Mechanism for Oxidation of High-Molecular-Weight Substrates by a Fungal Versatile Peroxidase, MnP2

Abstract

Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn 2؉ and H 2 O 2 were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.Pleurotus ostreatus, known as the oyster mushroom, is a white rot basidiomycete that degrades plant cell wall lignin effectively (33). It secrets a series of isozymes of extracellular oxidizing enzymes belonging to manganese peroxidase (MnP)

Bioremediation of Direct Blue 14 and Extracellular Ligninolytic Enzyme Production by White Rot Fungi:PleurotusSpp.

“…The laccase [16] and MnP [25] play a major role in complete oxidation of DB14. It was observed that laccase and peroxidases can act as starters of a chain reaction which leads to dye degradation by generating highly active free radicals (e.g., Mn 3+ , lipid, hydroxyl, and peroxy-radicals) [26, 27].…”

EPR and LC-MS studies on the mechanism of industrial dye decolorization by versatile peroxidase from Bjerkandera adusta

“…VP shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn(II) and high redox potential aromatic compounds, respectively (Tsukihara et al 2008;Ruiz-Dueñas et al 2009). This results suggest that the dye substrate interacts with a different enzyme active site that do not compete with Mn(II).…”

Cloning and Homologous Expression of Novel Lignin Peroxidase Genes in the White-Rot FungusPhanerochaete sordidaYK-624

“…Homologous expression of several lignin degrading peroxidases has been reported in some fungi, especially P. chrysosporium 31,32) and Pleurotus ostreatus. 33,41) This expression system can be used to investigate the structure and function these enzymes, especially high oxidation activities against lignin model compounds, by site-directed mutagenesis. Moreover, molecular breeding of the superior lignin degrading fungi applied in the pretreatment of wood in the production of bio-ethanol is possible using this homologous expression system, since P. sordida YK-624 degrades lignin more effectively and selectively than P. chrysosporium or T. versicolor.…”

“…[31][32][33] In our previous report, a gene transformation system for P. sordida YK-624 was constructed, and expression of the enhanced green fluorescent protein (EGFP) was successful. 34) Development of a homologous gene expression system for this fungus is important to elucidate the reason it shows high lignin-degrading activity and selectivity.…”