| Autor: |
Moriwaki Y; The Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan., Yato M, Terada T; The Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan., Saito S; Department of Medical Management and Informatics , Hokkaido Information University , 59-2, Nishi Nopporo , Ebetsu, Hokkaido 069-8585 , Japan.; Genaris, Inc. , 75-1 Ono-cho , Tsurumi-ku, Yokohama , Kanagawa 230-0046 , Japan., Nukui N; Genaris, Inc. , 75-1 Ono-cho , Tsurumi-ku, Yokohama , Kanagawa 230-0046 , Japan., Iwasaki T; Genaris, Inc. , 75-1 Ono-cho , Tsurumi-ku, Yokohama , Kanagawa 230-0046 , Japan., Nishi T; Genaris, Inc. , 75-1 Ono-cho , Tsurumi-ku, Yokohama , Kanagawa 230-0046 , Japan., Kawaguchi Y; Department of Biochemistry and Molecular Biology , Nippon Medical School , 1-1-5 Sendagi , Bunkyo-Ku, Tokyo 113-8602 , Japan., Okamoto K; Department of Biochemistry and Molecular Biology , Nippon Medical School , 1-1-5 Sendagi , Bunkyo-Ku, Tokyo 113-8602 , Japan., Arakawa T; The Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan., Yamada C; The Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan., Fushinobu S; The Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan., Shimizu K; The Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan. |
| Abstrakt: |
p -Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of p -hydroxybenzoate ( p -OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to p -OHB; however, hydroxylation does not occur on 3,4-DOHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. In this study, we report how the L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3-fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate by producing H 2 O 2 . To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining the crystal structure of the Y385F·3,4-DOHB complex. The simulations showed that the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-DOHB, which is necessary for initiating hydroxylation. Moreover, the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB. Overall, this study provides a consistent explanation for the change in the catalytic activity of PHBH caused by mutations, which will enable us to better design an enzyme with different activities. |
