title: 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.
authors.affiliation: Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Tsukihara
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Honda
authors.forename: Ryota
authors.initials: R
authors.lastname: Sakai
authors.forename: Takahito
authors.initials: T
authors.lastname: Watanabe
authors.forename: Takashi
authors.initials: T
authors.lastname: Watanabe
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000880:Anthraquinones / Q000378:metabolism*; D001592:Benzyl Alcohols / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020076:Pleurotus / Q000235:genetics / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000880:Anthraquinones; D001592:Benzyl Alcohols; D004271:DNA, Fungal; D005656:Fungal Proteins; D050505:Mutant Proteins; D011108:Polymers; D008345:Manganese; C017971:poly R-478; D006861:Hydrogen Peroxide; D010544:Peroxidases; C051129:manganese peroxidase; D012259:Ribonuclease, Pancreatic; C042197:veratryl alcohol