Heterologous expression and reconstitution of fungal Mn peroxidase.
Identifieur interne : 000C47 ( Main/Exploration ); précédent : 000C46; suivant : 000C48Heterologous expression and reconstitution of fungal Mn peroxidase.
Auteurs : R. Whitwam [États-Unis] ; M. TienSource :
- Archives of biochemistry and biophysics [ 0003-9861 ] ; 1996.
Descripteurs français
- KwdFr :
- Agaricales (enzymologie), Calcium (pharmacologie), Chlorure de potassium (pharmacologie), Cinétique (MeSH), Concentration en ions d'hydrogène (MeSH), Disulfure de glutathion (MeSH), Dénaturation des protéines (MeSH), Glutathion (analogues et dérivés), Glutathion (pharmacologie), Glycérol (pharmacologie), Hémine (pharmacologie), Isoenzymes (biosynthèse), Isoenzymes (composition chimique), Isoenzymes (métabolisme), Manganèse (pharmacologie), Peroxidases (biosynthèse), Peroxidases (composition chimique), Peroxidases (métabolisme), Pliage des protéines (MeSH), Protéines recombinantes (biosynthèse), Protéines recombinantes (composition chimique), Protéines recombinantes (métabolisme), Urée (pharmacologie).
- MESH :
- analogues et dérivés : Glutathion.
- biosynthèse : Isoenzymes, Peroxidases, Protéines recombinantes.
- composition chimique : Isoenzymes, Peroxidases, Protéines recombinantes.
- enzymologie : Agaricales.
- métabolisme : Isoenzymes, Peroxidases, Protéines recombinantes.
- pharmacologie : Calcium, Chlorure de potassium, Glutathion, Glycérol, Hémine, Manganèse, Urée.
- Cinétique, Concentration en ions d'hydrogène, Disulfure de glutathion, Dénaturation des protéines, Pliage des protéines.
English descriptors
- KwdEn :
- Agaricales (enzymology), Calcium (pharmacology), Glutathione (analogs & derivatives), Glutathione (pharmacology), Glutathione Disulfide (MeSH), Glycerol (pharmacology), Hemin (pharmacology), Hydrogen-Ion Concentration (MeSH), Isoenzymes (biosynthesis), Isoenzymes (chemistry), Isoenzymes (metabolism), Kinetics (MeSH), Manganese (pharmacology), Peroxidases (biosynthesis), Peroxidases (chemistry), Peroxidases (metabolism), Potassium Chloride (pharmacology), Protein Denaturation (MeSH), Protein Folding (MeSH), Recombinant Proteins (biosynthesis), Recombinant Proteins (chemistry), Recombinant Proteins (metabolism), Urea (pharmacology).
- MESH :
- chemical , analogs & derivatives : Glutathione.
- chemical , biosynthesis : Isoenzymes, Peroxidases, Recombinant Proteins.
- chemical , chemistry : Isoenzymes, Peroxidases, Recombinant Proteins.
- chemical , metabolism : Isoenzymes, Peroxidases, Recombinant Proteins.
- chemical , pharmacology : Calcium, Glutathione, Glycerol, Hemin, Manganese, Potassium Chloride, Urea.
- enzymology : Agaricales.
- chemical : Glutathione Disulfide, Hydrogen-Ion Concentration, Kinetics, Protein Denaturation, Protein Folding.
Abstract
We have optimized the conditions under which recombinant Mn peroxidase from the white-rot fungus Phanerochaete chrysosporium can be expressed in Escherichia coli. A bacterial expression vector for the cDNA of Mn peroxidase isozyme H4 (lambda MP1) was constructed (R. E. Whitwam, I. G. Gazarian, and M. Tien, Biochem. Biophys. Res. Commun. 216, 1013-1017, 1995) whose expression in E. coli results in the formation of catalytically inactive polypeptide which can be refolded to active enzyme. The refolded enzyme was purified to homogeneity. Refolding was most efficient in 2 M urea, pH 8.0, and was absolutely dependent upon the presence of CaCl2, hemin, and oxidized glutathione. The recombinant enzyme had the same spectral and kinetic properties as the native fungal enzyme. The Km of recombinant Mn peroxidase for substrates H2O2 and the Mn2+/oxalate complex are 100 and 52 microM, respectively. The kcat as measured by Mn3+/oxalate formation is 450 s-1. These are essentially the same values as seen with the native fungal enzyme. The rate of formation of compound I, the two-electron-oxidized state of the enzyme, is 4.0 x 10(6) M-1 s-1, identical to the rate of the native fungal Mn peroxidase. The reaction of compound I with Mn2+ is too fast to measure at pH 4.5 in the recombinant Mn peroxidase. At a suboptimal pH of 2.5 a rate of 4.2 x 10(4) M-1 s-1 is obtained for the recombinant enzyme. The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn2+/oxalate has a Kd of 13 microM and a first-order rate constant of 230 s-1 in the recombinant enzyme. These rates are essentially the same as those seen with the native fungal MnP. These results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase.
