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Title

Manganese oxidation site in Pleurotus eryngii versatile peroxidase: A site-directed mutagenesis, kinetic, and crystallographic study

AuthorsRuiz-Dueñas, F. J. ; Morales, María ; Pérez-Boada, Marta ; Choinowski, Thomas; Martínez, María Jesús ; Piontek, Klaus; Martínez, Ángel T.
Issue Date1-Sep-2007
PublisherAmerican Chemical Society
CitationBiochemistry 46(1): 66-77(2007)
AbstractThe 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
Description12 páginas, 6 figuras, 4 tablas -- PAGS nros. 66-77
Publisher version (URL)http://dx.doi.org/10.1021/bi061542h
URI10261/48376
DOI10.1021/bi061542h
ISSN0006-2960
E-ISSN1520-4995
Appears in Collections:(CIB) Artículos
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