>
<author><name sortKey="Lipman, Dj" uniqKey="Lipman D">DJ Lipman</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Shannon, P" uniqKey="Shannon P">P Shannon</name>
</author>
<author><name sortKey="Markiel, A" uniqKey="Markiel A">A Markiel</name>
</author>
<author><name sortKey="Ozier, O" uniqKey="Ozier O">O Ozier</name>
</author>
<author><name sortKey="Baliga, Ns" uniqKey="Baliga N">NS Baliga</name>
</author>
<author><name sortKey="Wang, Jt" uniqKey="Wang J">JT Wang</name>
</author>
<author><name sortKey="Ramage, D" uniqKey="Ramage D">D Ramage</name>
</author>
<author><name sortKey="Amin, N" uniqKey="Amin N">N Amin</name>
</author>
<author><name sortKey="Schwikowski, B" uniqKey="Schwikowski B">B Schwikowski</name>
</author>
<author><name sortKey="Ideker, T" uniqKey="Ideker T">T Ideker</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Edgar, Rc" uniqKey="Edgar R">RC Edgar</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wu, M" uniqKey="Wu M">M Wu</name>
</author>
<author><name sortKey="Chatterji, S" uniqKey="Chatterji S">S Chatterji</name>
</author>
<author><name sortKey="Eisen, Ja" uniqKey="Eisen J">JA Eisen</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Stamatakis, A" uniqKey="Stamatakis A">A Stamatakis</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Yang, Z" uniqKey="Yang Z">Z Yang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Yang, Z" uniqKey="Yang Z">Z Yang</name>
</author>
<author><name sortKey="Rannala, B" uniqKey="Rannala B">B Rannala</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Holm, L" uniqKey="Holm L">L Holm</name>
</author>
<author><name sortKey="Rosenstrom, P" uniqKey="Rosenstrom P">P Rosenstrom</name>
</author>
</analytic>
</biblStruct>
<biblStruct></biblStruct>
<biblStruct><analytic><author><name sortKey="Shindyalov, In" uniqKey="Shindyalov I">IN Shindyalov</name>
</author>
<author><name sortKey="Bourne, Pe" uniqKey="Bourne P">PE Bourne</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Baker, Na" uniqKey="Baker N">NA Baker</name>
</author>
<author><name sortKey="Sept, D" uniqKey="Sept D">D Sept</name>
</author>
<author><name sortKey="Joseph, S" uniqKey="Joseph S">S Joseph</name>
</author>
<author><name sortKey="Holst, Mj" uniqKey="Holst M">MJ Holst</name>
</author>
<author><name sortKey="Mccammon, Ja" uniqKey="Mccammon J">JA McCammon</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Biotechnol Biofuels</journal-id>
<journal-id journal-id-type="iso-abbrev">Biotechnol Biofuels</journal-id>
<journal-title-group><journal-title>Biotechnology for Biofuels</journal-title>
</journal-title-group>
<issn pub-type="epub">1754-6834</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
<publisher-loc>London</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">25161697</article-id>
<article-id pub-id-type="pmc">4144037</article-id>
<article-id pub-id-type="publisher-id">512</article-id>
<article-id pub-id-type="doi">10.1186/1754-6834-7-109</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research</subject>
</subj-group>
</article-categories>
<title-group><article-title>Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Book</surname>
<given-names>Adam J</given-names>
</name>
<address><email>ajbook@wisc.edu</email>
</address>
<xref ref-type="aff" rid="Aff49"></xref>
<xref ref-type="aff" rid="Aff50"></xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Yennamalli</surname>
<given-names>Ragothaman M</given-names>
</name>
<address><email>ragothaman@rice.edu</email>
</address>
<xref ref-type="aff" rid="Aff51"></xref>
<xref ref-type="aff" rid="Aff52"></xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Takasuka</surname>
<given-names>Taichi E</given-names>
</name>
<address><email>ttakasuka@glbrc.wisc.edu</email>
</address>
<xref ref-type="aff" rid="Aff49"></xref>
<xref ref-type="aff" rid="Aff51"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Currie</surname>
<given-names>Cameron R</given-names>
</name>
<address><email>currie@bact.wisc.edu</email>
</address>
<xref ref-type="aff" rid="Aff49"></xref>
<xref ref-type="aff" rid="Aff50"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Phillips</surname>
<given-names>George N</given-names>
<suffix>Jr</suffix>
</name>
<address><email>georgep@rice.edu</email>
</address>
<xref ref-type="aff" rid="Aff49"></xref>
<xref ref-type="aff" rid="Aff51"></xref>
<xref ref-type="aff" rid="Aff52"></xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Fox</surname>
<given-names>Brian G</given-names>
</name>
<address><email>bgfox@biochem.wisc.edu</email>
</address>
<xref ref-type="aff" rid="Aff49"></xref>
<xref ref-type="aff" rid="Aff51"></xref>
</contrib>
<aff id="Aff49"><label></label>
Department of Energy, Great Lakes Bioenergy Research Center, Madison, 1552 University Avenue, Madison, WI 53726 USA</aff>
<aff id="Aff50"><label></label>
Department of Bacteriology, University of Wisconsin-Madison, Microbial Sciences Building, 1550 Linden Dr., Madison, WI 53706 USA</aff>
<aff id="Aff51"><label></label>
Department of Biochemistry, University of Wisconsin-Madison, Biochemistry Addition, 433 Babcock Dr., Madison, WI 53706 USA</aff>
<aff id="Aff52"><label></label>
Biosciences at Rice, Rice University, George R. Brown Hall, Houston, TX 77005 USA</aff>
</contrib-group>
<pub-date pub-type="epub"><day>6</day>
<month>8</month>
<year>2014</year>
</pub-date>
<pub-date pub-type="collection"><year>2014</year>
</pub-date>
<volume>7</volume>
<elocation-id>109</elocation-id>
<history><date date-type="received"><day>14</day>
<month>3</month>
<year>2014</year>
</date>
<date date-type="accepted"><day>7</day>
<month>7</month>
<year>2014</year>
</date>
</history>
<permissions><copyright-statement>© Book et al.; licensee BioMed Central Ltd. 2014</copyright-statement>
<license license-type="open-access"><license-p>This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0">http://creativecommons.org/licenses/by/4.0</ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>
) applies to the data made available in this article, unless otherwise stated.</license-p>
</license>
</permissions>
<abstract id="Abs1"><sec><title>Background</title>
<p>Understanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families.</p>
</sec>
<sec><title>Results</title>
<p>Comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site.</p>
</sec>
<sec><title>Conclusions</title>
<p>The structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.</p>
</sec>
<sec><title>Electronic supplementary material</title>
<p>The online version of this article (doi:10.1186/1754-6834-7-109) contains supplementary material, which is available to authorized users.</p>
</sec>
</abstract>
<kwd-group xml:lang="en"><title>Keywords</title>
<kwd>Lytic polysaccharide monooxygenase</kwd>
<kwd>LPMO</kwd>
<kwd>Cellulase</kwd>
<kwd>Chitinase</kwd>
<kwd>Streptomyces</kwd>
<kwd>AA9</kwd>
<kwd>AA10</kwd>
<kwd>Enzyme evolution</kwd>
<kwd>Biofuels</kwd>
</kwd-group>
<custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name>
<meta-value>© The Author(s) 2014</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><sec id="Sec1"><title>Background</title>
<p>The two most abundant polysaccharides in nature are cellulose and chitin [<xref ref-type="bibr" rid="CR1">1</xref>
]. Plants, insects, crustaceans, molluscs, and fungi all utilize these two highly stable polymers as primary components of their cell walls. Deconstruction of polysaccharides is essential for ecosystem-level carbon and nitrogen cycling. Moreover, polysaccharides are potential energy sources that could help supplement the current massive demand for fossil fuels [<xref ref-type="bibr" rid="CR2">2</xref>
]. Intensive efforts worldwide focus on conversion of these energy-rich biomolecules into free sugars that can be fermented into biofuels or other value-added bioproducts. However, hydrolysis of these polymers is difficult due to their crystalline structure, the stability of the β-glucosidic bond, and their close association with hemicellulose, lignin, and other modifying molecules [<xref ref-type="bibr" rid="CR1">1</xref>
, <xref ref-type="bibr" rid="CR3">3</xref>
]. Cellulolytic and chitinolytic enzymes capable of this have been identified in a myriad of organisms, but most often in bacteria and fungi [<xref ref-type="bibr" rid="CR4">4</xref>
]. While the biochemical activities and mechanisms of hydrolytic enzymes have been known for decades, oxygenolytic pathways for deconstruction of chitin and cellulose have only recently been identified [<xref ref-type="bibr" rid="CR5">5</xref>
–<xref ref-type="bibr" rid="CR8">8</xref>
].</p>
<p>CBH1, one of the first representatives of what are now recognized to be lytic polysaccharides monooxygenases (LPMOs), was secreted by <italic>Streptomyces olivaceoviridis</italic>
and interacted with α-chitin, but since it lacked classical hydrolytic activity, it was thus considered to be a non-hydrolytic carbohydrate binding module (CBM) [<xref ref-type="bibr" rid="CR9">9</xref>
]. An ortholog of CBH1, chitin-binding protein 21 (CBP21) was identified in <italic>Serratia marcescens</italic>
[<xref ref-type="bibr" rid="CR10">10</xref>
] and initially classified as carbohydrate binding module 33 (CBM33, now systematically called Auxiliary Activity 10, AA10).<sup>a</sup>
The function of CBP21 was first demonstrated by Vaaje-Kolstad <italic>et al</italic>
. [<xref ref-type="bibr" rid="CR11">11</xref>
], who showed cleavage of crystalline chitin in an O<sub>2</sub>
-dependent reaction. Soon after this report, others showed that the eukaryotic counterpart, fungal glycoside hydrolase 61 (GH61, now systematically called Auxiliary Activity 9, AA9) was a Cu<sup>2+</sup>
-dependent enzyme [<xref ref-type="bibr" rid="CR11">11</xref>
–<xref ref-type="bibr" rid="CR14">14</xref>
]. An oxidative function has also been demonstrated for CelS2, an AA10 from <italic>Streptomyces coelicolor</italic>
[<xref ref-type="bibr" rid="CR6">6</xref>
], which reacts synergistically with hydrolytic cellobiohydrolases and endoglucanases [<xref ref-type="bibr" rid="CR15">15</xref>
], and more recently for BlAA10A from <italic>Bacillus licheniformis</italic>
and E8 from <italic>Thermobifida fusca</italic>
[<xref ref-type="bibr" rid="CR16">16</xref>
] which react with chitin and cellulose, respectively, giving four AA10 enzymes whose function has been determined.</p>
<p>AA9 and AA10 incorporate a single <sup>18</sup>
O from <sup>18</sup>
O<sub>2</sub>
into polysaccharide cleavage products, and so are now classified as LPMOs [<xref ref-type="bibr" rid="CR11">11</xref>
]. To date, structures of six AA9 and five AA10 enzymes have been solved, including one nuclear magnetic resonance (NMR) structure [<xref ref-type="bibr" rid="CR11">11</xref>
, <xref ref-type="bibr" rid="CR17">17</xref>
–<xref ref-type="bibr" rid="CR19">19</xref>
]. Overall, the two LPMO families share a conserved β-sandwich fold [<xref ref-type="bibr" rid="CR11">11</xref>
], and many residues on the substrate-binding surface are conserved. Moreover, Cu<sup>2+</sup>
has been identified in the active sites [<xref ref-type="bibr" rid="CR8">8</xref>
, <xref ref-type="bibr" rid="CR17">17</xref>
, <xref ref-type="bibr" rid="CR18">18</xref>
]. Although recent computational studies support the involvement of a copper-oxyl radical intermediate [<xref ref-type="bibr" rid="CR20">20</xref>
], the catalytic mechanism of this reaction is still largely unexplored.</p>
<p>Oxidative polysaccharide cleavage results in the formation of an aldonic acid from C1 oxidation [<xref ref-type="bibr" rid="CR21">21</xref>
] or a ketoaldose from C4 oxidation [<xref ref-type="bibr" rid="CR21">21</xref>
, <xref ref-type="bibr" rid="CR22">22</xref>
]. The monooxygenase reaction stoichiometry requires the addition of 2e<sup>-</sup>
from an oxidoreductase or other external electron donor. The presence of oxidoreductases has been reported in various cellulolytic fungi [<xref ref-type="bibr" rid="CR23">23</xref>
], though an actual, physiological electron partner for LPMOs has not been unambiguously determined.</p>
<p>In this study, we compared amino acid sequences and protein structures in order to explore the evolutionary relatedness of AA9 and AA10. Conserved sequence and structural features were correlated with potential substrate interactions and surfaces potentially used by electron donors. Phylogenetic analysis suggests that cellulose- and chitin-specific enzymes are distributed into different subclades within bacterial AA10, as has been recently reported for the fungal AA9 [<xref ref-type="bibr" rid="CR18">18</xref>
, <xref ref-type="bibr" rid="CR21">21</xref>
]. Potential evolutionarily pressures within the AA10 family were examined in order to understand how Darwinian selection might have influenced substrate specificity.</p>
</sec>
<sec id="Sec2" sec-type="results"><title>Results</title>
