Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.
Identifieur interne : 000724 ( Main/Exploration ); précédent : 000723; suivant : 000725Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.
Auteurs : Francisco J. Ruiz-Due As [Espagne] ; María Morales ; Marta Pérez-Boada ; Thomas Choinowski ; María Jesús Martínez ; Klaus Piontek ; Angel T. MartínezSource :
- Biochemistry [ 0006-2960 ] ; 2007.
Descripteurs français
- KwdFr :
- Cinétique (MeSH), Cristallisation (MeSH), Cristallographie aux rayons X (MeSH), Ligands (MeSH), Manganèse (composition chimique), Manganèse (métabolisme), Mutagenèse dirigée (MeSH), Mutation (MeSH), Oxydoréduction (MeSH), Peroxidases (composition chimique), Peroxidases (métabolisme), Pleurotus (enzymologie), Pleurotus (métabolisme), Protéines fongiques (composition chimique), Protéines fongiques (métabolisme), Sites de fixation (MeSH).
- MESH :
- composition chimique : Manganèse, Peroxidases, Protéines fongiques.
- enzymologie : Pleurotus.
- métabolisme : Manganèse, Peroxidases, Pleurotus, Protéines fongiques.
- Cinétique, Cristallisation, Cristallographie aux rayons X, Ligands, Mutagenèse dirigée, Mutation, Oxydoréduction, Sites de fixation.
English descriptors
- KwdEn :
- Binding Sites (MeSH), Crystallization (MeSH), Crystallography, X-Ray (MeSH), Fungal Proteins (chemistry), Fungal Proteins (metabolism), Kinetics (MeSH), Ligands (MeSH), Manganese (chemistry), Manganese (metabolism), Mutagenesis, Site-Directed (MeSH), Mutation (MeSH), Oxidation-Reduction (MeSH), Peroxidases (chemistry), Peroxidases (metabolism), Pleurotus (enzymology), Pleurotus (metabolism).
- MESH :
- chemical , chemistry : Fungal Proteins, Manganese, Peroxidases.
- chemical , metabolism : Fungal Proteins, Manganese, Peroxidases.
- enzymology : Pleurotus.
- metabolism : Pleurotus.
- Binding Sites, Crystallization, Crystallography, X-Ray, Kinetics, Ligands, Mutagenesis, Site-Directed, Mutation, Oxidation-Reduction.
Abstract
The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 A) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (<1% kcat). The affinity for Mn2+ also decreased ( approximately 25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained 30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in kcat in the case of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showed that introduction of a basic residue near Asp175 did not improve Mn2+ oxidation as found for MnP and ruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn2+. The structural and kinetic data obtained highlighted significant differences in the Mn2+ oxidation site of the new versatile enzyme compared to P. chrysosporium MnP.
DOI: 10.1021/bi061542h
PubMed: 17198376
Affiliations:
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Ruiz-Due As</name><affiliation wicri:level="2"><nlm:affiliation>Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid</wicri:regionArea><placeName><region nuts="2" type="communauté">Communauté de Madrid</region></placeName></affiliation></author><author><name sortKey="Morales, Maria" sort="Morales, Maria" uniqKey="Morales M" first="María" last="Morales">María Morales</name></author><author><name sortKey="Perez Boada, Marta" sort="Perez Boada, Marta" uniqKey="Perez Boada M" first="Marta" last="Pérez-Boada">Marta Pérez-Boada</name></author><author><name sortKey="Choinowski, Thomas" sort="Choinowski, Thomas" uniqKey="Choinowski T" first="Thomas" last="Choinowski">Thomas Choinowski</name></author><author><name sortKey="Martinez, Maria Jesus" sort="Martinez, Maria Jesus" uniqKey="Martinez M" first="María Jesús" last="Martínez">María Jesús Martínez</name></author><author><name sortKey="Piontek, Klaus" sort="Piontek, Klaus" uniqKey="Piontek K" first="Klaus" last="Piontek">Klaus Piontek</name></author><author><name sortKey="Martinez, Angel T" sort="Martinez, Angel T" uniqKey="Martinez A" first="Angel T" last="Martínez">Angel T. 