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M005811200v1
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Papers In Press, published online ahead of print August 23, 2000
J. Biol. Chem, 10.1074/jbc.M005811200
Submitted on July 3, 2000
Revised on August 4, 2000
Accepted on August 23, 2000

An Ala245Thr Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative substates

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

Pharmaceutical Chemistry, School of Pharmacy, S-926, University of California, San Francisco, San Francisco, CA 94143-0446

Corresponding Author: ortiz{at}cgl.ucsf.edu

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-, 11a-, 12-, and 16a-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.


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