Por favor, use este identificador para citar o enlazar a este item:
http://hdl.handle.net/10261/48376
COMPARTIR / EXPORTAR:
SHARE CORE BASE | |
Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL | DATACITE | |
Título: | Manganese oxidation site in Pleurotus eryngii versatile peroxidase: A site-directed mutagenesis, kinetic, and crystallographic study |
Autor: | Ruiz-Dueñas, F. J. CSIC ORCID ; Morales, María CSIC; Pérez-Boada, Marta CSIC ORCID ; Choinowski, Thomas; Martínez, María Jesús CSIC ORCID ; Piontek, Klaus; Martínez, Ángel T. CSIC ORCID | Fecha de publicación: | 1-sep-2007 | Editor: | American Chemical Society | Citación: | Biochemistry 46(1): 66-77(2007) | Resumen: | 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 | Descripción: | 12 páginas, 6 figuras, 4 tablas -- PAGS nros. 66-77 | Versión del editor: | http://dx.doi.org/10.1021/bi061542h | URI: | 10261/48376 | DOI: | 10.1021/bi061542h | ISSN: | 0006-2960 | E-ISSN: | 1520-4995 |
Aparece en las colecciones: | (CIB) Artículos |
Ficheros en este ítem:
Fichero | Descripción | Tamaño | Formato | |
---|---|---|---|---|
restringido.pdf | 21,67 kB | Adobe PDF | Visualizar/Abrir |
CORE Recommender
SCOPUSTM
Citations
77
checked on 19-mar-2024
WEB OF SCIENCETM
Citations
66
checked on 29-feb-2024
Page view(s)
288
checked on 17-mar-2024
Download(s)
80
checked on 17-mar-2024
Google ScholarTM
Check
Altmetric
Altmetric
NOTA: Los ítems de Digital.CSIC están protegidos por copyright, con todos los derechos reservados, a menos que se indique lo contrario.