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Ruiz-Due As</name><affiliation wicri:level="2"><nlm:affiliation>Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid</wicri:regionArea><placeName><region nuts="2" type="communauté">Communauté de Madrid</region></placeName></affiliation></author><author><name sortKey="Morales, Maria" sort="Morales, Maria" uniqKey="Morales M" first="María" last="Morales">María Morales</name></author><author><name sortKey="Perez Boada, Marta" sort="Perez Boada, Marta" uniqKey="Perez Boada M" first="Marta" last="Pérez-Boada">Marta Pérez-Boada</name></author><author><name sortKey="Choinowski, Thomas" sort="Choinowski, Thomas" uniqKey="Choinowski T" first="Thomas" last="Choinowski">Thomas Choinowski</name></author><author><name sortKey="Martinez, Maria Jesus" sort="Martinez, Maria Jesus" uniqKey="Martinez M" first="María Jesús" last="Martínez">María Jesús Martínez</name></author><author><name sortKey="Piontek, Klaus" sort="Piontek, Klaus" uniqKey="Piontek K" first="Klaus" last="Piontek">Klaus Piontek</name></author><author><name sortKey="Martinez, Angel T" sort="Martinez, Angel T" uniqKey="Martinez A" first="Angel T" last="Martínez">Angel T. Martínez</name></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><imprint><date when="2007" type="published">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Binding Sites (MeSH)</term><term>Crystallization (MeSH)</term><term>Crystallography, X-Ray (MeSH)</term><term>Fungal Proteins (chemistry)</term><term>Fungal Proteins (metabolism)</term><term>Kinetics (MeSH)</term><term>Ligands (MeSH)</term><term>Manganese (chemistry)</term><term>Manganese (metabolism)</term><term>Mutagenesis, Site-Directed (MeSH)</term><term>Mutation (MeSH)</term><term>Oxidation-Reduction (MeSH)</term><term>Peroxidases (chemistry)</term><term>Peroxidases (metabolism)</term><term>Pleurotus (enzymology)</term><term>Pleurotus (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cinétique (MeSH)</term><term>Cristallisation (MeSH)</term><term>Cristallographie aux rayons X (MeSH)</term><term>Ligands (MeSH)</term><term>Manganèse (composition chimique)</term><term>Manganèse (métabolisme)</term><term>Mutagenèse dirigée (MeSH)</term><term>Mutation (MeSH)</term><term>Oxydoréduction (MeSH)</term><term>Peroxidases (composition chimique)</term><term>Peroxidases (métabolisme)</term><term>Pleurotus (enzymologie)</term><term>Pleurotus (métabolisme)</term><term>Protéines fongiques (composition chimique)</term><term>Protéines fongiques (métabolisme)</term><term>Sites de fixation (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Fungal Proteins</term><term>Manganese</term><term>Peroxidases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Fungal Proteins</term><term>Manganese</term><term>Peroxidases</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Manganèse</term><term>Peroxidases</term><term>Protéines fongiques</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Pleurotus</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Pleurotus</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Pleurotus</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Manganèse</term><term>Peroxidases</term><term>Pleurotus</term><term>Protéines fongiques</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Binding Sites</term><term>Crystallization</term><term>Crystallography, X-Ray</term><term>Kinetics</term><term>Ligands</term><term>Mutagenesis, Site-Directed</term><term>Mutation</term><term>Oxidation-Reduction</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cinétique</term><term>Cristallisation</term><term>Cristallographie aux rayons X</term><term>Ligands</term><term>Mutagenèse dirigée</term><term>Mutation</term><term>Oxydoréduction</term><term>Sites de fixation</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 A) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (<1% kcat). The affinity for Mn2+ also decreased ( approximately 25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained 30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in kcat in the case of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showed that introduction of a basic residue near Asp175 did not improve Mn2+ oxidation as found for MnP and ruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn2+. The structural and kinetic data obtained highlighted significant differences in the Mn2+ oxidation site of the new versatile enzyme compared to P. chrysosporium MnP.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">17198376</PMID><DateCompleted><Year>2007</Year><Month>03</Month><Day>08</Day></DateCompleted><DateRevised><Year>2013</Year><Month>11</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0006-2960</ISSN><JournalIssue CitedMedium="Print"><Volume>46</Volume><Issue>1</Issue><PubDate><Year>2007</Year><Month>Jan</Month><Day>09</Day></PubDate></JournalIssue><Title>Biochemistry</Title><ISOAbbreviation>Biochemistry</ISOAbbreviation></Journal><ArticleTitle>Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.