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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Identification and characterization of two families of F<sub>420</sub>H<sub>2</sub>-dependent reductases from <italic>Mycobacteria</italic> that catalyse aflatoxin degradation</title><author><name sortKey="Taylor, Matthew C" sort="Taylor, Matthew C" uniqKey="Taylor M" first="Matthew C" last="Taylor">Matthew C. Taylor</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jackson, Colin J" sort="Jackson, Colin J" uniqKey="Jackson C" first="Colin J" last="Jackson">Colin J. Jackson</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="au2"><institution>Institute de Biologie Structurale</institution><addr-line>41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Tattersall, David B" sort="Tattersall, David B" uniqKey="Tattersall D" first="David B" last="Tattersall">David B. Tattersall</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="French, Nigel" sort="French, Nigel" uniqKey="French N" first="Nigel" last="French">Nigel French</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Peat, Thomas S" sort="Peat, Thomas S" uniqKey="Peat T" first="Thomas S" last="Peat">Thomas S. Peat</name><affiliation><nlm:aff id="au3"><institution>CSIRO Molecular and Health Technologies</institution><addr-line>343 Royal Parade, Parkville, Vic. 3052, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Newman, Janet" sort="Newman, Janet" uniqKey="Newman J" first="Janet" last="Newman">Janet Newman</name><affiliation><nlm:aff id="au3"><institution>CSIRO Molecular and Health Technologies</institution><addr-line>343 Royal Parade, Parkville, Vic. 3052, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Briggs, Lyndall J" sort="Briggs, Lyndall J" uniqKey="Briggs L" first="Lyndall J" last="Briggs">Lyndall J. Briggs</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lapalikar, Gauri V" sort="Lapalikar, Gauri V" uniqKey="Lapalikar G" first="Gauri V" last="Lapalikar">Gauri V. Lapalikar</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Campbell, Peter M" sort="Campbell, Peter M" uniqKey="Campbell P" first="Peter M" last="Campbell">Peter M. Campbell</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Scott, Colin" sort="Scott, Colin" uniqKey="Scott C" first="Colin" last="Scott">Colin Scott</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Russell, Robyn J" sort="Russell, Robyn J" uniqKey="Russell R" first="Robyn J" last="Russell">Robyn J. Russell</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Oakeshott, John G" sort="Oakeshott, John G" uniqKey="Oakeshott J" first="John G" last="Oakeshott">John G. Oakeshott</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20807200</idno><idno type="pmc">3034190</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3034190</idno><idno type="RBID">PMC:3034190</idno><idno type="doi">10.1111/j.1365-2958.2010.07356.x</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">002770</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">002770</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Identification and characterization of two families of F<sub>420</sub>H<sub>2</sub>-dependent reductases from <italic>Mycobacteria</italic> that catalyse aflatoxin degradation</title><author><name sortKey="Taylor, Matthew C" sort="Taylor, Matthew C" uniqKey="Taylor M" first="Matthew C" last="Taylor">Matthew C. Taylor</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jackson, Colin J" sort="Jackson, Colin J" uniqKey="Jackson C" first="Colin J" last="Jackson">Colin J. Jackson</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="au2"><institution>Institute de Biologie Structurale</institution><addr-line>41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Tattersall, David B" sort="Tattersall, David B" uniqKey="Tattersall D" first="David B" last="Tattersall">David B. Tattersall</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="French, Nigel" sort="French, Nigel" uniqKey="French N" first="Nigel" last="French">Nigel French</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Peat, Thomas S" sort="Peat, Thomas S" uniqKey="Peat T" first="Thomas S" last="Peat">Thomas S. Peat</name><affiliation><nlm:aff id="au3"><institution>CSIRO Molecular and Health Technologies</institution><addr-line>343 Royal Parade, Parkville, Vic. 3052, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Newman, Janet" sort="Newman, Janet" uniqKey="Newman J" first="Janet" last="Newman">Janet Newman</name><affiliation><nlm:aff id="au3"><institution>CSIRO Molecular and Health Technologies</institution><addr-line>343 Royal Parade, Parkville, Vic. 3052, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Briggs, Lyndall J" sort="Briggs, Lyndall J" uniqKey="Briggs L" first="Lyndall J" last="Briggs">Lyndall J. Briggs</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lapalikar, Gauri V" sort="Lapalikar, Gauri V" uniqKey="Lapalikar G" first="Gauri V" last="Lapalikar">Gauri V. Lapalikar</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Campbell, Peter M" sort="Campbell, Peter M" uniqKey="Campbell P" first="Peter M" last="Campbell">Peter M. Campbell</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Scott, Colin" sort="Scott, Colin" uniqKey="Scott C" first="Colin" last="Scott">Colin Scott</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Russell, Robyn J" sort="Russell, Robyn J" uniqKey="Russell R" first="Robyn J" last="Russell">Robyn J. Russell</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Oakeshott, John G" sort="Oakeshott, John G" uniqKey="Oakeshott J" first="John G" last="Oakeshott">John G. Oakeshott</name><affiliation><nlm:aff id="au1"><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">Molecular Microbiology</title><idno type="ISSN">0950-382X</idno><idno type="eISSN">1365-2958</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Aflatoxins are polyaromatic mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degradation, aflatoxins can be metabolized by certain species of <italic>Actinomycetales</italic>. However, the enzymatic basis for their breakdown has not been reported until now. We have identified nine <italic>Mycobacterium smegmatis</italic> enzymes that utilize the deazaflavin cofactor F<sub>420</sub>H<sub>2</sub> to catalyse the reduction of the α,β-unsaturated ester moiety of aflatoxins, activating the molecules for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F<sub>420</sub>H<sub>2</sub> dependent reductase (FDR-A and -B) families that are distantly related to the flavin mononucleotide (FMN) dependent pyridoxamine 5′-phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F<sub>420</sub>H<sub>2</sub> binding groove. A general role for these enzymes in xenobiotic metabolism is discussed, including the observation that the nitro-reductase Rv3547 from <italic>Mycobacterium tuberculosis</italic> that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR-A family.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Alberts, Jf" uniqKey="Alberts J">JF Alberts</name></author><author><name sortKey="Engelbrecht, Y" uniqKey="Engelbrecht Y">Y Engelbrecht</name></author><author><name sortKey="Steyn, Ps" uniqKey="Steyn P">PS Steyn</name></author><author><name sortKey="Holzapfel, Wh" uniqKey="Holzapfel W">WH Holzapfel</name></author><author><name sortKey="Van Zyl, Wh" uniqKey="Van Zyl W">WH van Zyl</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bashiri, G" uniqKey="Bashiri G">G Bashiri</name></author><author><name sortKey="Squire, Cj" uniqKey="Squire C">CJ 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Mol Microbiol</journal-id><journal-id journal-id-type="publisher-id">mmi</journal-id><journal-title-group><journal-title>Molecular Microbiology</journal-title></journal-title-group><issn pub-type="ppub">0950-382X</issn><issn pub-type="epub">1365-2958</issn><publisher><publisher-name>Blackwell Publishing Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">20807200</article-id><article-id pub-id-type="pmc">3034190</article-id><article-id pub-id-type="doi">10.1111/j.1365-2958.2010.07356.x</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Articles</subject></subj-group></article-categories><title-group><article-title>Identification and characterization of two families of F<sub>420</sub>H<sub>2</sub>-dependent reductases from <italic>Mycobacteria</italic> that catalyse aflatoxin degradation</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Taylor</surname><given-names>Matthew C</given-names></name><xref ref-type="aff" rid="au1">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Jackson</surname><given-names>Colin J</given-names></name><xref ref-type="aff" rid="au1">1</xref><xref ref-type="aff" rid="au2">2</xref></contrib><contrib contrib-type="author"><name><surname>Tattersall</surname><given-names>David B</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>French</surname><given-names>Nigel</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Peat</surname><given-names>Thomas S</given-names></name><xref ref-type="aff" rid="au3">3</xref></contrib><contrib contrib-type="author"><name><surname>Newman</surname><given-names>Janet</given-names></name><xref ref-type="aff" rid="au3">3</xref></contrib><contrib contrib-type="author"><name><surname>Briggs</surname><given-names>Lyndall J</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lapalikar</surname><given-names>Gauri V</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Campbell</surname><given-names>Peter M</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Scott</surname><given-names>Colin</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Russell</surname><given-names>Robyn J</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Oakeshott</surname><given-names>John G</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><aff id="au1"><label>1</label><institution>CSIRO Ecosystem Sciences</institution><addr-line>GPO Box 1700, Canberra, ACT 2601, Australia</addr-line></aff><aff id="au2"><label>2</label><institution>Institute de Biologie Structurale</institution><addr-line>41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France</addr-line></aff><aff id="au3"><label>3</label><institution>CSIRO Molecular and Health Technologies</institution><addr-line>343 Royal Parade, Parkville, Vic. 3052, Australia</addr-line></aff></contrib-group><author-notes><corresp id="cor1">*For correspondence. E-mail <email>m.taylor@csiro.au</email>; Tel. (+61) 2 6246 4404; Fax (+61) 2 6246 4094. <bold>Data deposition footnote:</bold> Coordinates have been deposited in the PDB under accession codes 3F7E (MSMEG_3380) and 3H96 (MSMEG_3356).