Site-directed mutagenesis of the heme axial ligands in the hemoflavoenzyme cellobiose dehydrogenase.
Identifieur interne : 000A23 ( Main/Exploration ); précédent : 000A22; suivant : 000A24Site-directed mutagenesis of the heme axial ligands in the hemoflavoenzyme cellobiose dehydrogenase.
Auteurs : F A Rotsaert [États-Unis] ; B. Li ; V. Renganathan ; M H GoldSource :
- Archives of biochemistry and biophysics [ 0003-9861 ] ; 2001.
Descripteurs français
- KwdFr :
- Carbohydrate dehydrogenases (génétique), Carbohydrate dehydrogenases (métabolisme), Catalyse (MeSH), Cellulose (métabolisme), Histidine (métabolisme), Hème (métabolisme), Ligands (MeSH), Masse moléculaire (MeSH), Mutagenèse dirigée (MeSH), Méthionine (métabolisme), Phanerochaete (enzymologie), Phanerochaete (métabolisme), Substitution d'acide aminé (MeSH).
- MESH :
- enzymologie : Phanerochaete.
- génétique : Carbohydrate dehydrogenases.
- métabolisme : Carbohydrate dehydrogenases, Cellulose, Histidine, Hème, Méthionine, Phanerochaete.
- Catalyse, Ligands, Masse moléculaire, Mutagenèse dirigée, Substitution d'acide aminé.
English descriptors
- KwdEn :
- Amino Acid Substitution (MeSH), Carbohydrate Dehydrogenases (genetics), Carbohydrate Dehydrogenases (metabolism), Catalysis (MeSH), Cellulose (metabolism), Heme (metabolism), Histidine (metabolism), Ligands (MeSH), Methionine (metabolism), Molecular Weight (MeSH), Mutagenesis, Site-Directed (MeSH), Phanerochaete (enzymology), Phanerochaete (metabolism).
- MESH :
- chemical , genetics : Carbohydrate Dehydrogenases.
- chemical , metabolism : Carbohydrate Dehydrogenases, Cellulose, Heme, Histidine, Methionine.
- enzymology : Phanerochaete.
- metabolism : Phanerochaete.
- Amino Acid Substitution, Catalysis, Ligands, Molecular Weight, Mutagenesis, Site-Directed.
Abstract
Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium is an extracellular 90-kDa hemoflavoenzyme, organized into an N-terminal heme domain and a C-terminal flavin domain. The amino acid residues Met65 and His114 or His163 were suggested to be heme iron ligands. Mutations of these residues were made and mutant proteins were characterized. H114A mutant cultures produce a stable hemoflavoenzyme with spectral and kinetic characteristics similar to those of wild-type CDH. The M65A and H163A transformants secrete a 90-kDa hemoflavoenzyme, which oxidizes cellobiose in the presence of 2,6-dichlorophenol-indophenol (DCPIP), but is unable to reduce cytochrome c. The heme domains of the M65A and H163A CDH variants are, however, unstable and susceptible to degradation, both yielding a 70-kDa cellobiose-oxidizing flavoenzyme. The spectral and kinetic characteristics of these truncated variants suggest that they contain only their respective flavin domains. The yield of the 90-kDa proteins was low and the proteins could not be purified to homogeneity; however, absorption spectra indicate that the 90-kDa proteins do contain the heme domain. Like the truncated flavoenzymes, the 90-kDa variants reduce DCPIP but are unable to transfer electrons to cytochrome c, in contrast to wild-type CDH. These findings suggest that H163 and M65 are the axial heme ligands and that both ligands are required for the reactivity and structural integrity of the heme domain.