<sec id="Sec3"><title>Structural comparison of LPMO families AA9 and AA10</title>
<p>Figure <xref rid="Fig1" ref-type="fig">1</xref>
a shows five crystal structures from the AA9 family. These are from <italic>Hypocrea jecorina</italic>
(<italic>Trichoderma reesei</italic>
, Protein Data Bank (pdb) id: 2VTC) [<xref ref-type="bibr" rid="CR24">24</xref>
], <italic>Thielavia terrestris</italic>
(pdb id: 3EII) [<xref ref-type="bibr" rid="CR7">7</xref>
], <italic>Thermoascus aurantiacus</italic>
(pdb id: 2YET) [<xref ref-type="bibr" rid="CR13">13</xref>
], <italic>Neurospora crassa</italic>
PMO-2 (pdb id: 4EIR) [<xref ref-type="bibr" rid="CR18">18</xref>
], and <italic>N. crassa</italic>
PMO-3 (pdb id: 4EIS) [<xref ref-type="bibr" rid="CR18">18</xref>
]. Structures of four AA10 enzymes are also shown in Figure <xref rid="Fig1" ref-type="fig">1</xref>
b. These are from <italic>S. marcescens</italic>
(pdb id: 2BEM) [<xref ref-type="bibr" rid="CR25">25</xref>
], <italic>Vibrio cholerae O1 biovar EI Tor</italic>
(pdb id: 2XWX) [<xref ref-type="bibr" rid="CR26">26</xref>
], <italic>Burkholderia pseudomallei</italic>
(pdb id: 3UAM), and <italic>Enterococcus faecalis</italic>
(pdb id: 4A02) [<xref ref-type="bibr" rid="CR27">27</xref>
]. Both AA9 and AA10 have a conserved β-sandwich fold with three to four β-sheet strands (Figure <xref rid="Fig1" ref-type="fig">1</xref>
c and <xref rid="Fig1" ref-type="fig">1</xref>
d). The average root mean square (RMS) deviation of the aligned structures is approximately 3 Å (Table <xref rid="Tab1" ref-type="table">1</xref>
) In addition to the fold-level similarity between AA9 and AA10, two key histidine (His) residues that coordinate a Cu<sup>2+</sup>
ion at their active sites are also highly conserved in both families (Table <xref rid="Tab1" ref-type="table">1</xref>
and Figure <xref rid="Fig1" ref-type="fig">1</xref>
c). The structural superposition of the metal ligands suggests that this configuration is essential for activity (inset in Figure <xref rid="Fig1" ref-type="fig">1</xref>
c and <xref rid="Fig1" ref-type="fig">1</xref>
d). A notable difference between AA9 and AA10 is the third, non-coordinating active site residue; being primarily tyrosine in the former and primarily phenylalanine in the latter, with a relatively few exceptions presently also identified.<fig id="Fig1"><label>Figure 1</label>
<caption><p><bold>Electrostatic surface comparison between AA9 and AA10 lytic polysaccharide monooxygenases.</bold>
The images show the protein surface containing the active site as presented to the substrate. The yellow circles indicate the location of the catalytic residues and bound Cu<sup>2+</sup>
. <bold>(a)</bold>
The AA9 family has a strip of positively charged surface sandwiched in an overall negatively charged surface (shown in red). <bold>(b)</bold>
In the AA10 family, a patch of positively charged surface (shown in blue) is adjacent to the active site. <bold>(c)</bold>
Superposition and inset showing the active site residues of AA9 from <italic>Hypocrea jecorina</italic>
(pdb id: 2VTC), <italic>Thielavia terrestris</italic>
(pdb id: 3EII), <italic>Thermoascus aurantiacus</italic>
(pdb id: 3ZUD), and <italic>Neurospora crassa</italic>
(pdb id: 4EIS and 4EIR) in ribbon representation and colored with respect to secondary structure (helix-red; strand-yellow; loop-green). The residues involved in the active site are shown as sticks and colored blue for <italic>H. jecorina</italic>
, magenta for <italic>T. terrestris</italic>
, orange for <italic>T. aurantiacus</italic>
, and light brown and gray for <italic>N. crassa</italic>
. The divalent metal atoms (Ni, Zn, Cu) are shown as spheres. The active site residues labeled in the inset are colored the same as the intact structures. <bold>(d)</bold>
Superposition and inset showing the active site residues of AA10 from <italic>Serratia marcescens</italic>
(pdb id: 2BEM), <italic>Vibrio cholerae</italic>
(pdb id: 2XWX), <italic>Burkholderia pseudomallei</italic>
(pdb id: 3UAM), and <italic>Enterococcus faecalis</italic>
(pdb id: 4A02) shown in ribbon representation and colored with respect to secondary structure. The residues (inset) involved in the active site are shown as sticks and colored green for <italic>S. marcescens</italic>
, blue for <italic>V. cholerae</italic>
, magenta for <italic>B. pseudomallei</italic>
, and orange for <italic>E. faecalis.</italic>
</p>
</caption>
<graphic xlink:href="13068_2014_512_Fig1_HTML" id="d30e673"></graphic>
</fig>
</p>
<table-wrap id="Tab1"><label>Table 1</label>
<caption><p><bold>Structural homology of lytic polysaccharide monooxygenases</bold>
</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>PDB ID</th>
<th>% RMSD
<sup>1</sup>
</th>
<th>%id</th>
<th>Source structure</th>
<th>CAZy family</th>
<th>Source organism</th>
<th>Active site residues</th>
</tr>
</thead>
<tbody><tr><td>2BEM</td>
<td>0</td>
<td>100</td>
<td>X-ray</td>
<td>AA10</td>
<td><italic>Serratia marcescens</italic>
</td>
<td>H28-H114-F187</td>
</tr>
<tr><td>2XWX</td>
<td>0.8</td>
<td>51</td>
<td>X-ray</td>
<td>AA10</td>
<td><italic>Vibrio cholerae</italic>
</td>
<td>H24-H121-F193</td>
</tr>
<tr><td>4A02</td>
<td>1</td>
<td>52</td>
<td>X-ray</td>
<td>AA10</td>
<td><italic>Enterococcus faecalis</italic>
</td>
<td>H29-H114-F185</td>
</tr>
<tr><td>2LHS</td>
<td>1.4</td>
<td>100</td>
<td>NMR</td>
<td>AA10</td>
<td><italic>Serratia marcescens</italic>
</td>
<td>H28-H114-F187</td>
</tr>
<tr><td>3UAM</td>
<td>1.4</td>
<td>39</td>
<td>X-ray</td>
<td>AA10</td>
<td><italic>Burkholderia pseudomallei</italic>
</td>
<td>H19-H122-F205</td>
</tr>
<tr><td>2VTC</td>
<td>3.2</td>
<td>9</td>
<td>X-ray</td>
<td>AA9</td>
<td><italic>Hypocrea jecorina</italic>
</td>
<td>H1-H89-Y176</td>
</tr>
<tr><td>4EIR</td>
<td>2.8</td>
<td>9</td>
<td>X-ray</td>
<td>AA9</td>
<td><italic>Neurospora crassa</italic>
</td>
<td>H1-H84-Y168</td>
</tr>
<tr><td>3ZUD</td>
<td>3.3</td>
<td>12</td>
<td>X-ray</td>
<td>AA9</td>
<td><italic>Thermoascus aurantiacus</italic>
</td>
<td>H1-H86-Y175</td>
</tr>
<tr><td>3EII</td>
<td>3.2</td>
<td>11</td>
<td>X-ray</td>
<td>AA9</td>
<td><italic>Thielavia terrestris</italic>
</td>
<td>H1-H68-Y153</td>
</tr>
<tr><td>4EIS</td>
<td>2.8</td>
<td>7</td>
<td>X-ray</td>
<td>AA9</td>
<td><italic>Neurospora crassa</italic>
</td>
<td>H1-H82-Y171</td>
</tr>
</tbody>
</table>
<table-wrap-foot><p><sup>1</sup>
Root mean square deviation (RMSD) (%) for each structure from Protein Data Bank (pdb) compared to 2BEM determined by X-ray crystallography; %id indicates the percentage identity of each sequence to that of 2BEM. Three active site residues, His, His, and Phe/Tyr, are shown with residue numbers.</p>
</table-wrap-foot>
</table-wrap>
<p>Six AA10 structures from <italic>E. faecalis</italic>
released in the pdb show copper in the active site, and a recently published structure of AA9 from <italic>Phanerochaete chrysosporium</italic>
(pdb id: 4B5Q) also shows copper bound in the active site [<xref ref-type="bibr" rid="CR28">28</xref>
]. Copper binds with nanomolar affinity to AA10 [<xref ref-type="bibr" rid="CR8">8</xref>
, <xref ref-type="bibr" rid="CR17">17</xref>
]; its presence is consistent with O<sub>2</sub>
activation required for the LPMO reaction.</p>
</sec>
<sec id="Sec4"><title>Surface electrostatic potential on the binding surfaces of AA9 and AA10</title>
<p>To explore factors that may contribute to substrate specificity in the AA9 and AA10 families, we characterized the electrostatic potential present at the substrate-binding surface. In both families, the metal-binding histidine residues are part of a planar surface that constitutes the polysaccharide-binding surface [<xref ref-type="bibr" rid="CR18">18</xref>
]. Figure <xref rid="Fig1" ref-type="fig">1</xref>
a and <xref rid="Fig1" ref-type="fig">1</xref>
b show the surface electrostatic potential of representatives from both AA9 and AA10 families. For the AA9 proteins, which are biochemically characterized as cellulose monooxygenases, negatively charged residues (shown in red) prominently surround the active site (Figure <xref rid="Fig1" ref-type="fig">1</xref>
a, yellow circle). In contrast, the AA10 chitin monooxygenases contain both positively charged (shown in blue) and negatively charged residues (shown in red) surrounding the active site (Figure <xref rid="Fig1" ref-type="fig">1</xref>
b, yellow circle). Aachmann <italic>et al.</italic>
[<xref ref-type="bibr" rid="CR17">17</xref>
] used NMR to identify residues from the chitinolytic AA10 enzyme from <italic>S. marcescens</italic>
(pdb id: 2BEM) that are involved in chitin binding. These residues are Q53, Y54, E55, Q67, S58, L110, T111, A112, H114, and T116 [<xref ref-type="bibr" rid="CR17">17</xref>
]. The positions of the corresponding residues from the other AA10 enzymes that align with 2BEM are shown as yellow on a grey surface in the lower parts of Figure <xref rid="Fig1" ref-type="fig">1</xref>
a and <xref rid="Fig1" ref-type="fig">1</xref>
b. In the other members of the AA10 family, most of these structurally conserved residues are also surface-exposed (Figure <xref rid="Fig1" ref-type="fig">1</xref>
b, bottom). However, in the AA9 family, only a few are exposed at the polysaccharide-binding surface (Figure <xref rid="Fig1" ref-type="fig">1</xref>
a, bottom), indicating that different residues from the folded structures will be involved in substrate binding in the AA9 and AA10 families.</p>
</sec>
<sec id="Sec5"><title>Diversity of domain structures in AA9 and AA10 proteins</title>
<p>Another possible determinant of substrate specificity within the AA9 and AA10 families is the domain architecture. LPMO enzymes have a diverse composition of domains: they can be single catalytic domains, associated with various CBMs, or even associated with other catalytic domains (such as glycoside hydrolase (GH) domains). Figure <xref rid="Fig2" ref-type="fig">2</xref>
shows a Cytoscape (The Cytoscape Consortium, San Diego, CA) protein sequence homology network accounting for the variations in domains in the AA9 and AA10 families, where nodes represent enzymes or functional classes, and edges represent sequence similarity (bit score >200, evalue <1e<sup>-50</sup>
). In order to prepare this network, sequences were collected from CAZy, compared via pairwise BLAST analysis, and then annotated with secondary CAZy domains. Nodes are colored according to their phylum-level taxonomic identification. The network contains 184 AA9 sequences and 495 AA10 sequences. All AA9 proteins were from eukaryotes, with a vast majority (99%) from the fungal phyla Ascomycota (135 sequences) and Basidiomycota (34 sequences). Of the protein sequence in the AA9 family, 31% include a secondary carbohydrate binding module 1 (CBM1), which has been reported to bind cellulose [<xref ref-type="bibr" rid="CR29">29</xref>
]. Seven AA9 sequences are associated with CBM0, an unclassified CBM family [<xref ref-type="bibr" rid="CR30">30</xref>
, <xref ref-type="bibr" rid="CR31">31</xref>
].<fig id="Fig2"><label>Figure 2</label>
<caption><p><bold>Domain and sequence similarity networks for the LPMO superfamily.</bold>
Circles represent proteins from either the AA9 or AA10 families, diamonds represent CAZy annotations. Edges represent either BLAST similarity with a bit score greater than 200 (evalue > e<sup>-50</sup>
) or annotation to the indicated CAZy functional group. Colors represent taxonomic distribution of phyla of the source organisms. No sequence similarity above the indicated threshold was identified between the AA9 and AA10 superfamilies.</p>
</caption>
<graphic xlink:href="13068_2014_512_Fig2_HTML" id="d30e1077"></graphic>
</fig>
</p>
<p>The AA10 family is exclusively from prokaryotes, with 226 sequences from Proteobacteria, 145 from Actinobacteria, and 132 from Firmicutes (Figure <xref rid="Fig2" ref-type="fig">2</xref>
). There were no edges linking members of the AA9 and AA10 families at the similarity threshold of evalue <1e<sup>-50</sup>
. Furthermore, when the similarity threshold was relaxed to 1e<sup>-5</sup>
there were still no connections between the AA9 and AA10 families. While Figure <xref rid="Fig2" ref-type="fig">2</xref>
shows that the AA9 network contains interspersed sequences from Ascomycota and Basidiomycota, the AA10 family shows clear taxonomic groupings assembled from different bacterial phyla. These results also show that while the active site residues of the AA9 and AA10 families are mostly conserved (Table <xref rid="Tab1" ref-type="table">1</xref>
), these two families do not share any other significant sequence similarities or consistent linkages to other domains.</p>
<p>Figure <xref rid="Fig2" ref-type="fig">2</xref>
also shows that the AA10 family is combined with a variety of secondary CBM domains, with 31% of the total sequences including cellulose-binding domains CBM2 and CBM3 [<xref ref-type="bibr" rid="CR32">32</xref>
