Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.
Identifieur interne : 000752 ( Main/Corpus ); précédent : 000751; suivant : 000753Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.
Auteurs : Francisco J. Ruiz-Due As ; María Morales ; Marta Pérez-Boada ; Thomas Choinowski ; María Jesús Martínez ; Klaus Piontek ; Angel T. MartínezSource :
- Biochemistry [ 0006-2960 ] ; 2007.
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
Links to Exploration step
pubmed:17198376Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.</title>
<author><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>
<affiliation><nlm:affiliation>Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.</nlm:affiliation>
</affiliation>
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<author><name sortKey="Morales, Maria" sort="Morales, Maria" uniqKey="Morales M" first="María" last="Morales">María Morales</name>
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<author><name sortKey="Perez Boada, Marta" sort="Perez Boada, Marta" uniqKey="Perez Boada M" first="Marta" last="Pérez-Boada">Marta Pérez-Boada</name>
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<author><name sortKey="Choinowski, Thomas" sort="Choinowski, Thomas" uniqKey="Choinowski T" first="Thomas" last="Choinowski">Thomas Choinowski</name>
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<author><name sortKey="Martinez, Maria Jesus" sort="Martinez, Maria Jesus" uniqKey="Martinez M" first="María Jesús" last="Martínez">María Jesús Martínez</name>
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<author><name sortKey="Piontek, Klaus" sort="Piontek, Klaus" uniqKey="Piontek K" first="Klaus" last="Piontek">Klaus Piontek</name>
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<author><name sortKey="Martinez, Angel T" sort="Martinez, Angel T" uniqKey="Martinez A" first="Angel T" last="Martínez">Angel T. Martínez</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en">Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.</title>
<author><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>
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<author><name sortKey="Morales, Maria" sort="Morales, Maria" uniqKey="Morales M" first="María" last="Morales">María Morales</name>
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<author><name sortKey="Perez Boada, Marta" sort="Perez Boada, Marta" uniqKey="Perez Boada M" first="Marta" last="Pérez-Boada">Marta Pérez-Boada</name>
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<author><name sortKey="Choinowski, Thomas" sort="Choinowski, Thomas" uniqKey="Choinowski T" first="Thomas" last="Choinowski">Thomas Choinowski</name>
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<author><name sortKey="Martinez, Maria Jesus" sort="Martinez, Maria Jesus" uniqKey="Martinez M" first="María Jesús" last="Martínez">María Jesús Martínez</name>
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<author><name sortKey="Piontek, Klaus" sort="Piontek, Klaus" uniqKey="Piontek K" first="Klaus" last="Piontek">Klaus Piontek</name>
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<author><name sortKey="Martinez, Angel T" sort="Martinez, Angel T" uniqKey="Martinez A" first="Angel T" last="Martínez">Angel T. Martínez</name>
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<series><title level="j">Biochemistry</title>
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<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>
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<term>Crystallography, X-Ray</term>
<term>Kinetics</term>
<term>Ligands</term>
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<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>
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<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>
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