</corresp><fn><p>Re-use of this article is permitted in accordance with the Terms and Conditions set out at <ext-link ext-link-type="uri" xlink:href="http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms">http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms</ext-link></p></fn></author-notes><pub-date pub-type="ppub"><month>11</month><year>2010</year></pub-date><volume>78</volume><issue>3</issue><fpage>561</fpage><lpage>575</lpage><history><date date-type="accepted"><day>17</day><month>8</month><year>2010</year></date></history><permissions><copyright-statement>Copyright © 2010 Blackwell Publishing Ltd</copyright-statement><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.5/"><license-p>Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.</license-p></license></permissions><abstract><p>Aflatoxins are polyaromatic mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degradation, aflatoxins can be metabolized by certain species of <italic>Actinomycetales</italic>. However, the enzymatic basis for their breakdown has not been reported until now. We have identified nine <italic>Mycobacterium smegmatis</italic> enzymes that utilize the deazaflavin cofactor F<sub>420</sub>H<sub>2</sub> to catalyse the reduction of the α,β-unsaturated ester moiety of aflatoxins, activating the molecules for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F<sub>420</sub>H<sub>2</sub> dependent reductase (FDR-A and -B) families that are distantly related to the flavin mononucleotide (FMN) dependent pyridoxamine 5′-phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F<sub>420</sub>H<sub>2</sub> binding groove. A general role for these enzymes in xenobiotic metabolism is discussed, including the observation that the nitro-reductase Rv3547 from <italic>Mycobacterium tuberculosis</italic> that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR-A family.</p></abstract></article-meta></front><body><sec sec-type=""><title>Introduction</title><p>Aflatoxins are secondary metabolites of various <italic>Aspergillus</italic> species, particularly <italic>A. flavus</italic> and <italic>A. parasiticus</italic>, and are generally separated into two groups: the difurocoumarocyclopentenones (including AFB1 and AFB2) and the difurocoumarolactones (including AFG1 and AFG2) (<xref ref-type="fig" rid="fig01">Fig. 1</xref>). Humans can be exposed to aflatoxins via consumption of <italic>Aspergillus-</italic>contaminated maize or nuts, or the milk of animals fed contaminated crops. Recurrent consumption of aflatoxin-contaminated food has been shown to contribute to an increased risk of nutritional deficiencies, immune suppression and hepatocellular carcinoma in several developing nations (<xref ref-type="bibr" rid="b66">Wagacha and Muthomi, 2008</xref>). The levels of aflatoxins in internationally traded commodities are tightly regulated by the FAO/WHO and other regulatory bodies (<xref ref-type="bibr" rid="b13">van Egmond <italic>et al</italic>., 2007</xref>).</p><fig id="fig01" position="float"><label>Fig. 1</label><caption><p>Chemical structures of compounds used in this study.</p></caption><graphic xlink:href="mmi0078-0561-f1"></graphic></fig><p>Although higher organisms and many microorganisms have little ability to degrade aflatoxins, they are known to be metabolized by certain fungi and bacteria (<xref ref-type="bibr" rid="b9">Ciegler <italic>et al</italic>., 1966</xref>). The bacteria known to degrade aflatoxins are all <italic>Actinomycetales</italic> of the suborder <italic>Corynebacterineae</italic>, such as <italic>Nocardia corynebacterioides</italic> (<xref ref-type="bibr" rid="b9">Ciegler <italic>et al</italic>., 1966</xref>; <xref ref-type="bibr" rid="b25">Hormisch <italic>et al</italic>., 2004</xref>), <italic>Rhodococcus erythropolis</italic> and <italic>Mycobacterium fluorantheniorans</italic> sp. Nov DSM44556<sup>T</sup> (<xref ref-type="bibr" rid="b63">Teniola <italic>et al</italic>., 2005</xref>). There is evidence that aflatoxin degradation by these species is enzyme catalysed (<xref ref-type="bibr" rid="b57">Smiley and Draughon, 2000</xref>; <xref ref-type="bibr" rid="b1">Alberts <italic>et al</italic>., 2006</xref>), although the reaction products and the enzymes involved have not previously been described. Because the degradation products of aflatoxins are non-toxic (<xref ref-type="bibr" rid="b9">Ciegler <italic>et al</italic>., 1966</xref>; <xref ref-type="bibr" rid="b63">Teniola <italic>et al</italic>., 2005</xref>), there is considerable interest in identifying and characterizing the enzymes involved.</p><p>Herein, we describe the isolation and characterization of nine F<sub>420</sub>H<sub>2</sub>-dependent reductases (FDRs) from <italic>Mycobacterium smegmatis</italic> that utilize an F<sub>420</sub>H<sub>2</sub> cofactor to catalyse the reduction of aflatoxins, leading to their spontaneous breakdown. Structural and phylogenetic analysis suggests that these nine enzymes fall into two hitherto unclassified enzyme families that are distantly related to the well characterized FMN-dependent pyridoxamine 5′-phosphate oxidase (PNPOx) family (<xref ref-type="bibr" rid="b53">di Salvo <italic>et al</italic>., 2003</xref>). The mechanism of aflatoxin breakdown appears to involve an initial, enzyme-catalysed reduction of the double bond of the α,β-unsaturated ester moiety, followed by spontaneous hydrolysis. The possible cellular functions of these enzymes in xenobiotic metabolism are discussed, as are their potential uses, both as an enzymatic means by which aflatoxins may be detoxified and as potential drug targets, owing to their exclusive presence in <italic>Archea, Actinomycetales</italic> and some species of <italic>γ-Proteobacteria.</italic></p></sec><sec sec-type=""><title>Results</title><sec><title>Identification of aflatoxin degrading bacterial strains</title><p>Seven bacterial strains (<italic>Mycobacterium smegmatis</italic> mc<sup>2</sup>155, <italic>Mycobacterium</italic> sp. ESD, <italic>Arthrobacter</italic> KW-ES, <italic>Arthrobacter</italic> RW thio, <italic>Arthrobacter</italic> RLH#41, <italic>Agrobacterium radiobacter</italic> P230 and <italic>Pseudomonas monteilii</italic> C11) known to degrade various xenobiotics (<xref ref-type="bibr" rid="b59">Sutherland <italic>et al</italic>., 2002</xref>; <xref ref-type="bibr" rid="b26">Horne <italic>et al</italic>., 2002a</xref>,<xref ref-type="bibr" rid="b27">b</xref>) were tested for their ability to degrade aflatoxin AFG1 in liquid culture. Live cultures and cell lysates of the two <italic>Mycobacterium</italic> strains were found to degrade AFG1, while no aflatoxin degradation was observed in live cultures or cell lysates of the other strains tested. The activity of the <italic>Mycobacterium</italic> lysates was lost upon heating, consistent with an enzyme-catalysed reaction, as shown by thin-layer chromatography (TLC; <xref ref-type="supplementary-material" rid="SD1">Fig. S1</xref>). Although other <italic>Actinomycetales</italic> have previously been shown to degrade aflatoxins (<xref ref-type="bibr" rid="b9">Ciegler <italic>et al</italic>., 1966</xref>; <xref ref-type="bibr" rid="b25">Hormisch <italic>et al</italic>., 2004</xref>; <xref ref-type="bibr" rid="b63">Teniola <italic>et al</italic>., 2005</xref>; <xref ref-type="bibr" rid="b1">Alberts <italic>et al</italic>., 2006</xref>), the failure of the <italic>Arthrobacter</italic> cultures tested to degrade AFG1 demonstrates that aflatoxins are not degraded by all <italic>Actinomycetales</italic>. Because the genome of <italic>M. smegmatis</italic> mc<sup>2</sup>155 has been sequenced and annotated, and methodologies for its manipulation in the laboratory are well established, it was used in all subsequent experiments to isolate and identify the gene/enzyme systems responsible for aflatoxin degradation.