, <xref ref-type="bibr" rid="CR33">33</xref>
] or chitin-binding domains CBM5 and CBM12 [<xref ref-type="bibr" rid="CR34">34</xref>
]. Further phylogenetic binning of AA10 showed expansion within the genera of <italic>Streptomyces</italic>
, <italic>Bacillus</italic>
, and <italic>Vibrio</italic>
(Additional file <xref rid="MOESM1" ref-type="media">1</xref>
: Figure S1). Interestingly, 94% of the AA10 sequences that included a cellulose-binding CBM were from the phylum Actinobacteria, whereas 95% of sequences including a chitin-binding CBM were from the phyla Firmicutes and Proteobacteria. Finally, two genes were identified that also encoded a glycoside hydrolase domain, suggesting a rare but possibly synergistic pairing of glycoside hydrolase and LPMO catalytic activities in a single enzyme.</p>
</sec>
<sec id="Sec6"><title>Phylogenic analysis of LPMO families</title>
<p>To gain further insight into the evolutionary relationship and possible functional roles of the distinct LPMO families, we created phylogenetic trees representing the AA9 and AA10 families (Figures <xref rid="Fig3" ref-type="fig">3</xref>
and <xref rid="Fig4" ref-type="fig">4</xref>
, respectively). Briefly, sequences were collected, curated to remove redundant sequences with 100% identity, aligned, trimmed to the conserved catalytic domain, and then the tree was constructed by MrBayes phylogenetic analysis [<xref ref-type="bibr" rid="CR35">35</xref>
]. The resulting consensus tree was midpoint rooted and annotated with associated carbohydrate-binding modules in addition to the AA9 or AA10 catalytic domains. The five crystal structures determined for AA9, 2YET, 2VTC, 4EIS, 4EIR, and 3EII, were mapped onto the phylogenetic tree. In Figure <xref rid="Fig3" ref-type="fig">3</xref>
, the surfaces of these structures have been colored to identify highly conserved residues shared across the AA9 family. The tree was also annotated to indicate whenever a putative cellobiose dehydrogenase (AA3 family enzymes) was present in the host genome using a cutoff criterion of 35% identity to <italic>N. crassa</italic>
CDH1. The ability of CDH to act as the proximal electron donor for LPMO in cellulose oxidative cleavage has been demonstrated in this organism [<xref ref-type="bibr" rid="CR18">18</xref>
, <xref ref-type="bibr" rid="CR36">36</xref>
–<xref ref-type="bibr" rid="CR38">38</xref>
].<fig id="Fig3"><label>Figure 3</label>
<caption><p><bold>Phylogenetic analysis of the AA9 LPMO superfamily.</bold>
MrBayes phylogenetic tree for 254 AA9 protein sequences. The tree was generated using the catalytic domain of the AA9 protein only. Additional carbohydrate-binding domains that are present in the full protein sequence are indicated in the CBM column, but were not included in the calculation of the tree structure. Source organisms were searched for the presence of a homolog to <italic>Neurospora crassa</italic>
cellobiose dehydrogenase (CDH1). Protein identity scores are indicated in the CDH column, and colors range from 30% identity (green) to 100% identity (red). Solved structures have been mapped onto the tree and colors represent conservation of residues across the whole AA9 family.</p>
</caption>
<graphic xlink:href="13068_2014_512_Fig3_HTML" id="d30e1166"></graphic>
</fig>
<fig id="Fig4"><label>Figure 4</label>
<caption><p><bold>Phylogenetic analysis of the AA10 LPMO superfamily.</bold>
MrBayes phylogenetic tree for the 374 AA10 protein sequences. The tree was generated using the catalytic domain of the AA10 protein only. Additional carbohydrate-binding domains that are present in the full protein sequence are indicated in the CBM column, but were not included in the calculation of the tree structure. Solved structures have been mapped onto the tree and colors represent conservation of residues across the whole AA9 family, and in the three modeled structures for <italic>Streptomyces</italic>
sp. SirexAA-E and AA10 enzymes. Gene expression data for the six AA10 isoforms from SirexAA-E showing fold change in transcripts from glucose grown cells to either cellulose or chitin grown cells.</p>
</caption>
<graphic xlink:href="13068_2014_512_Fig4_HTML" id="d30e1181"></graphic>
</fig>
</p>
<p>The AA9 LPMOs have been classified into four functional types based on their reaction products [<xref ref-type="bibr" rid="CR21">21</xref>
]. These are shown in Figure <xref rid="Fig3" ref-type="fig">3</xref>
as red boxes. LPMO1 enzymes hydroxylate the C1 position of pyranose rings and produce an aldonolactone [<xref ref-type="bibr" rid="CR18">18</xref>
, <xref ref-type="bibr" rid="CR21">21</xref>
], while LPMO2 enzymes hydroxylate the C4 position of pyranose rings and produce a 4-ketoaldose [<xref ref-type="bibr" rid="CR21">21</xref>
, <xref ref-type="bibr" rid="CR22">22</xref>
]. LPMO3 enzymes are less specific [<xref ref-type="bibr" rid="CR13">13</xref>
, <xref ref-type="bibr" rid="CR19">19</xref>
, <xref ref-type="bibr" rid="CR21">21</xref>
, <xref ref-type="bibr" rid="CR39">39</xref>
], and produce both aldonolactone and non-reducing end oxidized products, while LPMO3* produce only aldonic acids [<xref ref-type="bibr" rid="CR21">21</xref>
].Mapping of the four LPMO subgroups onto the global AA9 phylogeny showed that the LPMO2, LPMO3, and LPMO3* subgroups are monophyletic, with each having a single phylogenetic clade that corresponds to distinct functional classes (red boxes). In contrast, LPMO1 enzymes span a major evolutionary division as two branches cross into this functional class, indicating more sequence diversity in the LPMO1 family. Examples where all four LPMO functional types were fused to additional CBM domains are identified in Figure <xref rid="Fig3" ref-type="fig">3</xref>
. Moreover, Figure <xref rid="Fig3" ref-type="fig">3</xref>
also shows that the majority of AA9 proteins come from organisms that also contain a cellobiose dehydrogenase homolog.</p>
<p>The AA10 phylogenetic tree was generated in a similar manner using the catalytic domains of all non-redundant sequences present in the CAZy database. The AA10 tree shown in Figure <xref rid="Fig4" ref-type="fig">4</xref>
represents 374 non-redundant sequences that are entirely bacterial in origin. The tree was annotated with secondary CBM domains (central column), and divided into two major clades (clade I and clade II) that could be subdivided into four additional subclades (A through D). The biochemically characterized cellulose-oxidizing LPMOs from <italic>S. coelicolor</italic>
(A3) and <italic>T. fusca</italic>
are present in subclade A,<sup>b</sup>
while all other LPMOs with experimental confirmation of their reaction with chitin are present in subclades C and D [<xref ref-type="bibr" rid="CR6">6</xref>
].The tree was also annotated with microarray-based gene expression data for the six variants of AA10 present in <italic>Streptomyces</italic>
sp. SirexAA-E (SirexAA-E) [<xref ref-type="bibr" rid="CR40">40</xref>
]. Clade I contains a delineated mixture of phyla, with subclade C containing sequences only from Actinobacteria and with subclade D containing sequences from Firmicutes and Proteobacteria. Clade II is primarily composed of Actinobacteria and separates into subclades A and B. Subclades A and B contain only cellulose-binding CBMs (CBM2 and CBM3) associated with the catalytic AA10 domain, whereas subclades C and D contain only chitin-binding CBMs (CBM5 and 12). Furthermore, expression data from SirexAA-E shows that genes from subclades A and B were selectively upregulated only when cells were grown in medium containing cellulose as the sole carbon source, while genes from subclade C were upregulated only during growth on chitin [<xref ref-type="bibr" rid="CR40">40</xref>
].</p>
<p>The cellulose-oxidizing LPMOs from AA10 are primarily present in Actinobacteria, an aerobic filamentous bacterial phyla found in soil, but also associated with insects and other animals [<xref ref-type="bibr" rid="CR40">40</xref>
]. In Figure <xref rid="Fig4" ref-type="fig">4</xref>
, the structures of four AA10 enzymes are mapped to the phylogenetic tree: 3UAM, 4A02, 2BEM, and 2XWX. Additionally, predicted protein structures for expressed AA10 from SirexAA-E are mapped onto the tree. There is high amino-acid sequence identity among the AA10 proteins whose structures have been determined, with the highest sequence conservation observed at the active site (magenta color). Interestingly, homology models consistently predict an additional surface exposed loop region on the same side of the protein as the active site in clade II proteins (chitin oxidation), but not in clade I (cellulose oxidation). The position of this loop can be recognized in pdb id: 4GBO, the E7 enzyme from <italic>T. fusca</italic>
[<xref ref-type="bibr" rid="CR16">16</xref>
]
<italic>.</italic>
Recently, Vu <italic>et al</italic>
. have identified a role for these extra loops in substrate recognition and control of specificity of reaction in the AA9 family [<xref ref-type="bibr" rid="CR21">21</xref>
].</p>
</sec>
<sec id="Sec7"><title>Homology modeling of AA10 proteins and conserved sequence motif in LPMOs</title>
<p>Several LPMOs from within the AA10 family have been experimentally verified to be either chitin or cellulose monooxygenases (such as CBP21 and BlAA10A) which react with chitin, and CelS2 and E8, which react with cellulose [<xref ref-type="bibr" rid="CR6">6</xref>
, <xref ref-type="bibr" rid="CR25">25</xref>
].<sup>b</sup>
To further explore structural determinants that control substrate specificity, we compared homology models for 43 proteins that spanned the AA10 family (Figure <xref rid="Fig5" ref-type="fig">5</xref>
) across the clade I and clade II sequences shown in Figure <xref rid="Fig4" ref-type="fig">4</xref>
. Homology modeling using I_TASSER [<xref ref-type="bibr" rid="CR41">41</xref>
], followed by superposition of the modeled structures showed that the most significant structural differences were located in the substrate binding region (Figure <xref rid="Fig5" ref-type="fig">5</xref>
, Additional file <xref rid="MOESM2" ref-type="media">2</xref>
: Table S1). Specifically, the positions of loops (shown for illustration purposes only) on the substrate-binding side of the protein had more variations than other parts of the modeled structures. Correspondingly, the insertion observed in the sequence alignments mapped to loops on the substrate-binding side of the AA10 family. Given the structural variability of clades I and II, and differences in measured catalytic functions, it is likely that these structural differences help to modulate substrate selectivity between chitin and cellulose in AA10, as now predicted for AA9.<fig id="Fig5"><label>Figure 5</label>
<caption><p><bold>Three Multiple Em for Motif Elicitation (MEME) motifs mapped to the predicted structures of AA10.</bold>
MEME was used to identify motifs from four separate subclades A to D from the phylogenetic tree of AA10. The loops are shown for illustration purposes only. The number of sequences in each clade is as follows: subclade A (68 sequences), clade B (29 sequences), clade C (77 sequences), and clade D (122 sequences). These motifs were mapped to the structures that were predicted using iterative threading assembly refinement algorithm (I-TASSER), as shown in <bold>(a)</bold>
. Motif1 is shown in cyan color, motif2 is shown in blue and motif3 is shown in red. <bold>(b)</bold>
MEME motifs mapped to the sequences from the available crystallographic structures of AA10 and AA9, showing the distribution of three motifs in the sequence. Sequence logo of <bold>(c)</bold>
motif1 (cyan), <bold>(d)</bold>
motif2 (blue), and <bold>(e)</bold>
motif3 (red) in the available structures.</p>
</caption>
<graphic xlink:href="13068_2014_512_Fig5_HTML" id="d30e1333"></graphic>
</fig>
</p>
<p>To improve our mapping of potential functional determinants onto the modeled structures, Multiple EM for Motif Elicitation (MEME) [<xref ref-type="bibr" rid="CR42">42</xref>
] was used. This approach identified three sequence motifs among the 43 AA10 proteins (Figure <xref rid="Fig5" ref-type="fig">5</xref>
). These motifs were mapped back onto the structures and homology models. Simultaneously, MEME was used to determine whether there were significant motifs observed in the published structures of AA9 (Figure <xref rid="Fig5" ref-type="fig">5</xref>
).In the homology-modeled proteins (shown in Figure <xref rid="Fig5" ref-type="fig">5</xref>
corresponding to the four AA10 clades shown in Figure <xref rid="Fig4" ref-type="fig">4</xref>
), the three MEME motifs ranged from about 25 to 41 residues in length. Motif1 was present in both AA9 and AA10, and contained the variable insertion regions that possibly yield substrate selectivity in the AA10 family. Motif2 and motif3 were observed only in AA10 (Figure <xref rid="Fig5" ref-type="fig">5</xref>
