Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2

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Takahisa Tsukihara1, Yoichi Honda, Ryota Sakai

  • 1Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan.

Applied and Environmental Microbiology
|March 11, 2008
PubMed

Insights

Pleurotus ostreatus MnP2 directly oxidizes high-molecular-weight compounds via tryptophan 170. Mutations near this site affect substrate access, revealing the mechanism for fungal peroxidase action on polymers.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Fungal peroxidases, like Pleurotus ostreatus MnP2, typically oxidize small aromatic compounds.
  • MnP2 exhibits a unique ability to directly oxidize high-molecular-weight substrates, such as Poly R-478 and RNase A.
  • Understanding the mechanism of this direct oxidation is crucial for applications in bioremediation and biocatalysis.

Purpose of the Study:

  • To elucidate the mechanism by which Pleurotus ostreatus MnP2 directly oxidizes high-molecular-weight polymeric substrates.
  • To identify key amino acid residues involved in the direct oxidation of polymers.
  • To investigate the role of protein structure and post-translational modifications in MnP2 activity.

Main Methods:

  • Site-directed mutagenesis of Pleurotus ostreatus MnP2 using a homologous gene expression system.
  • Characterization of mutant enzyme reactivity towards veratryl alcohol, Poly R-478, and RNase A.
  • Kinetic analysis of Mn(2+) and H(2)O(2) interactions.
  • Three-dimensional modeling of mutant enzymes.
  • Analysis of protein modifications, including N-glycosylation.

Main Results:

  • A W170A mutant showed drastically reduced oxidation activity for both veratryl alcohol and high-molecular-weight substrates, indicating W170's direct role.
  • Mutations near W170 (Q266F, V166/168L) decreased activity specifically for high-molecular-weight substrates, suggesting steric hindrance.
  • An R263N mutant with added N-glycosylation exhibited increased reactivity towards both veratryl alcohol and high-molecular-weight substrates.
  • This study provides the first direct mechanistic insight into high-molecular-weight substrate oxidation by a fungal peroxidase using a homologous expression system.

Conclusions:

  • Tryptophan 170 (W170) is directly involved in the oxidation of both veratryl alcohol and high-molecular-weight substrates by Pleurotus ostreatus MnP2.
  • Steric factors around W170 influence the accessibility of polymeric substrates.
  • Additional N-glycosylation can enhance the catalytic efficiency of MnP2.
  • The findings offer a deeper understanding of fungal peroxidase mechanisms for degrading complex substrates.

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