</p></sec><sec><title>Aflatoxin degradation is cofactor F<sub>420</sub>-dependent</title><p>Transposon mutagenesis was used as a first approach to identify the genes/enzymes involved in the degradation of aflatoxin by <italic>M. smegmatis</italic>. After mutagenesis with the EZ::TN transposon, 3160 viable isolates were assayed by TLC for their ability to degrade AFG1 over 4 days (<xref ref-type="supplementary-material" rid="SD1">Fig. S1</xref>). Only five of the 3160 isolates did not degrade AFG1. Sequencing of the region surrounding the transposon insertion site in these colonies showed that four of the insertions disrupted the <italic>fbiC</italic> gene, which catalyses the final step in the biosynthesis of the deazaflavin, Fo, from intermediates of the riboflavin synthesis and tyrosine metabolic pathways (<xref ref-type="supplementary-material" rid="SD1">Fig. S2</xref>) (<xref ref-type="bibr" rid="b7">Choi <italic>et al</italic>., 2002</xref>; <xref ref-type="bibr" rid="b19">Graham <italic>et al</italic>., 2003</xref>). Fo is the precursor for the production of the cofactor F<sub>420</sub> (<xref ref-type="bibr" rid="b21">Graupner and White, 2001</xref>). The fifth transposon insertion disrupted the <italic>fgd</italic> gene, which encodes the F<sub>420</sub>-dependent glucose 6-phosphate dehydrogenase (FGD) (<xref ref-type="bibr" rid="b48">Purwantini and Daniels, 1998</xref>; <xref ref-type="bibr" rid="b2">Bashiri <italic>et al</italic>., 2008</xref>), which is known to recycle F<sub>420</sub> from its oxidized to its reduced F<sub>420</sub>H<sub>2</sub> form in <italic>Mycobacteria</italic> (<xref ref-type="supplementary-material" rid="SD1">Fig. S2</xref>). These data are consistent with an essential role for F<sub>420</sub> in the degradation of aflatoxins in <italic>M. smegmatis.</italic> Significantly, all the bacterial genera previously shown to degrade aflatoxins (see above) are also known to synthesize F<sub>420</sub> (<xref ref-type="bibr" rid="b29">Isabelle <italic>et al</italic>., 2002</xref>).</p> <p>To test whether FGD catalysed aflatoxin degradation, it was heterologously expressed in and purified from <italic>Escherichia coli.</italic> As expected, FGD could catalyse the reduction of F<sub>420</sub> to F<sub>420</sub>H<sub>2</sub> in the presence of glucose 6-phosphate (<xref ref-type="bibr" rid="b48">Purwantini and Daniels, 1998</xref>; <xref ref-type="bibr" rid="b2">Bashiri <italic>et al</italic>., 2008</xref>). However, neither FGD in the presence of F<sub>420</sub>/F<sub>420</sub>H<sub>2</sub>, nor F<sub>420</sub>H<sub>2</sub> in isolation, were able to catalyse aflatoxin breakdown at detectable levels. Thus, although F<sub>420</sub>H<sub>2</sub> is clearly essential to the reaction, it is most likely involved as a cofactor for another enzyme not identified in this screen.</p></sec><sec><title>Identification and kinetic analysis of two families of F<sub>420</sub>H<sub>2</sub>-dependent aflatoxin degrading enzymes</title><p>The enzymes responsible for aflatoxin degradation in <italic>M. smegmatis</italic> were purified from soluble cell extracts. Two protocols were used, one involved ammonium sulphate precipitation, hydrophobic interaction chromatography, anion exchange chromatography and SDS-PAGE (<xref ref-type="fig" rid="fig02">Fig. 2</xref>). The second involved ammonium sulphate precipitation, hydrophobic interaction, gel filtration and anion exchange chromatography. Proteins were then identified from active fractions in the final purification steps via tandem mass spectrometry of peptides obtained by tryptic digests and comparison of these fragments to predicted proteins from the <italic>M. smegmatis</italic> genome (<xref ref-type="supplementary-material" rid="SD1">Table S1</xref>). Only four proteins were identified in both purifications; MSMEG_3380, 3004, 2027 and 5717. The observation that multiple enzymes with the same activity were identified provides an explanation as to why they were not identified by transposon mutagenesis, since they would all need to be simultaneously inactivated to prevent AFG1 degradation.</p><fig id="fig02" position="float"><label>Fig. 2</label><caption><p>Purification protocol. Protein fractions that showed F<sub>420</sub>H<sub>2</sub> dependent AFG1 degradation as measured by TLC were separated from <italic>M. smegmatis</italic> extracts. The ammonium sulphate precipitated proteins were first purified by hydrophobic interaction chromatography (HIC). Active fractions were further purified by anion exchange chromatography before separation by SDS-PAGE. Bands were cut from SDS-PAGE gels, digested with trypsin and analysed by LC/MS/MS. Peptides were identified from the annotated <italic>M. smegmatis</italic> genome sequence and corresponding results are shown for some of the excised bands. In the TLCs ‘-ve’ denotes no enzyme negative control and ‘sm’ denotes <italic>M. smegmatis</italic> cell extract positive control.</p></caption><graphic xlink:href="mmi0078-0561-f2"></graphic></fig><p>The four proteins identified above were all predicted to be related to the PNPOx family by the protein fold recognition server <italic>PHYRE</italic> (<xref ref-type="bibr" rid="b33">Kelley and Sternberg, 2009</xref>), although their relationship is distant (less than 15% amino acid identity). The four proteins have predicted molecular weights, based on their sequences, of approximately 16kDa (<xref ref-type="supplementary-material" rid="SD1">Table S1</xref>), while proteins in the active <italic>M. smegmatis</italic> fractions were calculated to be approximately 30 kDa by size exclusion chromatography (<xref ref-type="supplementary-material" rid="SD1">Fig. S3</xref>), suggesting that these four proteins most likely oligomerize as dimers in solution. These results are consistent with the observation that the PNPOx enzymes form homodimers (<xref ref-type="bibr" rid="b51">di Salvo <italic>et al</italic>., 1998</xref>). Phylogenetic analysis of 146 PNPOx-like sequences, including 28 present in <italic>M. smegmatis</italic> (<xref ref-type="fig" rid="fig03">Fig. 3</xref> and <xref ref-type="supplementary-material" rid="SD1">Fig. S4</xref>), shows that the four proteins belong to two distinct families (< 12% amino acid identity), hereafter denoted FDR-A and -B (F<sub>420</sub>H<sub>2</sub> dependent reductase -A and -B), which are both clearly differentiated from the functionally defined PNPOx family. Although the PNPOx family is widely distributed among both prokaryotes and eukaryotes, the FDR-A and FDR-B families appear to be limited to the <italic>Actinomycetales, Archaea</italic> and some <italic>Proteobacteria,</italic> all of which possess F<sub>420</sub>. Furthermore, the two families are unrelated to other previously characterized F<sub>420</sub>H<sub>2</sub> dependent reductases, including sulphite reductase (<xref ref-type="bibr" rid="b32">Johnson and Mukhopadhyay, 2005</xref>), dinitrophenol reductase (<xref ref-type="bibr" rid="b12">Ebert <italic>et al</italic>., 2001</xref>) and the methylenetetrahydromethanopterin reductase complex involved in methanogenesis in <italic>Archaea</italic> (<xref ref-type="bibr" rid="b10">Deppenmeier, 2002</xref>; <xref ref-type="bibr" rid="b18">Graham and White, 2002</xref>). Interestingly, the only other functionally characterized enzyme that is related to either of these families is the bicyclic nitroimidazole prodrug activating reductase from <italic>M. tuberculosis</italic>, Rv3547 (<xref ref-type="bibr" rid="b36">Manjunatha <italic>et al</italic>., 2006</xref>), which has 49% sequence identity to MSMEG_5998 of the FDR-A family (<xref ref-type="fig" rid="fig03">Fig. 3</xref>).</p><fig id="fig03" position="float"><label>Fig. 3</label><caption><p>Phylogenetic relationship between the PNPOx, FDR-A and FDR-B families. A condensed tree was constructed from 146 protein sequences of FDRs/PNPOxs from seven species of <italic>Actinomycetales</italic> (<italic>M. smegmatis</italic>, <italic>M. tuberculosis H37Rv</italic>, <italic>M. vanbaalenii</italic>, <italic>Rhodococcus sp. RH1</italic>, <italic>Arthrobacter sp. FB24</italic>, <italic>S. coelicolor</italic>, <italic>Frankia alni</italic> and <italic>Nocardioides</italic> sp. JS614), plus the known PNPOx enzymes from <italic>H. sapiens</italic>, <italic>E. coli</italic>, <italic>Saccharomyces cerevisiae</italic>, <italic>Caenorhabditis elegans</italic> and <italic>Mus musculus</italic>. Solid red circles denote the FDRs described in this paper, and open red circles denote other potential FDRs from <italic>M. smegmatis</italic> (TIGR locus number shown). For clarity, only enzymes from other species that have been previously characterized are labelled: blue triangles denote <italic>M. tuberculosis</italic> enzymes with known structures or previously described functions and black squares denote previously described PNPOxs.</p></caption><graphic xlink:href="mmi0078-0561-f3"></graphic></fig><p>The next phase of our work focused on the four <italic>M. smegmatis</italic> enzymes identified by chromatography plus five other members of the FDR-A and FDR-B families in the <italic>M. smegmatis</italic> genome. These five enzymes were identified as close paralogues both by <sc>blast</sc> analysis and by their retention of a highly conserved loop motif and a putative phosphate binding motif (<xref ref-type="supplementary-material" rid="SD1">Fig. S4</xref>). The nine genes were cloned, expressed, purified and assayed against the four aflatoxins in the presence of F<sub>420</sub>H<sub>2</sub> (<xref ref-type="table" rid="tbl1">Table 1</xref>). All nine enzymes catalysed measurable degradation of all four aflatoxins tested (AFG1, AFG2, AFB1 and AFB2). AFG1 was found to be the best substrate for all nine enzymes: for example MSMEG_3380 has nearly 50-fold more activity with AFG1 than AFB2. However, the specific activities of the various enzymes differed by more than four orders of magnitude for AFG1. The four enzymes originally identified in the chromatographic experiments were not the most active, perhaps because of purification, expression or stability issues. The three most active enzymes were from the FDR-A family (MSMEG_5998, 2850 and 3356). Kinetic parameters were obtained for MSMEG_5998 with AFB1 (<italic>K</italic><sub>M</sub> = 47 ± 6 µM, <italic>K</italic><sub>cat</sub> = 63 ± 4 min<sup>−1</sup>); these values could not be obtained for other enzymes because enzyme activity with saturating substrate concentrations could not be measured, owing to the low solubility of the aflatoxins. The fact that all nine enzymes tested had aflatoxin degrading activity suggests that some, if not all, of the other 19 representatives of the two FDR families which we have found by <sc>blast</sc> analysis of the <italic>M. smegmatis</italic> genome might also catalyse aflatoxin breakdown.