b). It is interesting to note that the difference in the number of motifs identified in AA9 as compared to AA10 provides an additional line of evidence supporting the possibility of evolutionary selection in these two families. Although experimental evidence as to what these motifs (Figure <xref rid="Fig5" ref-type="fig">5</xref>
c-e) contribute is currently lacking, it is clear from the superposition of the homology-modeled structures (Figure <xref rid="Fig5" ref-type="fig">5</xref>
, cyan sequences) that motif1 is well-positioned to play a role in substrate binding and discrimination between binding to chitin or cellulose. Interestingly, motif2 and motif3 span the breadth of the protein and connect the substrate-binding surface to the opposite side of the protein where potential electron donor proteins might interact.</p>
</sec>
<sec id="Sec8"><title>Evolution of chitinolytic and cellulolytic subclades within the AA10 family</title>
<p>To evaluate the selective pressure on these functionally defined clades, the rates of non-synonymous and synonymous codon substitutions (dN and dS, respectively) of the catalytic domain were estimated (Figure <xref rid="Fig6" ref-type="fig">6</xref>
a). Pairwise comparisons were performed against all genes within either subclade A or subclade D. Estimations with dN values greater than 0.01 and dS values less than 1.5 were reported to allow sufficient mutational signal and to avoid the effect of back mutations that would artificially increase dS and reduce dN with increased sequence divergence [<xref ref-type="bibr" rid="CR43">43</xref>
]. Pairwise comparisons indicate that a group of the chitinolytic genes from subclade D are primarily under negative selection (Figure <xref rid="Fig6" ref-type="fig">6</xref>
, dN/dS <0.2), while a second group is under more neutral selection (1 > dN/dS >0.2). However, genes from subclade A have a significantly different distribution than from subclade D. Very few genes from cellulolytic subclade A show negative selection, while a significant proportion show increased positive selection (dN/dS >1). To confirm these results, site-specific dN/dS values were estimated for subclades A and D (Figure <xref rid="Fig6" ref-type="fig">6</xref>
b). The results show that a significant number of residues in subclade A were indeed positively selected, while residues in subclade D were all negatively or neutrally selected. When plotted on the protein structures, the negatively selected sites in both subclades A and D are primarily located around the active-site residues. In contrast, most of the positively selected residues in subclade A are surface exposed, including regions on the putative substrate-binding surface and along the interior of the protein traversing from the substrate-binding surface to the opposite surface of the protein. Interestingly, this latter region may provide a surface for interaction with accessory redox proteins such as cellobiose dehydrogenase (AA3 enzymes).<fig id="Fig6"><label>Figure 6</label>
<caption><p><bold>Evolution of chitinolytic and cellulolytic AA10 genes. (a)</bold>
Plot of pairwise estimated dN and dS for genes from cellulolytic and chitinolytic clades, clade A (circles) and clade D (diamonds), respectively. Values were calculated by pairwise comparison of all genes within each clade and filtered to remove insignificant values (see text). Also shown are trend lines for dN/dS ratios of 1 and 0.2, representing approximate thresholds for positive and negative selection. Site-specific estimation of dN/dS ratios for clade A <bold>(b)</bold>
and clade D <bold>(c)</bold>
. Positively selected residues are colored in red, neutral in grey, and negative in blue (x-axis corresponds to protein sequence and y-axis to the posterior probability of the estimation). dN/dS ratios were also mapped onto the CBP21 structure 2BEM, and the modeled structure for SACTE_3159. Colors correspond to selection rates described above.</p>
</caption>
<graphic xlink:href="13068_2014_512_Fig6_HTML" id="d30e1395"></graphic>
</fig>
</p>
</sec>
</sec>
<sec id="Sec9" sec-type="discussion"><title>Discussion</title>
<p>In this study, we analyzed the AA9 and AA10 families using available protein structures and sequence information to evaluate differences between and within the families, to explore features that influence substrate specificity, and to characterize selective pressures that may have led to functional diversification.</p>
<p>LPMOs share a common structural fold and a spatial conservation of active site residues, as seen by their low root mean square deviation (RMSD) values (ranging up to 3.3 Å, Table <xref rid="Tab1" ref-type="table">1</xref>
). While the core structural folds and the active site geometry of these two LPMO families are similar, there is low homology at the amino-acid sequence level, and the surface electrostatic potentials at the substrate-binding surface show considerable differences in charge distributions. Indeed, comparison of all AA9 and AA10 proteins available in the CAZy database failed to identify any sequences from across these two families that have significant homology (evalue <1e<sup>-5</sup>
). Our results indicate that although AA9 and AA10 families share structural similarities, they have so significantly diverged from a common ancestor that the only residue-level homology that remains is in the active site residues.</p>
<p>Due to the low sequence similarity between AA9 and AA10 families we analyzed their phylogenetic relationships separately. The AA9 phylogenetic tree is separated into three major evolutionarily related groups which partially correspond to the four types of enzyme activity observed for LPMOs [<xref ref-type="bibr" rid="CR21">21</xref>
]. LPMO2, LPMO3, and LPMO3* enzyme activities correspond to monophyletic clades, which suggests vertical inheritance and conserved enzyme functions within each clade. In contrast, LPMO1 enzymes are present in a polyphyletic clade, indicating a more diverse sequence space and potentially varied enzyme function. Sequences from Ascomycetes and Basidiomycetes are scattered throughout the three major evolutionarily related groups in AA9, suggesting an ancestral sequence that was shared before these two phyla separated.</p>
<p>The AA10 phylogenetic tree was separated into two major phylogenetic groups. When annotated with known activities, the two clades appear to separate enzymes with different substrate specificities. Clade I contains all biochemically defined chitin monooxygenases, while clade II contains subclades that are either cellulose or chitin monooxygenases. Gene expression data from SirexAA-E grown on either chitin or cellulose as the sole carbon source further corroborates this assessment [<xref ref-type="bibr" rid="CR40">40</xref>
]. We also observed that CBM domain composition varies between clade I and II. Clade I is dominated by CBM5 and 12 domains, which are primarily chitin binding, but possibly can have a lignin-binding function as well [<xref ref-type="bibr" rid="CR44">44</xref>
]. Clade II is enriched in CBM2 domains, which are primarily associated with cellulose binding. Most recently, Forsberg <italic>et al</italic>
. showed the binding specificity of CelS2 either with or without the associated CBM2 domain [<xref ref-type="bibr" rid="CR16">16</xref>
]. Interestingly, although this CBM2 domain was tightly bound to either α or β-chitin, the corresponding AA10 domain (CelS2) only reacted with cellulose. Further biochemical verification will be necessary to extend these observations more broadly into phylogenetic space.</p>
<p>To identify sequence and structural features that may contribute to clade II activity against cellulose, we generated homology-modeled structures for 43 sequences that span the phylogeny in AA10. Using MEME, these homology-modeled structures were identified to have three highly significant motifs, where motif1 shows the largest structural variability. Specifically, this variable motif is contained in a loop of un-modeled sequence at the substrate-binding surface and is only found in subclade A. Subclade A of AA10 contains biochemically characterized cellulose monooxygenases, and also contains the most highly upregulated AA10 enzyme when SirexAA-E is grown on cellulose [<xref ref-type="bibr" rid="CR40">40</xref>
]. We hypothesize that this additional sequence at the binding surface is a defining feature of cellulose-active AA10 enzymes, paralleling the identification of a loop-modulating reaction specificity in the AA9 enzymes [<xref ref-type="bibr" rid="CR21">21</xref>
]. Motif2 and motif3, which span the breadth of the protein, connect the substrate-binding surface to the opposite side of the protein. This suggests a possibility for modulation of electron donor interactions.</p>
<p>Finally, we explored the selective pressures within two clades of the AA10 family to understand how diversification may be distributed in this enzyme family. The results show that chitinolytic enzymes in subclade D (chitinolytic enzymes) have mostly negative selection at both the whole gene and site-specific levels. In contrast, subclade A (both chitinolytic and cellulolytic enzymes) contains more genes with diversifying selection at both the whole gene and site-specific levels. This result indicates that subclade A may have undergone a change in substrate specificity and that genes within this clade are potentially being selected for increased activity. Together, these data suggest that the ancestral form of AA10 may have been a chitin monooxygenase, and that clade II has apparently further specialized for cellulose oxidation. Selection may be towards more favorable substrate binding, better interactions with accessory redox proteins, such as cellobiose dehydrogenase enzymes, or perhaps both.</p>
</sec>
<sec id="Sec10" sec-type="conclusions"><title>Conclusions</title>
<p>In summary, this study provides a better understanding of the evolution of functional diversity within the recently discovered AA9 and AA10 LPMO families. Together, these data suggest that AA9 and AA10 families share a distant common ancestor. Furthermore, clades within the AA10 family are specialized for different substrates and subclade A has undergone diversifying selection at surface-exposed regions of the protein.</p>
</sec>
<sec id="Sec11" sec-type="materials|methods"><title>Materials and methods</title>
<sec id="Sec12"><title>Sequence similarity network</title>
<p>AA9 and AA10 protein-coding sequences were identified on the Carbohydrate-Active Enzyme (CAZy) database [<xref ref-type="bibr" rid="CR45">45</xref>
], and harvested from the National Center for Biotechnology Information (NCBI) protein database. All AA9 and AA10 sequences were compared against each other using BLAST [<xref ref-type="bibr" rid="CR46">46</xref>
] to identify similar proteins. All sequences were also re-annotated with CAZy families to identify the domain structure of each protein. This data was then used to build a similarity network using Cytoscape 2.8.0 [<xref ref-type="bibr" rid="CR47">47</xref>
], and visualized as an organic layout. Nodes in the network represent unique protein sequences and CAZy families. Edges represent a BLAST bit score of ≥200 (evalue ≥1 × e<sup>-50</sup>
), or an annotation to a CAZy category. Nodes were annotated with taxonomic information at the phylum level.</p>
</sec>
<sec id="Sec13"><title>Phylogenetic tree construction</title>
<p>AA9 and AA10 phylogenetic trees were constructed by first identifying proteins from the CAZy database, and the harvesting sequence from NCBI. Sequences from either AA9 or AA10 families were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) on the Cyberinfrastructure for Phylogenetic Research (CIPRES, <ext-link ext-link-type="uri" xlink:href="https://www.phylo.org/portal2/login!input.action">https://www.phylo.org/portal2/login!input.action</ext-link>
) Science Gateway [<xref ref-type="bibr" rid="CR48">48</xref>
]. Aligned sequences were then trimmed to retain only the AA9 or AA10 domain; sequences lacking the conserved active site His residues were removed from the alignment. Phylogenetic trees were generated using MrBayes code with a calculated standard deviation of ≤0.05. Non-default parameters were set to mcmc, ngen = 10,000,000, temp = 0.200, burninfrac = 0.25, stoprule = No, sump burnin = 4000, and sumt burnin = 4000. Resulting trees were annotated with pfam, phyla, solved structures, and cellobiose dehydrogenase homolog information.</p>
</sec>
<sec id="Sec14"><title>Evolutionary rate estimation</title>
