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Originally published In Press as doi:10.1074/jbc.M005811200 on August 23, 2000

J. Biol. Chem., Vol. 275, Issue 46, 35999-36006, November 17, 2000
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An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates*

Hong Xiang, Richard A. Tschirret-Guth, and Paul R. Ortiz de MontellanoDagger

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

Cytochrome P450eryF (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450eryF by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala245 by a threonine enables the oxidation of alternative substrates using either H2O2 or O2/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11alpha -, 12-, and 16alpha -hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450eryF-androstenedione complex. P450eryF is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450eryF mutants are also attractive templates for the engineering of novel P450 activities.

* This work was supported by National Institutes of Health Grant GM25515.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.

Dagger To whom correspondence should be addressed: School of Pharmacy, S-926, University of California, San Francisco, CA 94143-0446. Fax: 415-502-4728; E-mail ortiz@cgl.ucsf.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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