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><p>Specific activities of MSMEG proteins for aflatoxins</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th align="center" colspan="4" rowspan="1">Specific activity (nmol min<sup>−1</sup> µmol<sup>−1</sup> enzyme)<xref ref-type="table-fn" rid="tf1-1">a</xref></th></tr><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th align="left" colspan="4" rowspan="1"><hr></hr></th></tr><tr><th align="left" rowspan="1" colspan="1">Protein family</th><th align="left" rowspan="1" colspan="1">TIGR Locus<xref ref-type="table-fn" rid="tf1-2">b</xref></th><th align="center" rowspan="1" colspan="1">AFG1</th><th align="center" rowspan="1" colspan="1">AFG2</th><th align="center" rowspan="1" colspan="1">AFB1</th><th align="center" rowspan="1" colspan="1">AFB2</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">FDR-A</td><td align="left" rowspan="1" colspan="1">5998</td><td align="center" rowspan="1" colspan="1">83 000 ± 2000</td><td align="center" rowspan="1" colspan="1">6100 ± 200</td><td align="center" rowspan="1" colspan="1">9100 ± 700</td><td align="center" rowspan="1" colspan="1">7700 ± 700</td></tr><tr><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">2850</td><td align="center" rowspan="1" colspan="1">12 000 ± 2000</td><td align="center" rowspan="1" colspan="1">8000 ± 500</td><td align="center" rowspan="1" colspan="1">1300 ± 200</td><td align="center" rowspan="1" colspan="1">410 ± 60</td></tr><tr><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">3356</td><td align="center" rowspan="1" colspan="1">8080 ± 70</td><td align="center" rowspan="1" colspan="1">3100 ± 500</td><td align="center" rowspan="1" colspan="1">1200 ± 200</td><td align="center" rowspan="1" colspan="1">1300 ± 200</td></tr><tr><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">3004</td><td align="center" rowspan="1" colspan="1">1600 ± 100</td><td align="center" rowspan="1" colspan="1">1260 ± 50</td><td align="center" rowspan="1" colspan="1">310 ± 60</td><td align="center" rowspan="1" colspan="1">620 ± 20</td></tr><tr><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">2027</td><td align="center" rowspan="1" colspan="1">660 ± 40</td><td align="center" rowspan="1" colspan="1">310 ± 50</td><td align="center" rowspan="1" colspan="1">220 ± 50</td><td align="center" rowspan="1" colspan="1">160 ± 20</td></tr><tr><td align="left" rowspan="1" colspan="1">FDR-B</td><td align="left" rowspan="1" colspan="1">3380</td><td align="center" rowspan="1" colspan="1">1600 ± 100</td><td align="center" rowspan="1" colspan="1">320 ± 40</td><td align="center" rowspan="1" colspan="1">130 ± 10</td><td align="center" rowspan="1" colspan="1">33 ± 4</td></tr><tr><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">6848</td><td align="center" rowspan="1" colspan="1">900 ± 200</td><td align="center" rowspan="1" colspan="1">430 ± 50</td><td align="center" rowspan="1" colspan="1">100 ± 10</td><td align="center" rowspan="1" colspan="1">90 ± 3</td></tr><tr><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">5170</td><td align="center" rowspan="1" colspan="1">240 ± 40</td><td align="center" rowspan="1" colspan="1">140 ± 6</td><td align="center" rowspan="1" colspan="1">330 ± 50</td><td align="center" rowspan="1" colspan="1">90 ± 2</td></tr><tr><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">5717</td><td align="center" rowspan="1" colspan="1">3 ± 0.7</td><td align="center" rowspan="1" colspan="1">3 ± 0.5</td><td align="center" rowspan="1" colspan="1">3 ± 0.1</td><td align="center" rowspan="1" colspan="1">2 ± 0.2</td></tr></tbody></table><table-wrap-foot><fn id="tf1-1"><label>a</label><p>Units are expressed per µmole of the monomer for both FDR-A and -B.</p></fn><fn id="tf1-2"><label>b</label><p>The TIGR locus number is preceded by ‘MSMEG_’.</p></fn></table-wrap-foot></table-wrap><p>Although genome sequences of other <italic>Actinomycete</italic> strains that metabolize aflatoxins, such as <italic>R. erythropolis</italic>, <italic>N. corynebacterioides</italic> and <italic>M. fluoranthenivorans</italic> (<xref ref-type="bibr" rid="b9">Ciegler <italic>et al</italic>., 1966</xref>; <xref ref-type="bibr" rid="b25">Hormisch <italic>et al</italic>., 2004</xref>; <xref ref-type="bibr" rid="b63">Teniola <italic>et al</italic>., 2005</xref>), have not been published, our preliminary screening of several related <italic>Actinomycete</italic> genome sequences (<xref ref-type="supplementary-material" rid="SD1">Table S2</xref>) shows that each has at least 10 putative FDR-As and FDR-Bs. It thus seems likely that the aflatoxin degrading abilities of the FDRs shown in <italic>M. smegmatis</italic>, and quite likely many other <italic>Actinomycetales</italic>, may originate from FDRs of the A and B families. It is notable that there is only one annotated FDR-B and no annotated FDR-A sequences present in <italic>Arthrobacter</italic> genomes so far published (<xref ref-type="supplementary-material" rid="SD1">Table S2</xref>), which may explain the lack of aflatoxin degradation observed in the three <italic>Arthrobacter</italic> strains analysed in this study.</p></sec><sec><title>The PNPOx and FDR families are functionally distinct</title><p>Because the FDR families have significant sequence similarity to the PNPOxs and the respective cofactors F<sub>420</sub>H<sub>2</sub> and FMN/FMNH<sub>2</sub> are structurally related (<xref ref-type="supplementary-material" rid="SD1">Fig. S2</xref>), we investigated whether there was any cofactor, or catalytic, promiscuity between these enzyme families. Phylogenetic analysis (<xref ref-type="fig" rid="fig03">Fig. 3</xref>) identified MSMEG_5675 as the probable <italic>M. smegmatis</italic> PNPOx. In contrast to the expressed FDR-A and -B enzymes, preparations of purified MSMEG_5675 were an intense yellow colour with an absorption maximum at 450 nm, indicative of protein bound to FMN. MSMEG_5675 was confirmed to be a PNPOx by its capacity to catalyse the conversion of pyridoxamine 5′-phosphate (PMP) to pyridoxal 5′-phosphate (PLP) with a specific activity of 0.18 s<sup>−1</sup>, consistent with the catalytic activity of other PNPOxs (<xref ref-type="bibr" rid="b51">di Salvo <italic>et al</italic>., 1998</xref>; <xref ref-type="bibr" rid="b52">di Salvo <italic>et al</italic>., 2002</xref>). No activity was observed after incubating the FDRs overnight with 100 µM FMN and PMP, suggesting that these enzymes exclusively utilize F<sub>420</sub>H<sub>2</sub> as a cofactor. Moreover, once FMN was stripped from MSMEG_5675, the apo-enzyme was found to be unable to catalyse aflatoxin degradation in the presence of F<sub>420</sub>H<sub>2</sub> (data not shown). Thus, our data suggest that the PNPOx and FDR families are functionally distinct and they do not appear to have overlapping activities or cofactor preferences.</p></sec><sec><title>Structures of the FDR-A and FDR-B enzymes</title><p>To gain further insight into the function of these newly identified enzymes, crystal structures of members of the FDR-A (MSMEG_3356) and FDR-B families (MSMEG_3380) were solved to 2.0 and 1.2 Å resolution respectively. MSMEG_3356 and MSMEG_3380 both adopt the PNPOx-like split barrel fold, comprising a central split barrel surrounded by four helices (<xref ref-type="fig" rid="fig04">Fig. 4</xref>). MSMEG_3380 exists as a homodimer, while MSMEG_3356 is monomeric.</p><fig id="fig04" position="float"><label>Fig. 4</label><caption><p>The structures of FDR-A and FDR-B enzymes.</p><p>A. MSMEG_3356; from left to right, a topology diagram, showing the arrangement of the split barrel of anti-parallel β-sheets, surrounded by three large α-helices at the side and a short α-helix above, a cartoon diagram, and an electrostatic surface potential representation with F<sub>420</sub>H<sub>2</sub> docked.</p><p>B. MSMEG_3380; from left to right, a topology diagram of the structure, showing the arrangement of the split barrel of anti-parallel β-sheets, surrounded by one large α-helix at the side and three short α-helices above and below, a cartoon diagram of the 3380 dimer, and an electrostatic surface potential representation with F<sub>420</sub>H<sub>2</sub> docked.</p></caption><graphic xlink:href="mmi0078-0561-f4"></graphic></fig><p>The structures of MSMEG_3380 and MSMEG_3356 differ significantly, as would be expected given their low sequence similarity; only 88 amino acids could be structurally aligned with an r.m.s.d of 2.2 Å. A DALI (<xref ref-type="bibr" rid="b24">Holm <italic>et al</italic>., 2008</xref>) search indicated that the closest known structural homologue to the FDR-A MSMEG_3356 is a putative PNPOx from <italic>Agrobacterium tumefaciens</italic> (3DNH), with just 15% amino acid identity. Thus, it appears that MSMEG_3356 is the first characterized structure of a member of the FDR-A family. The closest relatives of MSMEG_3380 with known structures are the functionally uncharacterized proteins from <italic>M. tuberculosis</italic> Rv1155 and Rv2074 (<xref ref-type="bibr" rid="b3">Biswal <italic>et al</italic>., 2005</xref>; <xref ref-type="bibr" rid="b4">2006</xref>; <xref ref-type="bibr" rid="b6">Canaan <italic>et al</italic>., 2005</xref>), with amino acid identities of 18% and 24% respectively. These proteins have been putatively classified as PNPOxs, owing to their similarity to the PNPOx family, but no functional analysis has been performed. As shown in our phylogenetic analysis (<xref ref-type="fig" rid="fig03">Fig. 3</xref>), they have greater similarity to the FDR-B family than the PNPOxs and we propose that these proteins and MSMEG_3380 comprise the first structures of the FDR-B family.