<p>Coding sequences for subclades A-D from the AA10 family were collected and codon alignments were generated with MUSCLE. Sequences were trimmed to retain only the AA10 domain. Codon alignments were masked with Zorro (<ext-link ext-link-type="uri" xlink:href="http://phylogenomics.wordpress.com/software/zorro/">http://phylogenomics.wordpress.com/software/zorro/</ext-link>
) to generate quality scores for codon positions [<xref ref-type="bibr" rid="CR49">49</xref>
], and then a phylogenetic tree was generated with RAxML (<ext-link ext-link-type="uri" xlink:href="http://sco.h-its.org/exelixis/web/software/raxml/index.html">http://sco.h-its.org/exelixis/web/software/raxml/index.html</ext-link>
) using the masking scores [<xref ref-type="bibr" rid="CR50">50</xref>
]. Pairwise codon substitution models (dN/dS values) were estimated using the CODEML program in the PAML package [<xref ref-type="bibr" rid="CR51">51</xref>
, <xref ref-type="bibr" rid="CR52">52</xref>
]. Variables were set at CodonFreq = 0 and model = 0. Only pairwise dN values with values ≥0.01 and dS values ≤1.5 were reported so as to allow for sufficient mutational signal and to avoid the effects of back mutations. Site-specific codon substitution models were generated using the CODEML program in PAML, with model = 0, NSsites = 3, ncatG = 3 fix_kappa = 0, fix_omega = 0, cleandata = 1, and fix_blength = 2.</p>
</sec>
<sec id="Sec15"><title>Protein three-dimensional structure comparison</title>
<p>The Dali protein structure alignment database (<ext-link ext-link-type="uri" xlink:href="http://ekhidna.biocenter.helsinki.fi/dali_server/">http://ekhidna.biocenter.helsinki.fi/dali_server/</ext-link>
) was used to calculate %RMSD and %ID of LPMO enzymes whose structures are known using 2BEM as a query [<xref ref-type="bibr" rid="CR53">53</xref>
]. Structures with the ten best%RMSD are shown in Table <xref rid="Tab1" ref-type="table">1</xref>
.</p>
</sec>
<sec id="Sec16"><title>Homology modeling</title>
<p>The 43 sequences highlighted in Figure <xref rid="Fig4" ref-type="fig">4</xref>
were considered for prediction of three-dimensional structures using iterative threading assembly refinement algorithm (I-TASSER) (<ext-link ext-link-type="uri" xlink:href="http://zhanglab.ccmb.med.umich.edu/I-TASSER/">http://zhanglab.ccmb.med.umich.edu/I-TASSER/</ext-link>
) [<xref ref-type="bibr" rid="CR41">41</xref>
]. For each sequence, the signal peptides and other domains besides the Cu<sup>2+</sup>
-binding catalytic domain were included in the homology modeling. Alignments used for modeling are tabulated in Additional file <xref rid="MOESM2" ref-type="media">2</xref>
: Table S1. Models obtained with the highest C-score were retained for further analysis. The homology models have been deposited at Model Archive (doi:10.5452/ma-asp8e) [<xref ref-type="bibr" rid="CR54">54</xref>
].</p>
</sec>
<sec id="Sec17"><title>Structural analysis</title>
<p>Structural comparisons were done using the Combinatorial Extension algorithm [<xref ref-type="bibr" rid="CR55">55</xref>
] implemented in PyMOL (Schrödinger, Portland, OR). Protein surface electrostatics calculations were carried using Adaptive Poisson-Boltzmann Solver (APBS) [<xref ref-type="bibr" rid="CR56">56</xref>
], where an externally generated pdb (P) file with per-atom charge (Q) and radius (R) (PQR file) file was used to calculate the electrostatics. The parameters used were solvent and protein dielectrics of 78.0 and 2.0 respectively, solvent radius of 1.4, and a monovalent ion concentration of 0.15 M. The visualization was depicted in PyMOL with positive and negative molecular surface ranging from -2kT/e to 2kT/e.</p>
</sec>
<sec id="Sec18"><title>Motif identification</title>
<p>Sequence-based motifs were identified using Multiple Em for Motif Elicitation (MEME) (<ext-link ext-link-type="uri" xlink:href="http://meme.nbcr.net/meme/">http://meme.nbcr.net/meme/</ext-link>
) [<xref ref-type="bibr" rid="CR42">42</xref>
]. The occurrence of motifs in the sequence was assumed to be distributed either zero or one per sequence. Three motifs were identified for each set of sequences given. The phylogenetic tree of AA10 was divided into four subclades (A to D) based on major phylogenetic clades. For each clade, the motifs were identified using MEME.</p>
</sec>
<sec id="Sec19"><title>Endnotes</title>
<p><sup>a</sup>
CBM33 has recently been renamed as AA10; likewise GH61 has been renamed as AA9 [<xref ref-type="bibr" rid="CR11">11</xref>
]. These names will be used throughout.</p>
<p><sup>b</sup>
SACTE_3159 from the highly cellulolytic <italic>Streptomyces</italic>
sp. SirexAA-E, has also been confirmed to contain Cu<sup>2+</sup>
and have O<sub>2</sub>
-dependent cellulose oxidation activity (M. Mbughuni and BG Fox, unpublished data).</p>
</sec>
</sec>
<sec sec-type="supplementary-material"><title>Electronic supplementary material</title>
<sec id="Sec20"><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="13068_2014_512_MOESM1_ESM.zip"><caption><p>Additional file 1: Figure S1: Taxonomic diversity of AA10 sequences. AA10 sequences were collected from the CAZy database (608 sequences) and binned into taxonomic categories based on phylum and genus. Three phyla present were Proteobacteria, Firmicutes, and Actinobacteria (central pie chart). Smaller peripheral charts identify the number of sequences within each genus. (ZIP 1 MB)</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="MOESM2"><media xlink:href="13068_2014_512_MOESM2_ESM.xlsx"><caption><p>Additional file 2: Table S1: Sequence alignments used for structural modeling. (XLSX 29 KB)</p>
</caption>
</media>
</supplementary-material>
</sec>
</sec>
</body>
<back><glossary><title>Abbreviations</title>
<def-list><def-list><def-item><term>AA</term>
<def><p>Auxiliary activity</p>
</def>
</def-item>
<def-item><term>CBM</term>
<def><p>Carbohydrate-binding module</p>
</def>
</def-item>
<def-item><term>LPMO</term>
<def><p>Lytic polysaccharide monooxygenases</p>
</def>
</def-item>
<def-item><term>GH</term>
<def><p>Glycoside hydrolase.</p>
</def>
</def-item>
</def-list>
</def-list>
</glossary>
<fn-group><fn><p><bold>Competing interests</bold>
</p>
<p>The authors declare that they have no competing interests.</p>
</fn>
<fn><p><bold>Authors’ contributions</bold>
</p>
<p>AJB, RMY, and TET, designed and performed the experiments. All authors reviewed, discussed, and interpreted the results. AJB, RMY, TET, GNP, CRC, and BGF wrote and reviewed the manuscript. All authors read and approved the final manuscript.</p>
</fn>
</fn-group>
<ack><title>Acknowledgements</title>
<p>This work was funded by the Department of Energy Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC-02-07ER64494), the Department of Energy Bringing Advanced Computational Techniques to Energy Research program (DOE BACTER DE-FG02-04ER25627), the National Institute of Health Protein Structure Initiative (U01 GM098248), and the National Science Foundation GRAPE (CMMI-0941013).</p>
</ack>
<ref-list id="Bib1"><title>References</title>
<ref id="CR1"><label>1.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Himmel</surname>
<given-names>ME</given-names>
</name>
<name><surname>Ding</surname>
<given-names>SY</given-names>
</name>
<name><surname>Johnson</surname>
<given-names>DK</given-names>
</name>
<name><surname>Adney</surname>
<given-names>WS</given-names>
</name>
<name><surname>Nimlos</surname>
<given-names>MR</given-names>
</name>
<name><surname>Brady</surname>
<given-names>JW</given-names>
</name>
<name><surname>Foust</surname>
<given-names>TD</given-names>
</name>
</person-group>
<article-title>Biomass recalcitrance: engineering plants and enzymes for biofuels production</article-title>
<source>Science</source>
<year>2007</year>
<volume>315</volume>
<fpage>804</fpage>
<lpage>807</lpage>
<pub-id pub-id-type="doi">10.1126/science.1137016</pub-id>
<pub-id pub-id-type="pmid">17289988</pub-id>
</element-citation>
</ref>
<ref id="CR2"><label>2.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Faaij</surname>
<given-names>APC</given-names>
</name>
</person-group>
<article-title>Bio-energy in Europe: changing technology choices</article-title>
<source>Energ Policy</source>
<year>2006</year>
<volume>34</volume>
<fpage>322</fpage>
<lpage>342</lpage>
<pub-id pub-id-type="doi">10.1016/j.enpol.2004.03.026</pub-id>
</element-citation>
</ref>
<ref id="CR3"><label>3.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wilson</surname>
<given-names>DB</given-names>
</name>
</person-group>
<article-title>Microbial diversity of cellulose hydrolysis</article-title>
<source>Curr Opin Microbiol</source>
<year>2011</year>
<volume>14</volume>
<fpage>259</fpage>
<lpage>263</lpage>
<pub-id pub-id-type="doi">10.1016/j.mib.2011.04.004</pub-id>
<pub-id pub-id-type="pmid">21531609</pub-id>
</element-citation>
</ref>
<ref id="CR4"><label>4.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lynd</surname>
<given-names>LR</given-names>
</name>
<name><surname>Weimer</surname>
<given-names>PJ</given-names>
</name>
<name><surname>van Zyl</surname>
<given-names>WH</given-names>
</name>
<name><surname>Pretorius</surname>
<given-names>IS</given-names>
</name>
</person-group>
<article-title>Microbial cellulose utilization: fundamentals and biotechnology</article-title>
<source>Microbiol Mol Biol Rev</source>
<year>2002</year>
<volume>66</volume>
<fpage>506</fpage>
<lpage>577</lpage>
<pub-id pub-id-type="doi">10.1128/MMBR.66.3.506-577.2002</pub-id>
<pub-id pub-id-type="pmid">12209002</pub-id>
</element-citation>
</ref>
<ref id="CR5"><label>5.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Culpepper</surname>
<given-names>MA</given-names>
</name>
<name><surname>Rosenzweig</surname>
<given-names>AC</given-names>
</name>
</person-group>
<article-title>Architecture and active site of particulate methane monooxygenase</article-title>
<source>Crit Rev Biochem Mol Biol</source>
<year>2012</year>
<volume>47</volume>
<fpage>483</fpage>
<lpage>492</lpage>
<pub-id pub-id-type="doi">10.3109/10409238.2012.697865</pub-id>
<pub-id pub-id-type="pmid">22725967</pub-id>
</element-citation>
</ref>
<ref id="CR6"><label>6.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Forsberg</surname>
<given-names>Z</given-names>
</name>
<name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
<name><surname>Westereng</surname>
<given-names>B</given-names>
</name>
<name><surname>Bunaes</surname>
<given-names>AC</given-names>
</name>
<name><surname>Stenstrom</surname>
<given-names>Y</given-names>
</name>
<name><surname>MacKenzie</surname>
<given-names>A</given-names>
</name>
<name><surname>Sorlie</surname>
<given-names>M</given-names>
</name>
<name><surname>Horn</surname>
<given-names>SJ</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
</person-group>
<article-title>Cleavage of cellulose by a CBM33 protein</article-title>
<source>Protein Sci</source>
<year>2011</year>
<volume>20</volume>
<fpage>1479</fpage>
<lpage>1483</lpage>
<pub-id pub-id-type="doi">10.1002/pro.689</pub-id>
<pub-id pub-id-type="pmid">21748815</pub-id>
</element-citation>
</ref>
<ref id="CR7"><label>7.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Harris</surname>
<given-names>PV</given-names>
</name>
<name><surname>Welner</surname>
<given-names>D</given-names>
</name>
<name><surname>McFarland</surname>
<given-names>KC</given-names>
</name>
<name><surname>Re</surname>
<given-names>E</given-names>
</name>
<name><surname>Navarro Poulsen</surname>
<given-names>JC</given-names>
</name>
<name><surname>Brown</surname>
<given-names>K</given-names>
</name>
<name><surname>Salbo</surname>
<given-names>R</given-names>
</name>
<name><surname>Ding</surname>
<given-names>H</given-names>
</name>
<name><surname>Vlasenko</surname>
<given-names>E</given-names>
</name>
<name><surname>Merino</surname>
<given-names>S</given-names>
</name>
<name><surname>Xu</surname>
<given-names>F</given-names>
</name>
<name><surname>Cherry</surname>
<given-names>J</given-names>
</name>
<name><surname>Larsen</surname>
<given-names>S</given-names>
</name>
<name><surname>Lo Leggio</surname>
<given-names>L</given-names>
</name>
</person-group>
<article-title>Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family</article-title>
<source>Biochemistry</source>
<year>2010</year>
<volume>49</volume>
<fpage>3305</fpage>
<lpage>3316</lpage>
<pub-id pub-id-type="doi">10.1021/bi100009p</pub-id>
<pub-id pub-id-type="pmid">20230050</pub-id>
</element-citation>
</ref>
<ref id="CR8"><label>8.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hemsworth</surname>
<given-names>GR</given-names>
</name>
<name><surname>Taylor</surname>
<given-names>EJ</given-names>
</name>
<name><surname>Kim</surname>
<given-names>RQ</given-names>
</name>
<name><surname>Gregory</surname>
<given-names>RC</given-names>
</name>
<name><surname>Lewis</surname>
<given-names>SJ</given-names>
</name>
<name><surname>Turkenburg</surname>
<given-names>JP</given-names>
</name>
<name><surname>Parkin</surname>
