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Originally published In Press as doi:10.1074/jbc.M211575200 on January 7, 2003

J. Biol. Chem., Vol. 278, Issue 11, 9761-9767, March 14, 2003
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Substrate Recognition and Molecular Mechanism of Fatty Acid Hydroxylation by Cytochrome P450 from Bacillus subtilis
CRYSTALLOGRAPHIC, SPECTROSCOPIC, AND MUTATIONAL STUDIES*

Dong-Sun LeeDagger , Akari YamadaDagger , Hiroshi SugimotoDagger , Isamu Matsunaga§, Hisashi Ogura§, Kosuke Ichihara, Shin-ichi AdachiDagger , Sam-Yong ParkDagger ||, and Yoshitsugu ShiroDagger **

From the Dagger  RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, the § Department of Virology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan, and the  Osaka Seiki Women's College, 3-10-62 Aikawa, Higashiyodogawa-ku, Osaka 533-0007, Japan

Cytochrome P450 isolated from Bacillus subtilis (P450BSbeta ; molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the alpha - and beta -positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450BSbeta in the substrate-bound form, determined at a resolution of 2.1 Å. P450BSbeta exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg242 guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the alpha - and beta -positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg242 in the same fashion as has been reported for the active site of chloroperoxidase, His105-Glu183, which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450BSbeta is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.

* This work was supported in part by grants from the Structural Biology and the Molecular Ensemble Programs in RIKEN (to Y. S.) and Grant-in-aids from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to I. M. and Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1IZO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). .

|| Present address: Division of Protein Design, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama, 230-0045, Japan.

** To whom correspondence should be addressed. E-mail: yshiro@ mailman.riken.go.jp.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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