</p><p>Despite their low similarity, the structures of both the FDRs described here are characterized by large hydrophobic pockets formed by several aromatic residues and a positively charged groove that is principally formed by several lysine and arginine side-chains (<xref ref-type="fig" rid="fig04">Fig. 4</xref>). In terms of shape and electrostatic character, these pockets/channels are highly complementary to the F<sub>420</sub>H<sub>2</sub> cofactor, which possesses an aromatic deazaflavin group and a long, negatively charged, side-chain consisting of a phospho-lactate and a variable number of glutamates. In the absence of a structure of the holoenzyme, we have docked the cofactor, as shown in <xref ref-type="fig" rid="fig04">Fig. 4</xref>. Consistent with the level of electrostatic and hydrophobic interactions implied by the docking results, titration curves of activity versus cofactor concentration (<xref ref-type="fig" rid="fig05">Fig. 5</xref>) show that both MSMEG_3356 and MSMEG_3380 bind F<sub>420</sub>H<sub>2</sub> tightly, with apparent <italic>K</italic><sub>M</sub> values in the nanomolar range (260 nM and 130 nM, respectively).</p><fig id="fig05" position="float"><label>Fig. 5</label><caption><p>F<sub>420</sub>H<sub>2</sub> cofactor binding to enzymes 3356 and 3380. The apparent <italic>K</italic><sub>M</sub>'s for F<sub>420</sub>H<sub>2</sub> were calculated by measuring the velocity of the reaction with varying concentrations of F<sub>420</sub>H<sub>2</sub> from 0.05 to 10 µM while maintaining all other components at 100 µM aflatoxin, 1 µM enzyme. For clarity the velocity is expressed as percent of maximum velocity. Empty circles, 3356; filled circles, 3380.</p></caption><graphic xlink:href="mmi0078-0561-f5"></graphic></fig><p>Although the residues forming the putative F<sub>420</sub>H<sub>2</sub> binding channels and substrate binding pockets occur in homologous regions of the two families, the identities of the residues involved are not conserved (<xref ref-type="fig" rid="fig06">Fig. 6</xref> and <xref ref-type="supplementary-material" rid="SD1">Fig. S4</xref>). In MSMEG_3356, the deazaflavin/substrate pocket is formed by W7, N25, F26, M31, P48, M49, M50, F64, A68, W76, F121 and Y124, and the positively charged groove is formed by the side-chains of N8, H36, R39, K40, T41, K43, T47 and K67. In MSMEG_3380 the deazaflavin/substrate pocket is formed by M34, W35, Y95, L98 and L106 of monomer A and Y74 and Y76 of monomer B. The positively charged groove is formed by the side-chains of R24, Q30, N32, H47, R51, Q52, K53 and R55 from monomer A and R80 from monomer B. The hydrophobic aflatoxin/deazaflavin binding cavity of MSMEG_3380 is significantly larger than that of MSMEG_3356 (649 Å<sup>3</sup> versus 411 Å<sup>3</sup>), as measured by the 3V server (<xref ref-type="bibr" rid="b64">Voss and Gerstein, 2010</xref>), suggesting that these enzymes are likely to have different substrate ranges.</p><fig id="fig06" position="float"><label>Fig. 6</label><caption><p>The F<sub>420</sub>H<sub>2</sub> binding grooves of an FDR-A, MSMEG_3356, and an FDR-B, MSMEG_3380. The deazaflavin pockets are shown to be rich in aromatic and hydrophobic residues, while the side-chain binding grooves are rich in basic residues.</p></caption><graphic xlink:href="mmi0078-0561-f6"></graphic></fig></sec><sec><title>The catalytic mechanism of aflatoxin reduction</title><p>Liquid chromatography-mass spectrometry (LCMS) analysis of the aflatoxin degradation reaction showed that all of the FDRs tested appeared to catalyse aflatoxin breakdown via the same mechanism for all four substrates, with the ion species of the product differing from the corresponding reactant by 2.02 m/z in each instance (<xref ref-type="fig" rid="fig07">Fig. 7</xref>). This is consistent with the reduction of aflatoxin via transfer of two electrons from F<sub>420</sub>H<sub>2</sub>. The reaction product is unstable over time and several low abundance metabolites (possibly generated by spontaneous hydrolysis) appear after an hour. Previously characterized reactions involving AFB1 and AFG1 involved oxidation of the unsaturated carbons of the furan ring by mammalian liver CYP450's (<xref ref-type="bibr" rid="b39">Mishra and Das, 2003</xref>), or their spontaneous reduction to form AFB2 and AFG2 (<xref ref-type="fig" rid="fig01">Fig. 1</xref>). As the FDRs described here also catalyse reduction of AFB2 and AFG2, it is unlikely that it is the furan moiety that is reduced by these enzymes.</p><fig id="fig07" position="float"><label>Fig. 7</label><caption><p>Proposed reaction mechanism of AFB1 and AFG1 reduction by reduced F<sub>420</sub>.</p><p>A. The proposed reduction mechanism of AFB1, with the mass spectrum of the substrate and product below the chemical structures.</p><p>B. The mass spectrum and chemical structure of AFG1 and the reduced product.</p></caption><graphic xlink:href="mmi0078-0561-f7"></graphic></fig><p>A second candidate moiety for the FDR catalysed reduction is the α,β-unsaturated ester between the lactone rings in AFG1/G2 and the lactone and cyclopentenone rings in AFB1/B2. Indeed, the LCMS-induced fragmentation of AFG1 (<xref ref-type="fig" rid="fig07">Fig. 7</xref>) produces ion species (287.08 and 259.05 m/z) that would result from a retro Diels-Alder fragmentation of the lactone ring (loss of CO<sub>2</sub> and C<sub>2</sub>H<sub>4</sub> respectively), consistent with destabilization of the ring resulting from reduction of the α,β-unsaturated ester. To test this hypothesis, MSMEG_3380 was incubated with coumarin and 4-methylumbelliferone. Coumarin and 4-methylumbelliferone both contain α,β-unsaturated ester moieties, and lack furan rings. However, the α,β-unsaturated ester moiety in 4-methylumbelliferone is stabilized by a methyl group (<xref ref-type="fig" rid="fig01">Fig. 1</xref>). Degradation of coumarin was observed at rates similar to that for aflatoxin (0.5 µmol min<sup>−1</sup> µmol<sup>−1</sup>), while no degradation of 4-methylumbelliferone was observed. Further experiments will be required to establish details of the catalytic mechanism, but these results suggest that it is the α,β-unsaturated ester moiety that is reduced by the FDRs.</p></sec></sec><sec sec-type=""><title>Discussion</title><sec><title>The possible physiological role of FDRs</title><p>Herein we describe two families of enzymes that utilize F<sub>420</sub>H<sub>2</sub> to degrade aflatoxin. F<sub>420</sub>/F<sub>420</sub>H<sub>2</sub> is a deazaflavin hydride carrier found in the <italic>Archea, Actinomycetales</italic> and some species of γ-<italic>Proteobacteria.</italic> Although non-essential in <italic>Actinomycetales</italic> in a laboratory setting (<xref ref-type="bibr" rid="b41">Nakano <italic>et al</italic>., 2004</xref>), an important role for the cofactor has been documented in a wide variety of metabolic processes. These include methanogenesis (<xref ref-type="bibr" rid="b18">Graham and White, 2002</xref>), sulphite reduction (<xref ref-type="bibr" rid="b32">Johnson and Mukhopadhyay, 2005</xref>), the degradation of nitroaromatic compounds (<xref ref-type="bibr" rid="b12">Ebert <italic>et al</italic>., 2001</xref>; <xref ref-type="bibr" rid="b36">Manjunatha <italic>et al</italic>., 2006</xref>; <xref ref-type="bibr" rid="b22">Guerra-Lopez <italic>et al</italic>., 2007</xref>; <xref ref-type="bibr" rid="b56">Singh <italic>et al</italic>., 2008</xref>), antibiotic resistance (<xref ref-type="bibr" rid="b23">Hasan <italic>et al</italic>., 2010</xref>), malachite green decolourization (<xref ref-type="bibr" rid="b22">Guerra-Lopez <italic>et al</italic>., 2007</xref>) and the biosynthesis of chlortetracyclines in <italic>Streptomyces</italic> (<xref ref-type="bibr" rid="b41">Nakano <italic>et al</italic>., 2004</xref>). It may be that the highly negative redox potential of F<sub>420</sub>H<sub>2</sub> (<xref ref-type="bibr" rid="b31">Jacobson and Walsh, 1984</xref>) is enabling for some of these energetically demanding reactions, including the degradation of aflatoxins by the FDR-A and -B families described here.</p><p>It seems unlikely that <italic>M. smegmatis</italic> would need between nine and 28 FDR enzymes specifically to degrade aflatoxin. Indeed, it is questionable as to whether aflatoxins are the primary physiological substrates for any of them; <italic>M. smegmatis</italic> has been found in soil where it may encounter aflatoxins but it is also known to be a human saprophyte where it would be very unlikely to encounter these carcinogens. The <italic>K</italic><sub>M</sub> values of these enzymes for aflatoxin were also higher than what might be expected for a physiological substrate; they were above the solubility limit for aflatoxin (approximately 100 µM) for all the enzymes except for the most active aflatoxin degrading FDR that we characterized, MSMEG_5998, which had a <italic>K</italic><sub>M</sub> of 47 ± 6 µM for AFB1. By comparison the <italic>K</italic><sub>M</sub> value for <italic>E. coli</italic> PNPOx for pyridoxine 5′-phosphate is 2 µM (<xref ref-type="bibr" rid="b53">di Salvo <italic>et al</italic>., 2003</xref>). Furthermore, there are significant differences between the active site structure of the FDR-A and -B enzymes, which suggest that they may have a broad substrate range. Our analysis of the substrate binding pockets of MSMEG_3356 and MSMEG_3380 reveal that while the pockets are formed by homologous regions the residues are not conserved. Also, the MSMEG_3380 cavity is significantly larger than that of MSMEG_3356 (649 Å<sup>3</sup> versus 411 Å<sup>3</sup>). The aflatoxin degrading activity we have characterized may thus reflect inherently broad substrate specificities of the FDR-A and -B families for polyaromatic compounds containing α,β-unsaturated lactones.