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Applied and Environmental Microbiology, May 2008, p. 2873-2881, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02080-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Takahisa Tsukihara, Yoichi Honda,* Ryota Sakai, Takahito Watanabe, and Takashi Watanabe

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

Received 12 September 2007/ Accepted 27 February 2008

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 Mn2+ and H2O2 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.

* Corresponding author. Mailing address: Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan. Phone: 81 774 38 3643. Fax: 81 774 38 3681. E-mail: yhonda{at}rish.kyoto-u.ac.jp

{triangledown} Published ahead of print on 7 March 2008.

Applied and Environmental Microbiology, May 2008, p. 2873-2881, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02080-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ruiz-Duenas, F. J., Morales, M., Garcia, E., Miki, Y., Martinez, M. J., Martinez, A. T. (2009). Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases. J Exp Bot 60: 441-452 [Abstract] [Full Text]  


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