300-Fold Increase in Production of the Zn2+-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization
Identifieur interne : 001754 ( Pmc/Corpus ); précédent : 001753; suivant : 001755300-Fold Increase in Production of the Zn2+-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization
Auteurs : Colin J. Jackson ; Christopher W. Coppin ; Paul D. Carr ; Alexey Aleksandrov ; Matthew Wilding ; Elena Sugrue ; Joanna Ubels ; Michael Paks ; Janet Newman ; Thomas S. Peat ; Robyn J. Russell ; Martin Field ; Martin Weik ; John G. Oakeshott ; Colin ScottSource :
- Applied and Environmental Microbiology [ 0099-2240 ] ; 2014.
Abstract
Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from
Url:
DOI: 10.1128/AEM.00916-14
PubMed: 24771025
PubMed Central: 4054219
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PMC:4054219Le document en format XML
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-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization</title>
<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="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</nlm:aff>
</affiliation>
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<author><name sortKey="Coppin, Christopher W" sort="Coppin, Christopher W" uniqKey="Coppin C" first="Christopher W." last="Coppin">Christopher W. Coppin</name>
<affiliation><nlm:aff id="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
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<author><name sortKey="Carr, Paul D" sort="Carr, Paul D" uniqKey="Carr P" first="Paul D." last="Carr">Paul D. Carr</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
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<author><name sortKey="Aleksandrov, Alexey" sort="Aleksandrov, Alexey" uniqKey="Aleksandrov A" first="Alexey" last="Aleksandrov">Alexey Aleksandrov</name>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wilding, Matthew" sort="Wilding, Matthew" uniqKey="Wilding M" first="Matthew" last="Wilding">Matthew Wilding</name>
<affiliation><nlm:aff id="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
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<author><name sortKey="Sugrue, Elena" sort="Sugrue, Elena" uniqKey="Sugrue E" first="Elena" last="Sugrue">Elena Sugrue</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ubels, Joanna" sort="Ubels, Joanna" uniqKey="Ubels J" first="Joanna" last="Ubels">Joanna Ubels</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Paks, Michael" sort="Paks, Michael" uniqKey="Paks M" first="Michael" last="Paks">Michael Paks</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
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<author><name sortKey="Newman, Janet" sort="Newman, Janet" uniqKey="Newman J" first="Janet" last="Newman">Janet Newman</name>
<affiliation><nlm:aff id="aff3">CSIRO Materials, Science and Engineering, Parkville, Victoria, Australia</nlm:aff>
</affiliation>
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<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="aff3">CSIRO Materials, Science and Engineering, Parkville, Victoria, Australia</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="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Field, Martin" sort="Field, Martin" uniqKey="Field M" first="Martin" last="Field">Martin Field</name>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Weik, Martin" sort="Weik, Martin" uniqKey="Weik M" first="Martin" last="Weik">Martin Weik</name>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</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="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
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<author><name sortKey="Scott, Colin" sort="Scott, Colin" uniqKey="Scott C" first="Colin" last="Scott">Colin Scott</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">300-Fold Increase in Production of the Zn<sup>2+</sup>
-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization</title>
<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="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Coppin, Christopher W" sort="Coppin, Christopher W" uniqKey="Coppin C" first="Christopher W." last="Coppin">Christopher W. Coppin</name>
<affiliation><nlm:aff id="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Carr, Paul D" sort="Carr, Paul D" uniqKey="Carr P" first="Paul D." last="Carr">Paul D. Carr</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Aleksandrov, Alexey" sort="Aleksandrov, Alexey" uniqKey="Aleksandrov A" first="Alexey" last="Aleksandrov">Alexey Aleksandrov</name>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wilding, Matthew" sort="Wilding, Matthew" uniqKey="Wilding M" first="Matthew" last="Wilding">Matthew Wilding</name>
<affiliation><nlm:aff id="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sugrue, Elena" sort="Sugrue, Elena" uniqKey="Sugrue E" first="Elena" last="Sugrue">Elena Sugrue</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ubels, Joanna" sort="Ubels, Joanna" uniqKey="Ubels J" first="Joanna" last="Ubels">Joanna Ubels</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Paks, Michael" sort="Paks, Michael" uniqKey="Paks M" first="Michael" last="Paks">Michael Paks</name>
<affiliation><nlm:aff id="aff1">Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</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="aff3">CSIRO Materials, Science and Engineering, Parkville, Victoria, Australia</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="aff3">CSIRO Materials, Science and Engineering, Parkville, Victoria, Australia</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="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Field, Martin" sort="Field, Martin" uniqKey="Field M" first="Martin" last="Field">Martin Field</name>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Weik, Martin" sort="Weik, Martin" uniqKey="Weik M" first="Martin" last="Weik">Martin Weik</name>