</p><p>Our phylogenetic and genomic analyses (<xref ref-type="fig" rid="fig03">Fig. 3</xref> and <xref ref-type="supplementary-material" rid="SD1">Table S2</xref>) show that the FDR-A and -B families are widespread among the <italic>Actinomycetales</italic>. Many taxa in this order inhabit environments where aflatoxins are unlikely to be found. However, the FDRs may be involved in other metabolic processes. We can suggest three possibilities. First, we note that other <italic>Actinomycetales</italic>, including some previously found to degrade aflatoxins (<xref ref-type="bibr" rid="b25">Hormisch <italic>et al</italic>., 2004</xref>; <xref ref-type="bibr" rid="b63">Teniola <italic>et al</italic>., 2005</xref>), have been isolated from environments contaminated with structurally similar polyaromatic hydrocarbons (<xref ref-type="bibr" rid="b68">Willumsen <italic>et al</italic>., 2001</xref>; <xref ref-type="bibr" rid="b38">Miller <italic>et al</italic>., 2004</xref>). The FDR enzymes may play a role in the degradation of these compounds and hence their utilization as potential carbon and energy sources. Second, some <italic>Actinomycetales</italic> are plant pathogens/symbionts (<xref ref-type="bibr" rid="b14">El-Tarabily and Sivasithamparam, 2006</xref>; <xref ref-type="bibr" rid="b55">Schrey and Tarkka, 2008</xref>) and many plant antimycobacterials are coumarinyl compounds that are structurally similar to aflatoxins and could be substrates for FDR-A and -B enzymes (<xref ref-type="bibr" rid="b58">Stavri and Gibbons, 2005</xref>). Finally, many antibiotic intermediates are potential substrates for these enzymes; for instance the aclacinomycin biosynthetic gene cluster in <italic>Streptomyces galilaeus</italic> contains an uncharacterized gene, <italic>AclJ</italic>, which our phylogenetic analysis suggests is an FDR-A enzyme (<xref ref-type="bibr" rid="b8">Chung <italic>et al</italic>., 2002</xref>). Some FDR-A and -B enzymes may therefore be involved in antibiotic biosynthesis.</p></sec><sec><title>Potential industrial and medical applications</title><p>The FDR-catalysed aflatoxin breakdown described in this work constitutes significant detoxification of these compounds, as previous studies have shown that removal of the lactone ring of AFB1 reduces its mutagenicity by 450-fold and its acute toxicity by 18-fold (<xref ref-type="bibr" rid="b34">Lee <italic>et al</italic>., 1981</xref>). Furthermore, pre-incubation with various <italic>Actinomycetales</italic> has been demonstrated to reduce the mutagenicity and toxicity of aflatoxin to animals (<xref ref-type="bibr" rid="b9">Ciegler <italic>et al</italic>., 1966</xref>; <xref ref-type="bibr" rid="b1">Alberts <italic>et al</italic>., 2006</xref>; <xref ref-type="bibr" rid="b62">Tejada-Castaneda <italic>et al</italic>., 2008</xref>). Accordingly, there is a potential use for these enzymes in decontaminating aflatoxin-contaminated food crops, either as a transgenic input trait for the crop or in a post-harvest application. There are precedents for the effective decontamination of two other mycotoxins (zearalenone and deoxynivalenol) in maize and rice by transgenic expression of the corresponding bacterial detoxifying enzymes (<xref ref-type="bibr" rid="b28">Igawa <italic>et al</italic>., 2007</xref>; <xref ref-type="bibr" rid="b43">Ohsato <italic>et al</italic>., 2007</xref>). Coexpression of the genes required for the biosynthesis of reduced F<sub>420</sub> would also be required for the FDRs to be effective as an input trait and there are now many examples of plants being engineered with multiple genes (<xref ref-type="bibr" rid="b61">Taverniers <italic>et al</italic>., 2008</xref>). The identification of non-pathogenic strains of <italic>Actinomycetales</italic> that have the capacity to degrade aflatoxin may provide a less technologically intensive biocontrol approach. Indeed, inoculation of <italic>Aspergillus</italic> growth media with <italic>Streptomyces</italic> species has been shown to protect peanuts from aflatoxin contamination in the laboratory (<xref ref-type="bibr" rid="b70">Zucchi <italic>et al</italic>., 2008</xref>).</p><p>The exclusivity of these FDRs to certain <italic>Actinobacteria</italic> is an important ‘point of difference’ that can be targeted in the development of drugs, particularly considering the presence of several human pathogens within this phylum, including <italic>M. tuberculosis</italic>. For instance, the candidate anti-tuberculosis drug PA-824 is now known to be a prodrug that is metabolized to its active form through the activity of an FDR-A (Rv3547) (<xref ref-type="bibr" rid="b36">Manjunatha <italic>et al</italic>., 2006</xref>). Our identification and structural characterization of the first members of these FDR families thus raises the possibility of rational design of improved prodrugs with even greater activity and specificity. However, before detailed analysis of the catalytic mechanism is possible, structures of the holoenzymes (F<sub>420</sub>H<sub>2</sub> : Enz) and, ideally, a ternary complex will be required, since substantial conformational changes are often seen in cofactor and substrate binding in related enzymes, such as PNPOx (<xref ref-type="bibr" rid="b50">Safo <italic>et al</italic>., 2005</xref>).</p></sec><sec><title>Comparison between the FDRs and PNPOxs</title><p>Structurally and phylogenetically, the FDRs and PNPOxs appear to have a common evolutionary origin, yet these enzyme families have evolved to utilize different cofactors. Notwithstanding their structural similarities there are at least two important chemical differences between the two cofactors. One is the lower redox midpoint potential of F<sub>420</sub> in solvent at pH 7.5 (−350 mV for F<sub>420</sub> versus −230 mV for FMN) (<xref ref-type="bibr" rid="b11">Draper and Ingraham, 1968</xref>; <xref ref-type="bibr" rid="b31">Jacobson and Walsh, 1984</xref>). The other is that F<sub>420</sub> is not stable as a semiquinone and may only catalyse a two electron reduction, while FMN may catalyse one or two electron reduction and oxidation reactions (<xref ref-type="bibr" rid="b17">Goldberg <italic>et al</italic>., 1981</xref>). Indeed, owing to this, the chemistry that is generally catalysed by the two enzyme families is opposite (oxidation by the PNPOxs versus reduction by the FDRs). We suggest it is this difference in catalytic activity that has resulted in the evolutionary expansion of the FDRs in <italic>M. smegmatis</italic> (28 genes) compared with the single PNPOx gene; the reducing power of F<sub>420</sub>H<sub>2</sub> may have allowed these enzymes to catalyse the reduction of a wide variety of compounds, particularly xenobiotics, and provided a selective pressure for duplication and specialization.</p><p>There are also significant differences in the mechanisms of cofactor recycling between the FDRs and the PNPOxs. PNPOxs bind FMN with high affinity, <italic>K</italic><sub>D</sub> 30 nM (<xref ref-type="bibr" rid="b65">Wada and Snell, 1961</xref>), and require molecular oxygen as the electron acceptor (<xref ref-type="bibr" rid="b46">Pogell, 1958</xref>; <xref ref-type="bibr" rid="b42">Notheis <italic>et al</italic>., 1995</xref>). In contrast, there is a requirement for F<sub>420</sub> to be enzymatically reduced by FGD, at least in <italic>Mycobacteria</italic> and <italic>Nocardia</italic> (<xref ref-type="bibr" rid="b49">Purwantini <italic>et al</italic>., 1997</xref>). It is currently impossible to determine whether a common evolutionary progenitor of the FDRs and PNPOxs would have used either FMN, F<sub>420</sub>H<sub>2</sub>, or perhaps a different molecule again (such as a common phosphorylated intermediate from the biosynthetic pathways of FMN and F<sub>420</sub>; <xref ref-type="supplementary-material" rid="SD1">Fig. S2</xref>) as a cofactor, but these families of enzymes have the potential to advance our understanding of how enzymes evolve to utilize the catalytic power that different cofactors provide.</p></sec></sec><sec sec-type="methods"><title>Experimental procedures</title><sec><title>Bacterial strains, chemicals</title><p>Aflatoxins B<sub>1</sub>, B<sub>2</sub>, G<sub>1</sub> and G<sub>2</sub> were obtained from Sigma-Aldrich and Fermentek (Israel). All bacterial strains were previously isolated in our laboratory (<xref ref-type="bibr" rid="b59">Sutherland <italic>et al</italic>., 2002</xref>; <xref ref-type="bibr" rid="b26">Horne <italic>et al</italic>., 2002a</xref>,<xref ref-type="bibr" rid="b27">b</xref>), except for <italic>M. smegmatis</italic> mc<sup>2</sup>155, which was obtained from Dr H. Billman-Jacob (University of Melbourne, Australia). F<sub>420</sub> was purified from <italic>M. smegmatis</italic> soluble fractions according to previously published methods (<xref ref-type="bibr" rid="b29">Isabelle <italic>et al</italic>., 2002</xref>).