English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/10247
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:

Protein radicals in fungal versatile peroxidase: Catalytic tryptophan radical in both Compound-I and Compound-II and studies on W164Y, W164H and W164S variants

Other TitlesProtein Radicals in Versatile Peroxidase [Running title]
AuthorsRuiz-Dueñas, F. J. ; Pogni, Rebecca; Morales, María ; Giansanti, Stefania; Maté, María J. ; Romero, Antonio ; Martínez, María Jesús ; Basosi, Riccardo; Martínez, Ángel T.
Issue Date20-Mar-2009
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJ Biol Chem. 284(12): 7986–7994 (2009)
AbstractLignin-degrading peroxidases, a group of biotechnologically-interesting enzymes,?oxidize high redox-potential aromatics via an exposed protein radical. Low-temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp164) by tyrosine and histidine residues. No changes in the crystal structure were observed indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k2app) by veratryl alcohol (VA), compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k2) was more affected than the dissociation constant (KD2). Moreover, VPII reduction by VA was impaired by the above mutations revealing that the Trp164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k3) values were similar for the W164Y, W164H and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed revealing that Trp164 is involved in this autocatalytic process.
Description9 p.-4 fig.-4 tab.
Publisher version (URL)http://dx.doi.org/10.1074/jbc.M808069200
Appears in Collections:(CIB) Artículos
Files in This Item:
File Description SizeFormat 
Protein radicals_2009.pdf433,22 kBAdobe PDFThumbnail
Show full item record

Related articles:

WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.