</ArticleTitle><Pagination><MedlinePgn>66-77</MedlinePgn></Pagination><Abstract><AbstractText>The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 A) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (<1% kcat). The affinity for Mn2+ also decreased ( approximately 25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained 30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in kcat in the case of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showed that introduction of a basic residue near Asp175 did not improve Mn2+ oxidation as found for MnP and ruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn2+. The structural and kinetic data obtained highlighted significant differences in the Mn2+ oxidation site of the new versatile enzyme compared to P. chrysosporium MnP.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Ruiz-Dueñas</LastName><ForeName>Francisco J</ForeName><Initials>FJ</Initials><AffiliationInfo><Affiliation>Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Morales</LastName><ForeName>María</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Pérez-Boada</LastName><ForeName>Marta</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Choinowski</LastName><ForeName>Thomas</ForeName><Initials>T</Initials></Author><Author ValidYN="Y"><LastName>Martínez</LastName><ForeName>María Jesús</ForeName><Initials>MJ</Initials></Author><Author ValidYN="Y"><LastName>Piontek</LastName><ForeName>Klaus</ForeName><Initials>K</Initials></Author><Author ValidYN="Y"><LastName>Martínez</LastName><ForeName>Angel T</ForeName><Initials>AT</Initials></Author></AuthorList><Language>eng</Language><DataBankList CompleteYN="Y"><DataBank><DataBankName>PDB</DataBankName><AccessionNumberList><AccessionNumber>2BOQ</AccessionNumber></AccessionNumberList></DataBank></DataBankList><PublicationTypeList><PublicationType UI="D003160">Comparative Study</PublicationType><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochemistry</MedlineTA><NlmUniqueID>0370623</NlmUniqueID><ISSNLinking>0006-2960</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005656">Fungal Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D008024">Ligands</NameOfSubstance></Chemical><Chemical><RegistryNumber>42Z2K6ZL8P</RegistryNumber><NameOfSubstance UI="D008345">Manganese</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></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001665" MajorTopicYN="N">Binding Sites</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003460" MajorTopicYN="N">Crystallization</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D018360" MajorTopicYN="N">Crystallography, X-Ray</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005656" MajorTopicYN="N">Fungal 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MajorTopicYN="N">Peroxidases</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D020076" MajorTopicYN="N">Pleurotus</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2007</Year><Month>1</Month><Day>3</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2007</Year><Month>3</Month><Day>9</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2007</Year><Month>1</Month><Day>3</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">17198376</ArticleId><ArticleId IdType="doi">10.1021/bi061542h</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Espagne</li></country><region><li>Communauté de Madrid</li></region></list><tree><noCountry><name sortKey="Choinowski, Thomas" sort="Choinowski, Thomas" uniqKey="Choinowski T" first="Thomas" last="Choinowski">Thomas Choinowski</name><name sortKey="Martinez, Angel T" sort="Martinez, Angel T" uniqKey="Martinez A" first="Angel T" last="Martínez">Angel T. Martínez</name><name sortKey="Martinez, Maria Jesus" sort="Martinez, Maria Jesus" uniqKey="Martinez M" first="María Jesús" last="Martínez">María Jesús Martínez</name><name sortKey="Morales, Maria" sort="Morales, Maria" uniqKey="Morales M" first="María" last="Morales">María Morales</name><name sortKey="Perez Boada, Marta" sort="Perez Boada, Marta" uniqKey="Perez Boada M" first="Marta" last="Pérez-Boada">Marta Pérez-Boada</name><name sortKey="Piontek, Klaus" sort="Piontek, Klaus" uniqKey="Piontek K" first="Klaus" last="Piontek">Klaus Piontek</name></noCountry><country name="Espagne"><region name="Communauté de Madrid"><name sortKey="Ruiz Due As, Francisco J" sort="Ruiz Due As, Francisco J" uniqKey="Ruiz Due As F" first="Francisco J" last="Ruiz-Due As">Francisco J. Ruiz-Due As</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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