DOI: 10.1006/abbi.2001.2362
PubMed: 11396923
Affiliations:
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Li</name></author><author><name sortKey="Renganathan, V" sort="Renganathan, V" uniqKey="Renganathan V" first="V" last="Renganathan">V. Renganathan</name></author><author><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2001">2001</date><idno type="RBID">pubmed:11396923</idno><idno type="pmid">11396923</idno><idno type="doi">10.1006/abbi.2001.2362</idno><idno type="wicri:Area/Main/Corpus">000A50</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000A50</idno><idno type="wicri:Area/Main/Curation">000A50</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">000A50</idno><idno type="wicri:Area/Main/Exploration">000A50</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Site-directed mutagenesis of the heme axial ligands in the hemoflavoenzyme cellobiose dehydrogenase.</title><author><name sortKey="Rotsaert, F A" sort="Rotsaert, F A" uniqKey="Rotsaert F" first="F A" last="Rotsaert">F A Rotsaert</name><affiliation wicri:level="1"><nlm:affiliation>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921</wicri:regionArea><wicri:noRegion>Oregon 97006-8921</wicri:noRegion></affiliation></author><author><name sortKey="Li, B" sort="Li, B" uniqKey="Li B" first="B" last="Li">B. Li</name></author><author><name sortKey="Renganathan, V" sort="Renganathan, V" uniqKey="Renganathan V" first="V" last="Renganathan">V. Renganathan</name></author><author><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name></author></analytic><series><title level="j">Archives of biochemistry and biophysics</title><idno type="ISSN">0003-9861</idno><imprint><date when="2001" type="published">2001</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Substitution (MeSH)</term><term>Carbohydrate Dehydrogenases (genetics)</term><term>Carbohydrate Dehydrogenases (metabolism)</term><term>Catalysis (MeSH)</term><term>Cellulose (metabolism)</term><term>Heme (metabolism)</term><term>Histidine (metabolism)</term><term>Ligands (MeSH)</term><term>Methionine (metabolism)</term><term>Molecular Weight (MeSH)</term><term>Mutagenesis, Site-Directed (MeSH)</term><term>Phanerochaete (enzymology)</term><term>Phanerochaete (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Carbohydrate dehydrogenases (génétique)</term><term>Carbohydrate dehydrogenases (métabolisme)</term><term>Catalyse (MeSH)</term><term>Cellulose (métabolisme)</term><term>Histidine (métabolisme)</term><term>Hème (métabolisme)</term><term>Ligands (MeSH)</term><term>Masse moléculaire (MeSH)</term><term>Mutagenèse dirigée (MeSH)</term><term>Méthionine (métabolisme)</term><term>Phanerochaete (enzymologie)</term><term>Phanerochaete (métabolisme)</term><term>Substitution d'acide aminé (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Carbohydrate Dehydrogenases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Carbohydrate Dehydrogenases</term><term>Cellulose</term><term>Heme</term><term>Histidine</term><term>Methionine</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Carbohydrate dehydrogenases</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Carbohydrate dehydrogenases</term><term>Cellulose</term><term>Histidine</term><term>Hème</term><term>Méthionine</term><term>Phanerochaete</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Substitution</term><term>Catalysis</term><term>Ligands</term><term>Molecular Weight</term><term>Mutagenesis, Site-Directed</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Catalyse</term><term>Ligands</term><term>Masse moléculaire</term><term>Mutagenèse dirigée</term><term>Substitution d'acide aminé</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium is an extracellular 90-kDa hemoflavoenzyme, organized into an N-terminal heme domain and a C-terminal flavin domain. The amino acid residues Met65 and His114 or His163 were suggested to be heme iron ligands. Mutations of these residues were made and mutant proteins were characterized. H114A mutant cultures produce a stable hemoflavoenzyme with spectral and kinetic characteristics similar to those of wild-type CDH. The M65A and H163A transformants secrete a 90-kDa hemoflavoenzyme, which oxidizes cellobiose in the presence of 2,6-dichlorophenol-indophenol (DCPIP), but is unable to reduce cytochrome c. The heme domains of the M65A and H163A CDH variants are, however, unstable and susceptible to degradation, both yielding a 70-kDa cellobiose-oxidizing flavoenzyme. The spectral and kinetic characteristics of these truncated variants suggest that they contain only their respective flavin domains. The yield of the 90-kDa proteins was low and the proteins could not be purified to homogeneity; however, absorption spectra indicate that the 90-kDa proteins do contain the heme domain. Like the truncated flavoenzymes, the 90-kDa variants reduce DCPIP but are unable to transfer electrons to cytochrome c, in contrast to wild-type CDH. These findings suggest that H163 and M65 are the axial heme ligands and that both ligands are required for the reactivity and structural integrity of the heme domain.