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A more recent version of this article appeared on March 7, 2003
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Papers In Press, published online ahead of print January 7, 2003
J. Biol. Chem, 10.1074/jbc.M211575200
Submitted on November 13, 2002
Revised on December 27, 2002
Accepted on January 7, 2003

Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis: Crystallographic, spectroscopic and mutational studies

Dong-Sun Lee, Akari Yamada, Hiroshi Sugimoto, Isamu Matsunaga, Hisashi Ogura, Kosuke Ichihara, Shin-ichi Adachi, Sam-Yong Park, and Yoshitsugu Shiro

RIKEN Harima Institute, Sayo, Hyogo 679-5148

Corresponding Author: yshiro{at}mailman.riken.go.jp

Cytochrome P450 isolated Bacillus subtilis (P450BSb, MW: 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the a- and b-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450BSb in the substrate-bound form, determined at a resolution of 2.1?. P450BSb 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 a- and b-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 P450BSb is proposed, in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.


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