2W23

Structure of mutant W169Y of Pleurotus eryngii versatile peroxidase (VP)

Summary for 2W23

• Entry DOI: 10.2210/pdb2w23/pdb
• Related: 2BOQ 2VKA
• Descriptor: VERSATILE PEROXIDASE VPL2, PROTOPORPHYRIN IX CONTAINING FE, CALCIUM ION, ... (6 entities in total)
• Functional Keywords: oxidoreductase, organic radical, protein radicals, lignin degradation, manganese peroxidase, electron transfer, hydrogen peroxide, iron, heme, calcium, zymogen, secreted, manganese, peroxidase, glycoprotein, metal-binding
• Biological source: PLEUROTUS ERYNGII
• Cellular location: Secreted: O94753
• Total number of polymer chains: 1
• Total formula weight: 34108.98

Authors

Ruiz-Duenas, F.J.,Pogni, R.,Morales, M.,Giansanti, S.,Mate, M.J.,Romero, A.,Martinez, M.J.,Basosi, R.,Martinez, A.T. (deposition date: 2008-10-23, release date: 2009-01-20, Last modification date: 2024-10-16)

Primary citation

Ruiz-Duenas, F.J.,Pogni, R.,Morales, M.,Giansanti, S.,Mate, M.J.,Romero, A.,Martinez, M.J.,Basosi, R.,Martinez, A.T.
Protein Radicals in Fungal Versatile Peroxidase: Catalytic Tryptophan Radical in Both Compound I and Compound II and Studies on W164Y, W164H, and W164S Variants.
J.Biol.Chem., 284:7986-, 2009

PubMed Abstract: Lignin-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 the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) 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 variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k(2app)) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k(2)) was more affected than the dissociation constant (K(D)(2)). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k(3)) 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 Trp-164 is involved in this autocatalytic process.

PubMed: 19158088
DOI: 10.1074/JBC.M808069200

Experimental method

X-RAY DIFFRACTION (1.94 Å)