<affiliation><nlm:aff id="aff4">Institut de Biologie Structurale, Grenoble, France</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="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</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="aff2">CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Applied and Environmental Microbiology</title>
<idno type="ISSN">0099-2240</idno>
<idno type="eISSN">1098-5336</idno>
<imprint><date when="2014">2014</date>
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<front><div type="abstract" xml:lang="en"><p>Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from <named-content content-type="genus-species">Arthrobacter aurescens</named-content>
with environmental applications that catalyzes the hydrolysis of triazine herbicides, in <named-content content-type="genus-species">Escherichia coli</named-content>
. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.</p>
</div>
</front>
</TEI>
<pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Appl Environ Microbiol</journal-id>
<journal-id journal-id-type="iso-abbrev">Appl. Environ. Microbiol</journal-id>
<journal-id journal-id-type="hwp">aem</journal-id>
<journal-id journal-id-type="pmc">aem</journal-id>
<journal-id journal-id-type="publisher-id">AEM</journal-id>
<journal-title-group><journal-title>Applied and Environmental Microbiology</journal-title>
</journal-title-group>
<issn pub-type="ppub">0099-2240</issn>
<issn pub-type="epub">1098-5336</issn>
<publisher><publisher-name>American Society for Microbiology</publisher-name>
<publisher-loc>1752 N St., N.W., Washington, DC</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">24771025</article-id>
<article-id pub-id-type="pmc">4054219</article-id>
<article-id pub-id-type="publisher-id">00916-14</article-id>
<article-id pub-id-type="doi">10.1128/AEM.00916-14</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Enzymology and Protein Engineering</subject>
</subj-group>
</article-categories>
<title-group><article-title>300-Fold Increase in Production of the Zn<sup>2+</sup>
-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Jackson</surname>
<given-names>Colin J.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
<xref ref-type="aff" rid="aff4"><sup>d</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Coppin</surname>
<given-names>Christopher W.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Carr</surname>
<given-names>Paul D.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Aleksandrov</surname>
<given-names>Alexey</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>d</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Wilding</surname>
<given-names>Matthew</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Sugrue</surname>
<given-names>Elena</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ubels</surname>
<given-names>Joanna</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Paks</surname>
<given-names>Michael</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Newman</surname>
<given-names>Janet</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>c</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Peat</surname>
<given-names>Thomas S.</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>c</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Russell</surname>
<given-names>Robyn J.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Field</surname>
<given-names>Martin</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>d</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Weik</surname>
<given-names>Martin</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>d</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Oakeshott</surname>
<given-names>John G.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Scott</surname>
<given-names>Colin</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<aff id="aff1"><label>a</label>
Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia</aff>
<aff id="aff2"><label>b</label>
CSIRO Ecosystems Sciences, Black Mountain, Canberra, Australian Capital Territory, Australia</aff>
<aff id="aff3"><label>c</label>
CSIRO Materials, Science and Engineering, Parkville, Victoria, Australia</aff>
<aff id="aff4"><label>d</label>
Institut de Biologie Structurale, Grenoble, France</aff>
</contrib-group>
<contrib-group><contrib contrib-type="editor"><name><surname>Kivisaar</surname>
<given-names>M.</given-names>
</name>
<role>Editor</role>
</contrib>
</contrib-group>
<author-notes><corresp id="cor1">Address correspondence to Colin J. Jackson, <email>colin.jackson@anu.edu.au</email>
, or Colin Scott, <email>colin.scott@csiro.au</email>
.</corresp>
</author-notes>
<pub-date pub-type="ppub"><month>7</month>
<year>2014</year>
</pub-date>
<volume>80</volume>
<issue>13</issue>
<fpage>4003</fpage>
<lpage>4011</lpage>
<history><date date-type="received"><day>17</day>
<month>3</month>
<year>2014</year>
</date>
<date date-type="accepted"><day>12</day>
<month>4</month>
<year>2014</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2014, American Society for Microbiology. All Rights Reserved.</copyright-statement>
<copyright-year>2014</copyright-year>
<copyright-holder>American Society for Microbiology</copyright-holder>
</permissions>
<self-uri xlink:title="pdf" xlink:type="simple" xlink:href="zam01314004003.pdf"></self-uri>
<abstract><p>Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from <named-content content-type="genus-species">Arthrobacter aurescens</named-content>
with environmental applications that catalyzes the hydrolysis of triazine herbicides, in <named-content content-type="genus-species">Escherichia coli</named-content>
. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.</p>
</abstract>
</article-meta>
</front>
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