<given-names>A</given-names>
</name>
<name><surname>Davies</surname>
<given-names>GJ</given-names>
</name>
<name><surname>Walton</surname>
<given-names>PH</given-names>
</name>
</person-group>
<article-title>The copper active site of CBM33 polysaccharide oxygenases</article-title>
<source>J Am Chem Soc</source>
<year>2013</year>
<volume>135</volume>
<fpage>6069</fpage>
<lpage>6077</lpage>
<pub-id pub-id-type="doi">10.1021/ja402106e</pub-id>
<pub-id pub-id-type="pmid">23540833</pub-id>
</element-citation>
</ref>
<ref id="CR9"><label>9.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schnellmann</surname>
<given-names>J</given-names>
</name>
<name><surname>Zeltins</surname>
<given-names>A</given-names>
</name>
<name><surname>Blaak</surname>
<given-names>H</given-names>
</name>
<name><surname>Schrempf</surname>
<given-names>H</given-names>
</name>
</person-group>
<article-title>The novel lectin-like protein CHB1 is encoded by a chitin-inducible Streptomyces olivaceoviridis gene and binds specifically to crystalline alpha-chitin of fungi and other organisms</article-title>
<source>Mol Microbiol</source>
<year>1994</year>
<volume>13</volume>
<fpage>807</fpage>
<lpage>819</lpage>
<pub-id pub-id-type="doi">10.1111/j.1365-2958.1994.tb00473.x</pub-id>
<pub-id pub-id-type="pmid">7815940</pub-id>
</element-citation>
</ref>
<ref id="CR10"><label>10.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Suzuki</surname>
<given-names>K</given-names>
</name>
<name><surname>Suzuki</surname>
<given-names>M</given-names>
</name>
<name><surname>Taiyoji</surname>
<given-names>M</given-names>
</name>
<name><surname>Nikaidou</surname>
<given-names>N</given-names>
</name>
<name><surname>Watanabe</surname>
<given-names>T</given-names>
</name>
</person-group>
<article-title>Chitin binding protein (CBP21) in the culture supernatant of Serratia marcescens 2170</article-title>
<source>Biosci Biotechnol Biochem</source>
<year>1998</year>
<volume>62</volume>
<fpage>128</fpage>
<lpage>135</lpage>
<pub-id pub-id-type="doi">10.1271/bbb.62.128</pub-id>
<pub-id pub-id-type="pmid">9501524</pub-id>
</element-citation>
</ref>
<ref id="CR11"><label>11.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
<name><surname>Westereng</surname>
<given-names>B</given-names>
</name>
<name><surname>Horn</surname>
<given-names>SJ</given-names>
</name>
<name><surname>Liu</surname>
<given-names>Z</given-names>
</name>
<name><surname>Zhai</surname>
<given-names>H</given-names>
</name>
<name><surname>Sorlie</surname>
<given-names>M</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
</person-group>
<article-title>An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides</article-title>
<source>Science</source>
<year>2010</year>
<volume>330</volume>
<fpage>219</fpage>
<lpage>222</lpage>
<pub-id pub-id-type="doi">10.1126/science.1192231</pub-id>
<pub-id pub-id-type="pmid">20929773</pub-id>
</element-citation>
</ref>
<ref id="CR12"><label>12.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Levasseur</surname>
<given-names>A</given-names>
</name>
<name><surname>Drula</surname>
<given-names>E</given-names>
</name>
<name><surname>Lombard</surname>
<given-names>V</given-names>
</name>
<name><surname>Coutinho</surname>
<given-names>PM</given-names>
</name>
<name><surname>Henrissat</surname>
<given-names>B</given-names>
</name>
</person-group>
<article-title>Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes</article-title>
<source>Biotechnol Biofuels</source>
<year>2013</year>
<volume>6</volume>
<fpage>41</fpage>
<pub-id pub-id-type="doi">10.1186/1754-6834-6-41</pub-id>
<pub-id pub-id-type="pmid">23514094</pub-id>
</element-citation>
</ref>
<ref id="CR13"><label>13.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Quinlan</surname>
<given-names>RJ</given-names>
</name>
<name><surname>Sweeney</surname>
<given-names>MD</given-names>
</name>
<name><surname>Lo Leggio</surname>
<given-names>L</given-names>
</name>
<name><surname>Otten</surname>
<given-names>H</given-names>
</name>
<name><surname>Poulsen</surname>
<given-names>JC</given-names>
</name>
<name><surname>Johansen</surname>
<given-names>KS</given-names>
</name>
<name><surname>Krogh</surname>
<given-names>KB</given-names>
</name>
<name><surname>Jorgensen</surname>
<given-names>CI</given-names>
</name>
<name><surname>Tovborg</surname>
<given-names>M</given-names>
</name>
<name><surname>Anthonsen</surname>
<given-names>A</given-names>
</name>
<name><surname>Tryfona</surname>
<given-names>T</given-names>
</name>
<name><surname>Walter</surname>
<given-names>CP</given-names>
</name>
<name><surname>Dupree</surname>
<given-names>P</given-names>
</name>
<name><surname>Xu</surname>
<given-names>F</given-names>
</name>
<name><surname>Davies</surname>
<given-names>GJ</given-names>
</name>
<name><surname>Walton</surname>
<given-names>PH</given-names>
</name>
</person-group>
<article-title>Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components</article-title>
<source>Proc Natl Acad Sci U S A</source>
<year>2011</year>
<volume>108</volume>
<fpage>15079</fpage>
<lpage>15084</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.1105776108</pub-id>
<pub-id pub-id-type="pmid">21876164</pub-id>
</element-citation>
</ref>
<ref id="CR14"><label>14.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Westereng</surname>
<given-names>B</given-names>
</name>
<name><surname>Ishida</surname>
<given-names>T</given-names>
</name>
<name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
<name><surname>Wu</surname>
<given-names>M</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
<name><surname>Igarashi</surname>
<given-names>K</given-names>
</name>
<name><surname>Samejima</surname>
<given-names>M</given-names>
</name>
<name><surname>Stahlberg</surname>
<given-names>J</given-names>
</name>
<name><surname>Horn</surname>
<given-names>SJ</given-names>
</name>
<name><surname>Sandgren</surname>
<given-names>M</given-names>
</name>
</person-group>
<article-title>The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose</article-title>
<source>PLoS One</source>
<year>2011</year>
<volume>6</volume>
<fpage>e27807</fpage>
<pub-id pub-id-type="doi">10.1371/journal.pone.0027807</pub-id>
<pub-id pub-id-type="pmid">22132148</pub-id>
</element-citation>
</ref>
<ref id="CR15"><label>15.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Moser</surname>
<given-names>F</given-names>
</name>
<name><surname>Irwin</surname>
<given-names>D</given-names>
</name>
<name><surname>Chen</surname>
<given-names>S</given-names>
</name>
<name><surname>Wilson</surname>
<given-names>DB</given-names>
</name>
</person-group>
<article-title>Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8</article-title>
<source>Biotechnol Bioeng</source>
<year>2008</year>
<volume>100</volume>
<fpage>1066</fpage>
<lpage>1077</lpage>
<pub-id pub-id-type="doi">10.1002/bit.21856</pub-id>
<pub-id pub-id-type="pmid">18553392</pub-id>
</element-citation>
</ref>
<ref id="CR16"><label>16.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Forsberg</surname>
<given-names>Z</given-names>
</name>
<name><surname>Rohr</surname>
<given-names>AK</given-names>
</name>
<name><surname>Mekasha</surname>
<given-names>S</given-names>
</name>
<name><surname>Andersson</surname>
<given-names>KK</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
<name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
<name><surname>Sorlie</surname>
<given-names>M</given-names>
</name>
</person-group>
<article-title>Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases</article-title>
<source>Biochemistry</source>
<year>2014</year>
<volume>53</volume>
<fpage>1647</fpage>
<lpage>1656</lpage>
<pub-id pub-id-type="doi">10.1021/bi5000433</pub-id>
<pub-id pub-id-type="pmid">24559135</pub-id>
</element-citation>
</ref>
<ref id="CR17"><label>17.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aachmann</surname>
<given-names>FL</given-names>
</name>
<name><surname>Sorlie</surname>
<given-names>M</given-names>
</name>
<name><surname>Skjak-Braek</surname>
<given-names>G</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
<name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
</person-group>
<article-title>NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions</article-title>
<source>Proc Natl Acad Sci U S A</source>
<year>2012</year>
<volume>109</volume>
<fpage>18779</fpage>
<lpage>18784</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.1208822109</pub-id>
<pub-id pub-id-type="pmid">23112164</pub-id>
</element-citation>
</ref>
<ref id="CR18"><label>18.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname>
<given-names>X</given-names>
</name>
<name><surname>Beeson</surname>
<given-names>WT</given-names>
</name>
<name><surname>Phillips</surname>
<given-names>CM</given-names>
</name>
<name><surname>Marletta</surname>
<given-names>MA</given-names>
</name>
<name><surname>Cate</surname>
<given-names>JH</given-names>
</name>
</person-group>
<article-title>Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases</article-title>
<source>Structure</source>
<year>2012</year>
<volume>20</volume>
<fpage>1051</fpage>
<lpage>1061</lpage>
<pub-id pub-id-type="doi">10.1016/j.str.2012.04.002</pub-id>
<pub-id pub-id-type="pmid">22578542</pub-id>
</element-citation>
</ref>
<ref id="CR19"><label>19.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Phillips</surname>
<given-names>CM</given-names>
</name>
<name><surname>Beeson</surname>
<given-names>WT</given-names>
</name>
<name><surname>Cate</surname>
<given-names>JH</given-names>
</name>
<name><surname>Marletta</surname>
<given-names>MA</given-names>
</name>
</person-group>
<article-title>Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa</article-title>
<source>ACS Chem Biol</source>
<year>2011</year>
<volume>6</volume>
<fpage>1399</fpage>
<lpage>1406</lpage>
<pub-id pub-id-type="doi">10.1021/cb200351y</pub-id>
<pub-id pub-id-type="pmid">22004347</pub-id>
</element-citation>
</ref>
<ref id="CR20"><label>20.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kim</surname>
<given-names>S</given-names>
</name>
<name><surname>Stahlberg</surname>
<given-names>J</given-names>
</name>
<name><surname>Sandgren</surname>
<given-names>M</given-names>
</name>
<name><surname>Paton</surname>
<given-names>RS</given-names>
</name>
<name><surname>Beckham</surname>
<given-names>GT</given-names>
</name>
</person-group>
<article-title>Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism</article-title>
<source>Proc Natl Acad Sci U S A</source>
<year>2014</year>
<volume>111</volume>
<fpage>149</fpage>
<lpage>154</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.1316609111</pub-id>
<pub-id pub-id-type="pmid">24344312</pub-id>
</element-citation>
</ref>
<ref id="CR21"><label>21.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vu</surname>
<given-names>VV</given-names>
</name>
<name><surname>Beeson</surname>
<given-names>WT</given-names>
</name>
<name><surname>Phillips</surname>
<given-names>CM</given-names>
</name>
<name><surname>Cate</surname>
<given-names>JHD</given-names>
</name>
<name><surname>Marletta</surname>
<given-names>MA</given-names>
</name>
</person-group>
<article-title>Determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases</article-title>
<source>J Am Chem Soc</source>
<year>2014</year>
<volume>136</volume>
<fpage>562</fpage>
<lpage>565</lpage>
<pub-id pub-id-type="doi">10.1021/ja409384b</pub-id>
<pub-id pub-id-type="pmid">24350607</pub-id>
</element-citation>
</ref>
<ref id="CR22"><label>22.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Isaksen</surname>
<given-names>T</given-names>
</name>
<name><surname>Westereng</surname>
<given-names>B</given-names>
</name>
<name><surname>Aachmann</surname>
<given-names>FL</given-names>
</name>
<name><surname>Agger</surname>
<given-names>JW</given-names>
</name>
<name><surname>Kracher</surname>
<given-names>D</given-names>
</name>
<name><surname>Kittl</surname>
<given-names>R</given-names>
</name>
<name><surname>Ludwig</surname>
<given-names>R</given-names>
</name>
<name><surname>Haltrich</surname>
<given-names>D</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
<name><surname>Horn</surname>
<given-names>SJ</given-names>
</name>
</person-group>
<article-title>A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides</article-title>
<source>J Biol Chem</source>
<year>2014</year>
<volume>289</volume>
<fpage>2632</fpage>
<lpage>2642</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.M113.530196</pub-id>
<pub-id pub-id-type="pmid">24324265</pub-id>
</element-citation>
</ref>
<ref id="CR23"><label>23.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hori</surname>
<given-names>C</given-names>
</name>
<name><surname>Gaskell</surname>
<given-names>J</given-names>
</name>
<name><surname>Igarashi</surname>
<given-names>K</given-names>
</name>
<name><surname>Samejima</surname>