</p></sec><sec><title>Identification of aflatoxin degrading bacteria</title><p>Bacterial strains were first grown on Luria–Bertani (LB) agar before inoculation into Peptone Yeast extract Broth (PYB; 9 g/l peptone, 4.5 g l<sup>−1</sup> yeast extract, 23 mM Na<sub>2</sub>HPO<sub>4</sub>, 88 mM KH<sub>2</sub>PO<sub>4</sub>, 9 mM NaCl, pH 6.0) supplemented with 4 µg ml<sup>−1</sup> AFG1 or 6 µg ml<sup>−1</sup> AFB1, and incubation at 28°C for 48 h on an orbital shaker (200 rpm). Five microlitres of each reaction mixture was spotted and dried onto silica gel 60 <italic>F</italic><sub>254</sub> TLC plates (Merck, Germany). Chloroform/acetone/acetic acid (40:10:1 by volume) was used as the developing solvent and aflatoxin fluorescence was detected by viewing under ultraviolet light (365 nm). Images of TLC plates were recorded by an AlphaImager 2200 Imaging System (Alpha Innotech, USA) fitted with an ethidium bromide bandpass filter.</p></sec><sec><title>Transposon mutagenesis of <italic>M. smegmatis</italic></title><p>Random insertion mutants of <italic>M. smegmatis</italic> mc<sup>2</sup>155 were generated with the EZ::TN insertion kit (Epicentre, USA). The EZ::TN tnp transposase complex (1 µl) was used to electroporate 100 µl of electrocompetent <italic>M. smegmatis</italic> mc<sup>2</sup>155 cells, which was then plated onto LB agar plates containing 20 µg ml<sup>−1</sup> kanamycin. Approximately 2000 mutants were obtained per transformation. Transposon insertion mutants were individually inoculated into 2 ml square wells of 96-deep-well growth blocks (Axygen, USA) containing 200 µl of PYB supplemented with 20 µg ml<sup>−1</sup> kanamycin and 4 µg ml<sup>−1</sup> aflatoxin G<sub>1</sub>. The growth blocks were sealed with silicone mats (Axygen) and incubated for 3 days (37°C at 200 rpm) before 5 µl of each culture was examined for aflatoxin degradation by TLC. Mutants that exhibited detectable growth but had reduced ability to degrade AFG1 compared with wild-type cells were selected for further analysis. The genomic regions of selected mutants containing the EZ::TN transposon were isolated by plasmid rescue. Genomic DNA was isolated using the Bactozol DNA isolation kit (Molecular Research Center, USA), digested with EcoRI, self-ligated and used to electroporate <italic>E. coli</italic> TransforMax EC100D pir-116 (Epicentre, USA). Transformants containing the transposon-interrupted <italic>M. smegmatis</italic> DNA were selected by plating onto LB agar containing 40 µg ml<sup>−1</sup> kanamycin. The resulting plasmids were isolated and the genomic regions flanking the transposon were sequenced using primers supplied with the EZ::TN insertion kit.</p> </sec><sec><title>Identification of AFG1-degrading proteins in <italic>M. smegmatis</italic></title><p>Protein fractions with AFG1 activity were purified from <italic>M. smegmatis</italic> by ammonium sulphate precipitation, hydrophobic interaction chromatography, anion exchange chromatography and gel filtration chromatography, as detailed below. Proteins from active fractions were visualized by SDS-PAGE and Coomasie staining. Mass spectrometry of tryptic digests with an Agilent XCT ion trap mass spectrometer (<xref ref-type="bibr" rid="b5">Campbell <italic>et al</italic>., 2008</xref>) was used to identify the proteins from protein bands excised from SDS-PAGE and active fractions from gel filtration chromatography. Agilent's Spectrum Mill software was used to match the data with annotated protein sequences in the <italic>M. smegmatis</italic> mc<sup>2</sup>155 CMR database (<xref ref-type="bibr" rid="b45">Peterson <italic>et al</italic>., 2001</xref>).</p></sec><sec><title>Chromatography of <italic>M. smegmatis</italic> proteins</title><p><italic>Mycobacterium smegmatis</italic> was grown for 4 days before centrifugation at 10 000 <italic>g</italic> and lysis via sonication with glass beads in 20 mM Tris-HCl, pH 7.5, with 1 mg ml<sup>−1</sup> lysozyme, 5 mM DTT and 1 mM PMSF. Saturated (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> solution (0°C) was added to the soluble fraction to a concentration of 40% before centrifugation at 20 000 <italic>g</italic>. A second (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> cut (70%) of the soluble fraction was made and the resulting pellet was resuspended in 1 M (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 20 mM Tris-HCl, pH 7.5, and clarified via filtration with a 0.22 µm filter. A Biologic HR FPLC system (Bio-Rad, USA) was used during the subsequent chromatographic separation steps. Hydrophobic interaction chromatography (HIC) was performed using a 200 ml phenyl sepharose high performance column (GE Healthcare, USA) equilibrated with 1 M (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 20 mM Tris-HCl, pH 7.5. After the resolubilized (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> pellet was loaded onto the column, proteins were eluted over a linear gradient [1 M–0 M (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>] over 100 ml. Active fractions were pooled and dialysed against 20 mM Tris-HCl, pH 7.5 prior to anion exchange chromatography with a MonoQ HR 5/5 column (GE Healthcare), equilibrated with 20 mM Tris-HCl, pH 7.5. The dialysed fractions were loaded onto the column and eluted over a linear gradient (0–0.5 M NaCl) over 20 column volumes. For size exclusion chromatography, as used in the second purification, HIC active fractions were pooled and loaded onto a Superdex 200 Hi Load 26/60 gel filtration column (GE Healthcare), equilibrated with 150 mM NaCl, 20 mM Tris-HCl, pH 7.5. Gel filtration chromatography standards (GE Healthcare) were run in parallel to allow estimation of the size of the proteins in solution.</p></sec><sec><title>Phylogeny</title><p>Homologues of the four aflatoxin degrading enzymes identified by reverse genetics were identified by searching the TIGR CMR peptide and NCBI protein databases, using the <sc>blast</sc> and CDD (<xref ref-type="bibr" rid="b37">Marchler-Bauer <italic>et al</italic>., 2007</xref>). MEGA4 (<xref ref-type="bibr" rid="b60">Tamura <italic>et al</italic>., 2007</xref>) was used for the phylogenetic analysis of a limited data set of 146 amino acid sequences from seven different <italic>Actinomycetales</italic> (<italic>M. smegmatis</italic>, <italic>M. tuberculosis</italic> H37Rv, <italic>M. vanbaalenii</italic>, <italic>Rhodococcus</italic> sp. RH1, <italic>Arthrobacter</italic> sp. FB24, <italic>S. coelicolor</italic>, <italic>Frankia alni</italic> and <italic>Nocardioides</italic> sp. JS614), plus the known PNPOx enzymes from <italic>H. sapiens</italic>, <italic>E. coli</italic>, <italic>Saccharomyces cerevisiae</italic>, <italic>Caenorhabditis elegans</italic> and <italic>Mus musculus</italic>. Protein sequences were aligned by the Gonnett algorithm and the tree constructed from the N-terminal sequences using the neighbour-joining method with pairwise deletion, poisson correction and 1000 bootstrap replicates. The 28 <italic>M. smegmatis</italic> PNPOx-like sequences are referred to by their TIGR locus numbers (MSMEG_).</p></sec><sec><title>Cloning, expression and purification of recombinant proteins</title><p>Genes were amplified from <italic>M. smegmatis</italic> mc<sup>2</sup>155 genomic DNA using Platinum high fidelity <italic>Taq</italic> polymerase (Invitrogen, USA) using the primer pairs in <xref ref-type="supplementary-material" rid="SD1">Table S3</xref>. The amplicons were recombined into the Gateway expression vector, pDEST17 (Invitrogen, USA) and the sequences confirmed by capillary electrophoresis (Micromon, Australia). Proteins were expressed in <italic>E. coli</italic> BL21-AI (Invitrogen) and purified by nickel agarose affinity chromatography using a 1 ml Ni-NTA superflow column (Qiagen, Germany). Proteins were stored at 4°C in 50 mM Tris-HCl pH 7.5. The FDR MSMEG_5998, which was purified as exclusion bodies, was refolded following the methods of <xref ref-type="bibr" rid="b67">Whitbread <italic>et al</italic>. (2005</xref>). Protein concentrations were determined by measuring absorbance at 280 nm using a NanoDrop Spectrophotometer ND1000 and calculated based on the extinction co-efficient for each protein determined using Vector NTI (Invitrogen). FMN was stripped from MSMEG_5675 by dialysis, as described previously (<xref ref-type="bibr" rid="b51">di Salvo <italic>et al</italic>., 1998</xref>), and this was confirmed by loss of the FMN absorption maxima at 450 nm. For protein crystallography, MSMEG_3380 and MSMEG_3356 were cloned into pDEST17, with a TEV-cleavage site designed into the forward primer (<xref ref-type="supplementary-material" rid="SD1">Table S3</xref>). Expression and purification were performed as described previously (<xref ref-type="bibr" rid="b30">Jackson <italic>et al</italic>., 2008</xref>). The <italic>fgd</italic> gene was cloned into the NdeI and BamHI sites of pET14b (Novagen, Germany) and expressed in Tuner cells (Novagen) by induction with 0.4 mM Isopropyl-β-D-thiogalactopyranoside (IPTG).