DOI: 10.1006/abbi.1996.0413
PubMed: 8809085
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
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Tien</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1996">1996</date><idno type="RBID">pubmed:8809085</idno><idno type="pmid">8809085</idno><idno type="doi">10.1006/abbi.1996.0413</idno><idno type="wicri:Area/Main/Corpus">000C34</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000C34</idno><idno type="wicri:Area/Main/Curation">000C34</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">000C34</idno><idno type="wicri:Area/Main/Exploration">000C34</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Heterologous expression and reconstitution of fungal Mn peroxidase.</title><author><name sortKey="Whitwam, R" sort="Whitwam, R" uniqKey="Whitwam R" first="R" last="Whitwam">R. Whitwam</name><affiliation wicri:level="4"><nlm:affiliation>Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802</wicri:regionArea><orgName type="university">Université d'État de Pennsylvanie</orgName><placeName><settlement type="city">University Park (Pennsylvanie)</settlement><region type="state">Pennsylvanie</region></placeName></affiliation></author><author><name sortKey="Tien, M" sort="Tien, M" uniqKey="Tien M" first="M" last="Tien">M. 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A bacterial expression vector for the cDNA of Mn peroxidase isozyme H4 (lambda MP1) was constructed (R. E. Whitwam, I. G. Gazarian, and M. Tien, Biochem. Biophys. Res. Commun. 216, 1013-1017, 1995) whose expression in E. coli results in the formation of catalytically inactive polypeptide which can be refolded to active enzyme. The refolded enzyme was purified to homogeneity. Refolding was most efficient in 2 M urea, pH 8.0, and was absolutely dependent upon the presence of CaCl2, hemin, and oxidized glutathione. The recombinant enzyme had the same spectral and kinetic properties as the native fungal enzyme. The Km of recombinant Mn peroxidase for substrates H2O2 and the Mn2+/oxalate complex are 100 and 52 microM, respectively. The kcat as measured by Mn3+/oxalate formation is 450 s-1. These are essentially the same values as seen with the native fungal enzyme. The rate of formation of compound I, the two-electron-oxidized state of the enzyme, is 4.0 x 10(6) M-1 s-1, identical to the rate of the native fungal Mn peroxidase. The reaction of compound I with Mn2+ is too fast to measure at pH 4.5 in the recombinant Mn peroxidase. At a suboptimal pH of 2.5 a rate of 4.2 x 10(4) M-1 s-1 is obtained for the recombinant enzyme. The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn2+/oxalate has a Kd of 13 microM and a first-order rate constant of 230 s-1 in the recombinant enzyme. These rates are essentially the same as those seen with the native fungal MnP. These results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">8809085</PMID><DateCompleted><Year>1996</Year><Month>11</Month><Day>04</Day></DateCompleted><DateRevised><Year>2013</Year><Month>11</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0003-9861</ISSN><JournalIssue CitedMedium="Print"><Volume>333</Volume><Issue>2</Issue><PubDate><Year>1996</Year><Month>Sep</Month><Day>15</Day></PubDate></JournalIssue><Title>Archives of biochemistry and biophysics</Title><ISOAbbreviation>Arch Biochem Biophys</ISOAbbreviation></Journal><ArticleTitle>Heterologous expression and reconstitution of fungal Mn peroxidase.</ArticleTitle><Pagination><MedlinePgn>439-46</MedlinePgn></Pagination><Abstract><AbstractText>We have optimized the conditions under which recombinant Mn peroxidase from the white-rot fungus Phanerochaete chrysosporium can be expressed in Escherichia coli. A bacterial expression vector for the cDNA of Mn peroxidase isozyme H4 (lambda MP1) was constructed (R. E. Whitwam, I. G. Gazarian, and M. Tien, Biochem. Biophys. Res. Commun. 216, 1013-1017, 1995) whose expression in E. coli results in the formation of catalytically inactive polypeptide which can be refolded to active enzyme. The refolded enzyme was purified to homogeneity. Refolding was most efficient in 2 M urea, pH 8.0, and was absolutely dependent upon the presence of CaCl2, hemin, and oxidized glutathione. The recombinant enzyme had the same spectral and kinetic properties as the native fungal enzyme. The Km of recombinant Mn peroxidase for substrates H2O2 and the Mn2+/oxalate complex are 100 and 52 microM, respectively. The kcat as measured by Mn3+/oxalate formation is 450 s-1. These are essentially the same values as seen with the native fungal enzyme. The rate of formation of compound I, the two-electron-oxidized state of the enzyme, is 4.0 x 10(6) M-1 s-1, identical to the rate of the native fungal