<given-names>M</given-names>
</name>
<name><surname>Hibbett</surname>
<given-names>D</given-names>
</name>
<name><surname>Henrissat</surname>
<given-names>B</given-names>
</name>
<name><surname>Cullen</surname>
<given-names>D</given-names>
</name>
</person-group>
<article-title>Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay</article-title>
<source>Mycologia</source>
<year>2013</year>
<volume>105</volume>
<fpage>1412</fpage>
<lpage>1427</lpage>
<pub-id pub-id-type="doi">10.3852/13-072</pub-id>
<pub-id pub-id-type="pmid">23935027</pub-id>
</element-citation>
</ref>
<ref id="CR24"><label>24.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Karkehabadi</surname>
<given-names>S</given-names>
</name>
<name><surname>Hansson</surname>
<given-names>H</given-names>
</name>
<name><surname>Kim</surname>
<given-names>S</given-names>
</name>
<name><surname>Piens</surname>
<given-names>K</given-names>
</name>
<name><surname>Mitchinson</surname>
<given-names>C</given-names>
</name>
<name><surname>Sandgren</surname>
<given-names>M</given-names>
</name>
</person-group>
<article-title>The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution</article-title>
<source>J Mol Biol</source>
<year>2008</year>
<volume>383</volume>
<fpage>144</fpage>
<lpage>154</lpage>
<pub-id pub-id-type="doi">10.1016/j.jmb.2008.08.016</pub-id>
<pub-id pub-id-type="pmid">18723026</pub-id>
</element-citation>
</ref>
<ref id="CR25"><label>25.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
<name><surname>Houston</surname>
<given-names>DR</given-names>
</name>
<name><surname>Riemen</surname>
<given-names>AH</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
<name><surname>van Aalten</surname>
<given-names>DM</given-names>
</name>
</person-group>
<article-title>Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21</article-title>
<source>J Biol Chem</source>
<year>2005</year>
<volume>280</volume>
<fpage>11313</fpage>
<lpage>11319</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.M407175200</pub-id>
<pub-id pub-id-type="pmid">15590674</pub-id>
</element-citation>
</ref>
<ref id="CR26"><label>26.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wong</surname>
<given-names>E</given-names>
</name>
<name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
<name><surname>Ghosh</surname>
<given-names>A</given-names>
</name>
<name><surname>Hurtado-Guerrero</surname>
<given-names>R</given-names>
</name>
<name><surname>Konarev</surname>
<given-names>PV</given-names>
</name>
<name><surname>Ibrahim</surname>
<given-names>AF</given-names>
</name>
<name><surname>Svergun</surname>
<given-names>DI</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
<name><surname>Chatterjee</surname>
<given-names>NS</given-names>
</name>
<name><surname>van Aalten</surname>
<given-names>DM</given-names>
</name>
</person-group>
<article-title>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</article-title>
<source>PLoS Pathog</source>
<year>2012</year>
<volume>8</volume>
<fpage>e1002373</fpage>
<pub-id pub-id-type="doi">10.1371/journal.ppat.1002373</pub-id>
<pub-id pub-id-type="pmid">22253590</pub-id>
</element-citation>
</ref>
<ref id="CR27"><label>27.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vaaje-Kolstad</surname>
<given-names>G</given-names>
</name>
<name><surname>Bohle</surname>
<given-names>LA</given-names>
</name>
<name><surname>Gaseidnes</surname>
<given-names>S</given-names>
</name>
<name><surname>Dalhus</surname>
<given-names>B</given-names>
</name>
<name><surname>Bjoras</surname>
<given-names>M</given-names>
</name>
<name><surname>Mathiesen</surname>
<given-names>G</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
</person-group>
<article-title>Characterization of the chitinolytic machinery of Enterococcus faecalis V583 and high-resolution structure of its oxidative CBM33 enzyme</article-title>
<source>J Mol Biol</source>
<year>2012</year>
<volume>416</volume>
<fpage>239</fpage>
<lpage>254</lpage>
<pub-id pub-id-type="doi">10.1016/j.jmb.2011.12.033</pub-id>
<pub-id pub-id-type="pmid">22210154</pub-id>
</element-citation>
</ref>
<ref id="CR28"><label>28.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wu</surname>
<given-names>M</given-names>
</name>
<name><surname>Beckham</surname>
<given-names>GT</given-names>
</name>
<name><surname>Larsson</surname>
<given-names>AM</given-names>
</name>
<name><surname>Ishida</surname>
<given-names>T</given-names>
</name>
<name><surname>Kim</surname>
<given-names>S</given-names>
</name>
<name><surname>Payne</surname>
<given-names>CM</given-names>
</name>
<name><surname>Himmel</surname>
<given-names>ME</given-names>
</name>
<name><surname>Crowley</surname>
<given-names>MF</given-names>
</name>
<name><surname>Horn</surname>
<given-names>SJ</given-names>
</name>
<name><surname>Westereng</surname>
<given-names>B</given-names>
</name>
<name><surname>Igarashi</surname>
<given-names>K</given-names>
</name>
<name><surname>Samejima</surname>
<given-names>M</given-names>
</name>
<name><surname>Ståhlberg</surname>
<given-names>J</given-names>
</name>
<name><surname>Eijsink</surname>
<given-names>VG</given-names>
</name>
<name><surname>Sandgren</surname>
<given-names>M</given-names>
</name>
</person-group>
<article-title>Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the Basidiomycota fungus Phanerochaete chrysosporium</article-title>
<source>J Biol Chem</source>
<year>2013</year>
<volume>288</volume>
<fpage>12828</fpage>
<lpage>12839</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.M113.459396</pub-id>
<pub-id pub-id-type="pmid">23525113</pub-id>
</element-citation>
</ref>
<ref id="CR29"><label>29.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Blake</surname>
<given-names>AW</given-names>
</name>
<name><surname>McCartney</surname>
<given-names>L</given-names>
</name>
<name><surname>Flint</surname>
<given-names>JE</given-names>
</name>
<name><surname>Bolam</surname>
<given-names>DN</given-names>
</name>
<name><surname>Boraston</surname>
<given-names>AB</given-names>
</name>
<name><surname>Gilbert</surname>
<given-names>HJ</given-names>
</name>
<name><surname>Knox</surname>
<given-names>JP</given-names>
</name>
</person-group>
<article-title>Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes</article-title>
<source>J Biol Chem</source>
<year>2006</year>
<volume>281</volume>
<fpage>29321</fpage>
<lpage>29329</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.M605903200</pub-id>
<pub-id pub-id-type="pmid">16844685</pub-id>
</element-citation>
</ref>
<ref id="CR30"><label>30.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hogg</surname>
<given-names>D</given-names>
</name>
<name><surname>Pell</surname>
<given-names>G</given-names>
</name>
<name><surname>Dupree</surname>
<given-names>P</given-names>
</name>
<name><surname>Goubet</surname>
<given-names>F</given-names>
</name>
<name><surname>Martin-Orue</surname>
<given-names>SM</given-names>
</name>
<name><surname>Armand</surname>
<given-names>S</given-names>
</name>
<name><surname>Gilbert</surname>
<given-names>HJ</given-names>
</name>
</person-group>
<article-title>The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation</article-title>
<source>Biochem J</source>
<year>2003</year>
<volume>371</volume>
<fpage>1027</fpage>
<lpage>1043</lpage>
<pub-id pub-id-type="doi">10.1042/BJ20021860</pub-id>
<pub-id pub-id-type="pmid">12523937</pub-id>
</element-citation>
</ref>
<ref id="CR31"><label>31.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname>
<given-names>N</given-names>
</name>
<name><surname>Zhang</surname>
<given-names>Y</given-names>
</name>
<name><surname>Wang</surname>
<given-names>Q</given-names>
</name>
<name><surname>Liu</surname>
<given-names>J</given-names>
</name>
<name><surname>Wang</surname>
<given-names>H</given-names>
</name>
<name><surname>Xue</surname>
<given-names>Y</given-names>
</name>
<name><surname>Ma</surname>
<given-names>Y</given-names>
</name>
</person-group>
<article-title>Gene cloning and characterization of a novel alpha-amylase from alkaliphilic Alkalimonas amylolytica</article-title>
<source>Biotechnol J</source>
<year>2006</year>
<volume>1</volume>
<fpage>1258</fpage>
<lpage>1265</lpage>
<pub-id pub-id-type="doi">10.1002/biot.200600098</pub-id>
<pub-id pub-id-type="pmid">17068753</pub-id>
</element-citation>
</ref>
<ref id="CR32"><label>32.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname>
<given-names>ES</given-names>
</name>
<name><surname>Wilson</surname>
<given-names>DB</given-names>
</name>
</person-group>
<article-title>Identification of a celE-binding protein and its potential role in induction of the celE gene in Thermomonospora fusca</article-title>
<source>J Bacteriol</source>
<year>1988</year>
<volume>170</volume>
<fpage>3843</fpage>
<lpage>3846</lpage>
<pub-id pub-id-type="pmid">3410818</pub-id>
</element-citation>
</ref>
<ref id="CR33"><label>33.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname>
<given-names>S</given-names>
</name>
<name><surname>Lao</surname>
<given-names>G</given-names>
</name>
<name><surname>Wilson</surname>
<given-names>DB</given-names>
</name>
</person-group>
<article-title>Characterization of a Thermomonospora fusca exocellulase</article-title>
<source>Biochemistry</source>
<year>1995</year>
<volume>34</volume>
<fpage>3386</fpage>
<lpage>3395</lpage>
<pub-id pub-id-type="doi">10.1021/bi00010a030</pub-id>
<pub-id pub-id-type="pmid">7880834</pub-id>
</element-citation>
</ref>
<ref id="CR34"><label>34.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huang</surname>
<given-names>L</given-names>
</name>
<name><surname>Garbulewska</surname>
<given-names>E</given-names>
</name>
<name><surname>Sato</surname>
<given-names>K</given-names>
</name>
<name><surname>Kato</surname>
<given-names>Y</given-names>
</name>
<name><surname>Nogawa</surname>
<given-names>M</given-names>
</name>
<name><surname>Taguchi</surname>
<given-names>G</given-names>
</name>
<name><surname>Shimosaka</surname>
<given-names>M</given-names>
</name>
</person-group>
<article-title>Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase</article-title>
<source>J Biosci Bioeng</source>
<year>2012</year>
<volume>113</volume>
<fpage>293</fpage>
<lpage>299</lpage>
<pub-id pub-id-type="doi">10.1016/j.jbiosc.2011.10.018</pub-id>
<pub-id pub-id-type="pmid">22178339</pub-id>
</element-citation>
</ref>
<ref id="CR35"><label>35.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ronquist</surname>
<given-names>F</given-names>
</name>
<name><surname>Teslenko</surname>
<given-names>M</given-names>
</name>
<name><surname>van der Mark</surname>
<given-names>P</given-names>
</name>
<name><surname>Ayres</surname>
<given-names>DL</given-names>
</name>
<name><surname>Darling</surname>
<given-names>A</given-names>
</name>
<name><surname>Hohna</surname>
<given-names>S</given-names>
</name>
<name><surname>Larget</surname>
<given-names>B</given-names>
</name>
<name><surname>Liu</surname>
<given-names>L</given-names>
</name>
<name><surname>Suchard</surname>
<given-names>MA</given-names>
</name>
<name><surname>Huelsenbeck</surname>
<given-names>JP</given-names>
</name>
</person-group>
<article-title>MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space</article-title>
<source>Syst Biol</source>
<year>2012</year>
<volume>61</volume>
<fpage>539</fpage>
<lpage>542</lpage>
<pub-id pub-id-type="doi">10.1093/sysbio/sys029</pub-id>
<pub-id pub-id-type="pmid">22357727</pub-id>
</element-citation>
</ref>
<ref id="CR36"><label>36.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bey</surname>
<given-names>M</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>S</given-names>
</name>
<name><surname>Poidevin</surname>
<given-names>L</given-names>
</name>
<name><surname>Henrissat</surname>
<given-names>B</given-names>
</name>
<name><surname>Coutinho</surname>
<given-names>PM</given-names>
</name>
<name><surname>Berrin</surname>
<given-names>JG</given-names>
</name>
<name><surname>Sigoillot</surname>
<given-names>JC</given-names>
</name>
</person-group>
<article-title>Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina</article-title>
<source>Appl Environ Microbiol</source>
<year>2013</year>
<volume>79</volume>
<fpage>488</fpage>
<lpage>496</lpage>
<pub-id pub-id-type="doi">10.1128/AEM.02942-12</pub-id>
<pub-id pub-id-type="pmid">23124232</pub-id>
</element-citation>
</ref>
<ref id="CR37"><label>37.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kittl</surname>
<given-names>R</given-names>
</name>
<name><surname>Kracher</surname>
<given-names>D</given-names>
</name>
<name><surname>Burgstaller</surname>
<given-names>D</given-names>
</name>
<name><surname>Haltrich</surname>
<given-names>D</given-names>
</name>
<name><surname>Ludwig</surname>
<given-names>R</given-names>
</name>
</person-group>
<article-title>Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay</article-title>