</p></sec><sec><title>Enzyme assays</title><p>The FGD activity was estimated using the previously described spectrophotometric assay for F<sub>420</sub> to F<sub>420</sub>H<sub>2</sub> reduction (<xref ref-type="bibr" rid="b47">Purwantini and Daniels, 1996</xref>). One unit of FGD activity was defined as the amount of enzyme required to reduce one µmole of F<sub>420</sub> per min. Assays of aflatoxin degradation activity were conducted in either 10 µl or 20 µl reaction volumes, in the dark, at room temperature, using reaction mix (30 µM aflatoxin, 10 µM F<sub>420</sub>, 0.2 U µl<sup>−1</sup> FGD, 2.5 mM glucose 6-phosphate, 20 mM Tris-HCl, pH 7.5) and enzyme. Reactions were incubated for 0.5 min to 24 h depending on the enzyme and aflatoxin used. The reaction was stopped by the addition of formic acid to a final concentration of 2% and incubated on ice for 15 min. Protein was pelleted by centrifugation for 5 min at 14 000 <italic>g</italic>. Samples were injected onto an Agilent Zorbax XDB C18 column (3.5 µm, 2.1 × 30 mm). Aflatoxin concentrations were quantified using an Agilent 1200 series binary HPLC running isocratically with 30% methanol and 0.5% acetic acid and detected at 365 nm and quantified using Chemstation software (Agilent). Results are given in specific activity units because of the low aqueous solubility of aflatoxin [∼100 µM for AFB1 (<xref ref-type="bibr" rid="b20">Grant and Phillips, 1998</xref>)], which prevented the estimation of kinetic parameters.</p></sec><sec><title>LC/MS assays</title><p>The reaction products of aflatoxin reduction were determined using an Agilent 1100 Series Binary LC with diode array detector and in-line Time of Flight Mass Spectrometer (MSD TOF). Samples were separated on an Agilent Zorbax XDB C18 column (3.5 µm, 2.1 × 30 mm) over a gradient of 5% acetonitrile (v/v) and 0.1% formic acid (v/v) from 0.5 min to 20% acetonitrile at 2 min, and then increased to 50% acetonitrile at 10 min, at a flow rate of 0.3 ml min<sup>−1</sup>. Reaction products were analysed using Analyst QS software.</p></sec><sec><title>Structural analysis</title><p>The crystallization and data collection of 3380 have been previously reported (<xref ref-type="bibr" rid="b30">Jackson <italic>et al</italic>., 2008</xref>). The crystallization of Seleno-Methionine 3356 was performed in 2 µl hanging drops with 20 mg ml<sup>−1</sup> enzyme and a reservoir solution of 36% PEG-MME 5000, 0.1 M sodium acetate, pH 5.5. Seleno-Methionine labelled crystals were soaked in cryoprotectant [35% PEG 10K, 10 mM Tris-HCl, pH 8.5 for MSMEG_3380 and reservoir solution with 10% glycerol and 10% ethylene glycol (v/v) for MSMEG_3356] before vitrification in liquid nitrogen. Both data sets were collected at the Australian Synchrotron, 3380 on the PX1 beamline and 3356 on the PX2 microfocus beamline. The use of the microfocus beamline for 3356 was necessitated by severe non-merohedral twinning of the crystals; crystals were scanned for regions displaying a minimum of twinning. Intensity data were collected at 100 K using a wavelength corresponding to the SeMet edge at 0.9793 Å and a 1.0° oscillation angle per image. Diffraction data were integrated and scaled using the programs <italic>MOSFLM</italic> (<xref ref-type="bibr" rid="b35">Leslie, 2006</xref>) and <italic>SCALA</italic> (<xref ref-type="bibr" rid="b16">Evans, 2006</xref>). Details of the data collection are given in <xref ref-type="table" rid="tbl2">Table 2</xref>. Based on systematic absences, the 3380 and 3356 crystals were assigned to the P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub> and P2<sub>1</sub> space groups respectively. The SHELX suite of programs (<xref ref-type="bibr" rid="b54">Schneider and Sheldrick, 2002</xref>) was used for experimental phasing. Four and eight selenium sites were found in the asymmetric units of 3380 and 3356, respectively, using SHELXD, and were used to calculate phases. After refinement and density modification, a significant amount of both structures, corresponding to a dimer in the asymmetric unit of MSMEG_3380 and a tetramer in the asymmetric unit of MSMEG_3358, could be auto-built using ARP/wARP (<xref ref-type="bibr" rid="b44">Perrakis <italic>et al</italic>., 2001</xref>), yielding a preliminary <italic>R</italic><sub>free</sub> values of 0.304 and 0.421 for 3380 and 3356 respectively. Further maximum-likelihood refinement for 3380, or twin-refinement with a twin fraction of 7% for 3356, using REFMAC5 (<xref ref-type="bibr" rid="b40">Murshudov <italic>et al</italic>., 1997</xref>), interspersed with manual building in COOT (<xref ref-type="bibr" rid="b15">Emsley and Cowtan, 2004</xref>), addition of water molecules and final anisotropic (for 3380) refinement bought the final <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub> values to 16.8/19.1 and 19.0/25.5 for 3380 and 3356 respectively. The F<sub>420</sub> cofactor of the FDRs was docked into the structures using the docking program CDOCKER (<xref ref-type="bibr" rid="b69">Wu <italic>et al</italic>., 2003</xref>), as implemented in the Accelrys Discovery Studio. Hydrogens were added to cofactor and enzyme and the CHARMm forcefield was used. Slight manual adjustment of the top hit was made using COOT. Analysis of cavity volumes was performed by submitting the coordinates to the 3V server (<ext-link ext-link-type="uri" xlink:href="http://3vee.molmovdb.org">http://3vee.molmovdb.org</ext-link>) (<xref ref-type="bibr" rid="b64">Voss and Gerstein, 2010</xref>), and all images were created using Pymol (<ext-link ext-link-type="uri" xlink:href="http://www.pymol.org">http://www.pymol.org</ext-link>).</p><table-wrap id="tbl2" position="float"><label>Table 2</label><caption><p>Data collection and refinement statistics for the crystallography</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Crystal</th><th align="left" rowspan="1" colspan="1">MSMEG_3356</th><th align="left" rowspan="1" colspan="1">MSMEG_3380</th></tr><tr><th align="left" colspan="3" rowspan="1"><hr></hr></th></tr><tr><th align="left" rowspan="1" colspan="1">PDB ID</th><th align="left" rowspan="1" colspan="1">3H96</th><th align="left" rowspan="1" colspan="1">3F7E</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Space group</td><td align="left" rowspan="1" colspan="1">P1211</td><td align="left" rowspan="1" colspan="1">P212121</td></tr><tr><td align="left" rowspan="1" colspan="1">Unit cell (Å)</td><td align="left" rowspan="1" colspan="1">a = 59.65, b = 71.15, c = 63.84</td><td align="left" rowspan="1" colspan="1">a = 56.86, b = 65.35, c = 69.74</td></tr><tr><td align="left" rowspan="1" colspan="1">Unit cell (°)</td><td align="left" rowspan="1" colspan="1">α = 90, β = 90.39, γ = 90</td><td align="left" rowspan="1" colspan="1">α = 90, β = 90, γ = 90</td></tr><tr><td align="left" rowspan="1" colspan="1">Data collection</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Resolution (Å)<xref ref-type="table-fn" rid="tf2-1">a</xref></td><td align="left" rowspan="1" colspan="1">45.7–2.0 (2.11–2.00)</td><td align="left" rowspan="1" colspan="1">22–1.23 (1.23–1.30)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Unique reflections</td><td align="left" rowspan="1" colspan="1">34129</td><td align="left" rowspan="1" colspan="1">73957</td></tr><tr><td align="left" rowspan="1" colspan="1"> Redundancy</td><td align="left" rowspan="1" colspan="1">7.2 (6.4)</td><td align="left" rowspan="1" colspan="1">6.4 (6.2)</td></tr><tr><td align="left" rowspan="1" colspan="1"> </td><td align="left" rowspan="1" colspan="1">5.7 (3.3)</td><td align="left" rowspan="1" colspan="1">6.8 (1.5)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Completeness (%)</td><td align="left" rowspan="1" colspan="1">94.5 (91.5)</td><td align="left" rowspan="1" colspan="1">97.6 (95.4)</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>sym</sub> (%)<xref ref-type="table-fn" rid="tf2-2">b</xref></td><td align="left" rowspan="1" colspan="1">10.5 (17.4)</td><td align="left" rowspan="1" colspan="1">7.2 (49.4)</td></tr><tr><td align="left" rowspan="1" colspan="1">Refinement</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> No. reflections work/free</td><td align="left" rowspan="1" colspan="1">31 389/2743</td><td align="left" rowspan="1" colspan="1">70 232/3725</td></tr><tr><td align="left" rowspan="1" colspan="1"> Resolution range</td><td align="left" rowspan="1" colspan="1">45.7–2.0 (2.05–2.00)</td><td align="left" rowspan="1" colspan="1">20–1.23 (1.26–1.23)</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub> (%)<xref ref-type="table-fn" rid="tf2-3">c</xref></td><td align="left" rowspan="1" colspan="1">19.0/25.5 (20.9/27.7)</td><td align="left" rowspan="1" colspan="1">16.8/19.1 (20.0/23.1)</td></tr><tr><td align="left" rowspan="1" colspan="1">R.m.s deviations</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.025</td><td align="left" rowspan="1" colspan="1">0.012</td></tr><tr><td align="left" rowspan="1" colspan="1"> Angles (°)</td><td align="left" rowspan="1" colspan="1">1.84</td><td align="left" rowspan="1" colspan="1">1.59</td></tr></tbody></table><table-wrap-foot><fn id="tf2-1"><label>a</label><p>Values in parenthesis are for the highest resolution shell.</p></fn><fn id="tf2-2"><label>b</label><p><italic>R</italic><sub>symm</sub> = |<italic>I<sub>j</sub></italic> – <italic>I<sub>j</sub></italic>|/<italic>I<sub>j</sub></italic>, where I<sub>j</sub> is the averaged intensity for symmetry related reflections.</p></fn><fn id="tf2-3"><label>c</label><p><italic>R</italic><sub>work</sub> = |<italic>F</italic><sub>(obs)</sub> – <italic>F</italic><sub>(calc)</sub>|/<italic>F</italic><sub>(obs)</sub>; 5% of the data that were excluded from the refinement were used to calculate <italic>R</italic><sub>free</sub>.</p></fn></table-wrap-foot></table-wrap></sec></sec></body><back><ack><p>We thank Dr Helen Billman-Jacob for kindly providing the <italic>Mycobacterium smegmatis</italic> mc<sup>2</sup>155 strain. We gratefully acknowledge Professor Chris Easton and Dr Mike Lacey for their assistance in interpreting the data. We thank the CSIRO C3 crystallization facility for assistance in obtaining protein crystals and the Australian Synchrotron for beamtime. 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