Mn peroxidase. The reaction of compound I with Mn2+ is too fast to measure at pH 4.5 in the recombinant Mn peroxidase. At a suboptimal pH of 2.5 a rate of 4.2 x 10(4) M-1 s-1 is obtained for the recombinant enzyme. The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn2+/oxalate has a Kd of 13 microM and a first-order rate constant of 230 s-1 in the recombinant enzyme. These rates are essentially the same as those seen with the native fungal MnP. These results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Whitwam</LastName><ForeName>R</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Tien</LastName><ForeName>M</ForeName><Initials>M</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>1-P42ES04922-01</GrantID><Acronym>ES</Acronym><Agency>NIEHS NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Arch Biochem Biophys</MedlineTA><NlmUniqueID>0372430</NlmUniqueID><ISSNLinking>0003-9861</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D007527">Isoenzymes</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>42Z2K6ZL8P</RegistryNumber><NameOfSubstance UI="D008345">Manganese</NameOfSubstance></Chemical><Chemical><RegistryNumber>660YQ98I10</RegistryNumber><NameOfSubstance UI="D011189">Potassium Chloride</NameOfSubstance></Chemical><Chemical><RegistryNumber>743LRP9S7N</RegistryNumber><NameOfSubstance UI="D006427">Hemin</NameOfSubstance></Chemical><Chemical><RegistryNumber>8W8T17847W</RegistryNumber><NameOfSubstance UI="D014508">Urea</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.11.1.-</RegistryNumber><NameOfSubstance UI="D010544">Peroxidases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.11.1.13</RegistryNumber><NameOfSubstance UI="C051129">manganese peroxidase</NameOfSubstance></Chemical><Chemical><RegistryNumber>GAN16C9B8O</RegistryNumber><NameOfSubstance UI="D005978">Glutathione</NameOfSubstance></Chemical><Chemical><RegistryNumber>PDC6A3C0OX</RegistryNumber><NameOfSubstance UI="D005990">Glycerol</NameOfSubstance></Chemical><Chemical><RegistryNumber>SY7Q814VUP</RegistryNumber><NameOfSubstance UI="D002118">Calcium</NameOfSubstance></Chemical><Chemical><RegistryNumber>ULW86O013H</RegistryNumber><NameOfSubstance UI="D019803">Glutathione Disulfide</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000363" MajorTopicYN="N">Agaricales</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D002118" MajorTopicYN="N">Calcium</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005978" MajorTopicYN="N">Glutathione</DescriptorName><QualifierName UI="Q000031" MajorTopicYN="N">analogs & derivatives</QualifierName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019803" MajorTopicYN="N">Glutathione Disulfide</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005990" MajorTopicYN="N">Glycerol</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006427" MajorTopicYN="N">Hemin</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006863" MajorTopicYN="N">Hydrogen-Ion Concentration</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007527" MajorTopicYN="N">Isoenzymes</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D007700" MajorTopicYN="N">Kinetics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008345" MajorTopicYN="N">Manganese</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010544" MajorTopicYN="N">Peroxidases</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011189" MajorTopicYN="N">Potassium Chloride</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011489" MajorTopicYN="N">Protein Denaturation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017510" MajorTopicYN="Y">Protein Folding</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014508" MajorTopicYN="N">Urea</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1996</Year><Month>9</Month><Day>15</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1996</Year><Month>9</Month><Day>15</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1996</Year><Month>9</Month><Day>15</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">8809085</ArticleId><ArticleId IdType="pii">S0003-9861(96)90413-0</ArticleId><ArticleId IdType="doi">10.1006/abbi.1996.0413</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>Pennsylvanie</li></region><settlement><li>University Park (Pennsylvanie)</li></settlement><orgName><li>Université d'État de Pennsylvanie</li></orgName></list><tree><noCountry><name sortKey="Tien, M" sort="Tien, M" uniqKey="Tien M" first="M" last="Tien">M. Tien</name></noCountry><country name="États-Unis"><region name="Pennsylvanie"><name sortKey="Whitwam, R" sort="Whitwam, R" uniqKey="Whitwam R" first="R" last="Whitwam">R. Whitwam</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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