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">11396923</PMID><DateCompleted><Year>2001</Year><Month>07</Month><Day>12</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>390</Volume><Issue>2</Issue><PubDate><Year>2001</Year><Month>Jun</Month><Day>15</Day></PubDate></JournalIssue><Title>Archives of biochemistry and biophysics</Title><ISOAbbreviation>Arch Biochem Biophys</ISOAbbreviation></Journal><ArticleTitle>Site-directed mutagenesis of the heme axial ligands in the hemoflavoenzyme cellobiose dehydrogenase.</ArticleTitle><Pagination><MedlinePgn>206-14</MedlinePgn></Pagination><Abstract><AbstractText>Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium is an extracellular 90-kDa hemoflavoenzyme, organized into an N-terminal heme domain and a C-terminal flavin domain. The amino acid residues Met65 and His114 or His163 were suggested to be heme iron ligands. Mutations of these residues were made and mutant proteins were characterized. H114A mutant cultures produce a stable hemoflavoenzyme with spectral and kinetic characteristics similar to those of wild-type CDH. The M65A and H163A transformants secrete a 90-kDa hemoflavoenzyme, which oxidizes cellobiose in the presence of 2,6-dichlorophenol-indophenol (DCPIP), but is unable to reduce cytochrome c. The heme domains of the M65A and H163A CDH variants are, however, unstable and susceptible to degradation, both yielding a 70-kDa cellobiose-oxidizing flavoenzyme. The spectral and kinetic characteristics of these truncated variants suggest that they contain only their respective flavin domains. The yield of the 90-kDa proteins was low and the proteins could not be purified to homogeneity; however, absorption spectra indicate that the 90-kDa proteins do contain the heme domain. Like the truncated flavoenzymes, the 90-kDa variants reduce DCPIP but are unable to transfer electrons to cytochrome c, in contrast to wild-type CDH. These findings suggest that H163 and M65 are the axial heme ligands and that both ligands are required for the reactivity and structural integrity of the heme domain.</AbstractText><CopyrightInformation>Copyright 2001 Academic Press.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Rotsaert</LastName><ForeName>F A</ForeName><Initials>FA</Initials><AffiliationInfo><Affiliation>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Li</LastName><ForeName>B</ForeName><Initials>B</Initials></Author><Author ValidYN="Y"><LastName>Renganathan</LastName><ForeName>V</ForeName><Initials>V</Initials></Author><Author ValidYN="Y"><LastName>Gold</LastName><ForeName>M H</ForeName><Initials>MH</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-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="D008024">Ligands</NameOfSubstance></Chemical><Chemical><RegistryNumber>42VZT0U6YR</RegistryNumber><NameOfSubstance UI="D006418">Heme</NameOfSubstance></Chemical><Chemical><RegistryNumber>4QD397987E</RegistryNumber><NameOfSubstance UI="D006639">Histidine</NameOfSubstance></Chemical><Chemical><RegistryNumber>9004-34-6</RegistryNumber><NameOfSubstance UI="D002482">Cellulose</NameOfSubstance></Chemical><Chemical><RegistryNumber>AE28F7PNPL</RegistryNumber><NameOfSubstance UI="D008715">Methionine</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.1.-</RegistryNumber><NameOfSubstance UI="D002237">Carbohydrate Dehydrogenases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.1.99.18</RegistryNumber><NameOfSubstance UI="C019859">cellobiose-quinone oxidoreductase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D019943" MajorTopicYN="N">Amino Acid Substitution</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002237" MajorTopicYN="N">Carbohydrate Dehydrogenases</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D002384" MajorTopicYN="N">Catalysis</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002482" MajorTopicYN="N">Cellulose</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006418" MajorTopicYN="N">Heme</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006639" MajorTopicYN="N">Histidine</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008024" MajorTopicYN="N">Ligands</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008715" MajorTopicYN="N">Methionine</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008970" MajorTopicYN="N">Molecular Weight</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D016297" MajorTopicYN="N">Mutagenesis, Site-Directed</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D020075" MajorTopicYN="N">Phanerochaete</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2001</Year><Month>6</Month><Day>9</Day><Hour>10</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2001</Year><Month>7</Month><Day>13</Day><Hour>10</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2001</Year><Month>6</Month><Day>9</Day><Hour>10</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">11396923</ArticleId><ArticleId IdType="doi">10.1006/abbi.2001.2362</ArticleId><ArticleId IdType="pii">S0003-9861(01)92362-8</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name><name sortKey="Li, B" sort="Li, B" uniqKey="Li B" first="B" last="Li">B. Li</name><name sortKey="Renganathan, V" sort="Renganathan, V" uniqKey="Renganathan V" first="V" last="Renganathan">V. Renganathan</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Rotsaert, F A" sort="Rotsaert, F A" uniqKey="Rotsaert F" first="F A" last="Rotsaert">F A Rotsaert</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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