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|Title:||Protein radicals in fungal versatile peroxidase: Catalytic tryptophan radical in both Coumpound-I and Coumpound-II and studies on W164Y, W164H and W164S variants||Authors:||RUIZ DUENAS F., J
MATE M., J
MARTINEZ M., J
Martinez, A. T.
|Keywords:||Protein radical; versatile peroxidase; tryptophan radical; oxizymes; EPR||Issue Date:||2009||Project:||None||Journal:||THE JOURNAL OF BIOLOGICAL CHEMISTRY||Abstract:||
Lignin-degrading peroxidases, a group of biotechnologicallyinteresting enzymes, oxidize high redox potential aromatics viaan exposed protein radical. Low temperature EPR of Pleurotuseryngii versatile peroxidase (VP) revealed, for the first time in afungal peroxidase, the presence of a tryptophanyl radical in boththe two-electron (VPI) and the one-electron (VPII) activatedforms of the enzyme. Site-directed mutagenesis was used to substitutethis tryptophan (Trp-164) by tyrosine and histidine residues.Nochanges in the crystal structure were observed, indicatingthat the modified behavior was due exclusively to themutations introduced. EPR revealed the formation of tyrosylradicals in both VPI and VPII of the W164Y variant. However,no protein radical was detected in the W164H variant, whoseVPI spectrum indicated a porphyrin radical identical to that ofthe inactive W164S variant. Stopped-flow spectrophotometryshowed that the W164Y mutation reduced 10-fold the apparentsecond-order rate constant for VPI reduction (k2app) by veratrylalcohol (VA), when compared with over 50-fold reduction inW164S, revealing some catalytic activity of the tyrosine radical.Its first-order rate constant (k2) was more affected than the dissociationconstant (KD2). Moreover, VPII reduction by VA wasimpaired by the above mutations, revealing that the Trp-164radical was involved in catalysis by both VPI and VPII. The lowfirst-order rate constant (k3) values were similar for the W164Y,W164H, and W164S variants, indicating that the tyrosyl radicalin VPII was not able to oxidize VA (in contrast with thatobserved for VPI). VPII self-reduction was also suppressed,revealing that Trp-164 is involved in this autocatalytic process.
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