<source>Biotechnol Biofuels</source>
<year>2012</year>
<volume>5</volume>
<fpage>79</fpage>
<pub-id pub-id-type="doi">10.1186/1754-6834-5-79</pub-id>
<pub-id pub-id-type="pmid">23102010</pub-id>
</element-citation>
</ref>
<ref id="CR38"><label>38.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Langston</surname>
<given-names>JA</given-names>
</name>
<name><surname>Shaghasi</surname>
<given-names>T</given-names>
</name>
<name><surname>Abbate</surname>
<given-names>E</given-names>
</name>
<name><surname>Xu</surname>
<given-names>F</given-names>
</name>
<name><surname>Vlasenko</surname>
<given-names>E</given-names>
</name>
<name><surname>Sweeney</surname>
<given-names>MD</given-names>
</name>
</person-group>
<article-title>Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61</article-title>
<source>Appl Environ Microbiol</source>
<year>2011</year>
<volume>77</volume>
<fpage>7007</fpage>
<lpage>7015</lpage>
<pub-id pub-id-type="doi">10.1128/AEM.05815-11</pub-id>
<pub-id pub-id-type="pmid">21821740</pub-id>
</element-citation>
</ref>
<ref id="CR39"><label>39.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Beeson</surname>
<given-names>WT</given-names>
</name>
<name><surname>Phillips</surname>
<given-names>CM</given-names>
</name>
<name><surname>Cate</surname>
<given-names>JH</given-names>
</name>
<name><surname>Marletta</surname>
<given-names>MA</given-names>
</name>
</person-group>
<article-title>Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases</article-title>
<source>J Am Chem Soc</source>
<year>2012</year>
<volume>134</volume>
<fpage>890</fpage>
<lpage>892</lpage>
<pub-id pub-id-type="doi">10.1021/ja210657t</pub-id>
<pub-id pub-id-type="pmid">22188218</pub-id>
</element-citation>
</ref>
<ref id="CR40"><label>40.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Takasuka</surname>
<given-names>TE</given-names>
</name>
<name><surname>Book</surname>
<given-names>AJ</given-names>
</name>
<name><surname>Lewin</surname>
<given-names>GR</given-names>
</name>
<name><surname>Currie</surname>
<given-names>CR</given-names>
</name>
<name><surname>Fox</surname>
<given-names>BG</given-names>
</name>
</person-group>
<article-title>Aerobic deconstruction of cellulosic biomass by an insect-associated Streptomyces</article-title>
<source>Sci Rep</source>
<year>2013</year>
<volume>3</volume>
<fpage>1030</fpage>
<pub-id pub-id-type="doi">10.1038/srep01030</pub-id>
<pub-id pub-id-type="pmid">23301151</pub-id>
</element-citation>
</ref>
<ref id="CR41"><label>41.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Roy</surname>
<given-names>A</given-names>
</name>
<name><surname>Kucukural</surname>
<given-names>A</given-names>
</name>
<name><surname>Zhang</surname>
<given-names>Y</given-names>
</name>
</person-group>
<article-title>I-TASSER: a unified platform for automated protein structure and function prediction</article-title>
<source>Nat Protoc</source>
<year>2010</year>
<volume>5</volume>
<fpage>725</fpage>
<lpage>738</lpage>
<pub-id pub-id-type="doi">10.1038/nprot.2010.5</pub-id>
<pub-id pub-id-type="pmid">20360767</pub-id>
</element-citation>
</ref>
<ref id="CR42"><label>42.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bailey</surname>
<given-names>TL</given-names>
</name>
<name><surname>Williams</surname>
<given-names>N</given-names>
</name>
<name><surname>Misleh</surname>
<given-names>C</given-names>
</name>
<name><surname>Li</surname>
<given-names>WW</given-names>
</name>
</person-group>
<article-title>MEME: discovering and analyzing DNA and protein sequence motifs</article-title>
<source>Nucleic Acids Res</source>
<year>2006</year>
<volume>34</volume>
<fpage>W369</fpage>
<lpage>W373</lpage>
<pub-id pub-id-type="doi">10.1093/nar/gkl198</pub-id>
<pub-id pub-id-type="pmid">16845028</pub-id>
</element-citation>
</ref>
<ref id="CR43"><label>43.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hurst</surname>
<given-names>LD</given-names>
</name>
</person-group>
<article-title>The Ka/Ks ratio: diagnosing the form of sequence evolution</article-title>
<source>Trends Genet</source>
<year>2002</year>
<volume>18</volume>
<fpage>486</fpage>
<pub-id pub-id-type="doi">10.1016/S0168-9525(02)02722-1</pub-id>
<pub-id pub-id-type="pmid">12175810</pub-id>
</element-citation>
</ref>
<ref id="CR44"><label>44.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bianchetti</surname>
<given-names>CM</given-names>
</name>
<name><surname>Harmann</surname>
<given-names>CH</given-names>
</name>
<name><surname>Takasuka</surname>
<given-names>TE</given-names>
</name>
<name><surname>Hura</surname>
<given-names>GL</given-names>
</name>
<name><surname>Dyer</surname>
<given-names>K</given-names>
</name>
<name><surname>Fox</surname>
<given-names>BG</given-names>
</name>
</person-group>
<article-title>Fusion of dioxygenase and lignin-binding domains in a novel secreted enzyme from cellulolytic streptomyces sp SirexAA-E</article-title>
<source>J Biol Chem</source>
<year>2013</year>
<volume>288</volume>
<fpage>18574</fpage>
<lpage>18587</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.M113.475848</pub-id>
<pub-id pub-id-type="pmid">23653358</pub-id>
</element-citation>
</ref>
<ref id="CR45"><label>45.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cantarel</surname>
<given-names>BL</given-names>
</name>
<name><surname>Coutinho</surname>
<given-names>PM</given-names>
</name>
<name><surname>Rancurel</surname>
<given-names>C</given-names>
</name>
<name><surname>Bernard</surname>
<given-names>T</given-names>
</name>
<name><surname>Lombard</surname>
<given-names>V</given-names>
</name>
<name><surname>Henrissat</surname>
<given-names>B</given-names>
</name>
</person-group>
<article-title>The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics</article-title>
<source>Nucleic Acids Res</source>
<year>2009</year>
<volume>37</volume>
<fpage>D233</fpage>
<lpage>D238</lpage>
<pub-id pub-id-type="doi">10.1093/nar/gkn663</pub-id>
<pub-id pub-id-type="pmid">18838391</pub-id>
</element-citation>
</ref>
<ref id="CR46"><label>46.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Altschul</surname>
<given-names>SF</given-names>
</name>
<name><surname>Madden</surname>
<given-names>TL</given-names>
</name>
<name><surname>Schaffer</surname>
<given-names>AA</given-names>
</name>
<name><surname>Zhang</surname>
<given-names>J</given-names>
</name>
<name><surname>Zhang</surname>
<given-names>Z</given-names>
</name>
<name><surname>Miller</surname>
<given-names>W</given-names>
</name>
<name><surname>Lipman</surname>
<given-names>DJ</given-names>
</name>
</person-group>
<article-title>Gapped BLAST and PSI-BLAST: a new generation of protein database search programs</article-title>
<source>Nucleic Acids Res</source>
<year>1997</year>
<volume>25</volume>
<fpage>3389</fpage>
<lpage>3402</lpage>
<pub-id pub-id-type="doi">10.1093/nar/25.17.3389</pub-id>
<pub-id pub-id-type="pmid">9254694</pub-id>
</element-citation>
</ref>
<ref id="CR47"><label>47.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shannon</surname>
<given-names>P</given-names>
</name>
<name><surname>Markiel</surname>
<given-names>A</given-names>
</name>
<name><surname>Ozier</surname>
<given-names>O</given-names>
</name>
<name><surname>Baliga</surname>
<given-names>NS</given-names>
</name>
<name><surname>Wang</surname>
<given-names>JT</given-names>
</name>
<name><surname>Ramage</surname>
<given-names>D</given-names>
</name>
<name><surname>Amin</surname>
<given-names>N</given-names>
</name>
<name><surname>Schwikowski</surname>
<given-names>B</given-names>
</name>
<name><surname>Ideker</surname>
<given-names>T</given-names>
</name>
</person-group>
<article-title>Cytoscape: a software environment for integrated models of biomolecular interaction networks</article-title>
<source>Genome Res</source>
<year>2003</year>
<volume>13</volume>
<fpage>2498</fpage>
<lpage>2504</lpage>
<pub-id pub-id-type="doi">10.1101/gr.1239303</pub-id>
<pub-id pub-id-type="pmid">14597658</pub-id>
</element-citation>
</ref>
<ref id="CR48"><label>48.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Edgar</surname>
<given-names>RC</given-names>
</name>
</person-group>
<article-title>MUSCLE: multiple sequence alignment with high accuracy and high throughput</article-title>
<source>Nucleic Acids Res</source>
<year>2004</year>
<volume>32</volume>
<fpage>1792</fpage>
<lpage>1797</lpage>
<pub-id pub-id-type="doi">10.1093/nar/gkh340</pub-id>
<pub-id pub-id-type="pmid">15034147</pub-id>
</element-citation>
</ref>
<ref id="CR49"><label>49.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wu</surname>
<given-names>M</given-names>
</name>
<name><surname>Chatterji</surname>
<given-names>S</given-names>
</name>
<name><surname>Eisen</surname>
<given-names>JA</given-names>
</name>
</person-group>
<article-title>Accounting for alignment uncertainty in phylogenomics</article-title>
<source>PLoS One</source>
<year>2012</year>
<volume>7</volume>
<fpage>e30288</fpage>
<pub-id pub-id-type="doi">10.1371/journal.pone.0030288</pub-id>
<pub-id pub-id-type="pmid">22272325</pub-id>
</element-citation>
</ref>
<ref id="CR50"><label>50.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stamatakis</surname>
<given-names>A</given-names>
</name>
</person-group>
<article-title>RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies</article-title>
<source>Bioinformatics</source>
<year>2014</year>
<volume>30</volume>
<fpage>1312</fpage>
<lpage>1313</lpage>
<pub-id pub-id-type="doi">10.1093/bioinformatics/btu033</pub-id>
<pub-id pub-id-type="pmid">24451623</pub-id>
</element-citation>
</ref>
<ref id="CR51"><label>51.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname>
<given-names>Z</given-names>
</name>
</person-group>
<article-title>PAML: a program package for phylogenetic analysis by maximum likelihood</article-title>
<source>Comput Appl Biosci</source>
<year>1997</year>
<volume>13</volume>
<fpage>555</fpage>
<lpage>556</lpage>
<pub-id pub-id-type="pmid">9367129</pub-id>
</element-citation>
</ref>
<ref id="CR52"><label>52.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname>
<given-names>Z</given-names>
</name>
<name><surname>Rannala</surname>
<given-names>B</given-names>
</name>
</person-group>
<article-title>Bayesian phylogenetic inference using DNA sequences: a Markov Chain Monte Carlo Method</article-title>
<source>Mol Biol Evol</source>
<year>1997</year>
<volume>14</volume>
<fpage>717</fpage>
<lpage>724</lpage>
<pub-id pub-id-type="doi">10.1093/oxfordjournals.molbev.a025811</pub-id>
<pub-id pub-id-type="pmid">9214744</pub-id>
</element-citation>
</ref>
<ref id="CR53"><label>53.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Holm</surname>
<given-names>L</given-names>
</name>
<name><surname>Rosenstrom</surname>
<given-names>P</given-names>
</name>
</person-group>
<article-title>Dali server: conservation mapping in 3D</article-title>
<source>Nucleic Acids Res</source>
<year>2010</year>
<volume>38</volume>
<fpage>W545</fpage>
<lpage>W549</lpage>
<pub-id pub-id-type="doi">10.1093/nar/gkq366</pub-id>
<pub-id pub-id-type="pmid">20457744</pub-id>
</element-citation>
</ref>
<ref id="CR54"><label>54.</label>
<mixed-citation publication-type="other"><bold>The Model Archive</bold>
<ext-link ext-link-type="uri" xlink:href="http://www.modelarchive.org/doi/10.5452/ma-asp8e">http://www.modelarchive.org/doi/10.5452/ma-asp8e</ext-link>
</mixed-citation>
</ref>
<ref id="CR55"><label>55.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shindyalov</surname>
<given-names>IN</given-names>
</name>
<name><surname>Bourne</surname>
<given-names>PE</given-names>
</name>
</person-group>
<article-title>Protein structure alignment by incremental combinatorial extension (CE) of the optimal path</article-title>
<source>Protein Eng</source>
<year>1998</year>
<volume>11</volume>
<fpage>739</fpage>
<lpage>747</lpage>
<pub-id pub-id-type="doi">10.1093/protein/11.9.739</pub-id>
<pub-id pub-id-type="pmid">9796821</pub-id>
</element-citation>
</ref>
<ref id="CR56"><label>56.</label>
<element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Baker</surname>
<given-names>NA</given-names>
</name>
<name><surname>Sept</surname>
<given-names>D</given-names>
</name>
<name><surname>Joseph</surname>
<given-names>S</given-names>
</name>
<name><surname>Holst</surname>
<given-names>MJ</given-names>
</name>
<name><surname>McCammon</surname>
<given-names>JA</given-names>
</name>
</person-group>
<article-title>Electrostatics of nanosystems: application to microtubules and the ribosome</article-title>
<source>Proc Natl Acad Sci U S A</source>
<year>2001</year>
<volume>98</volume>
<fpage>10037</fpage>
<lpage>10041</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.181342398</pub-id>
<pub-id pub-id-type="pmid">11517324</pub-id>
</element-citation>
</ref>
</ref-list>
</back>
</pmc>
</record>
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