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J. Biol. Chem., Vol. 275, Issue 46, 35999-36006, November 17, 2000

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An A245T Mutation Conveys on Cytochrome P450_eryF the Ability to Oxidize Alternative Substrates^*

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

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

Cytochrome P450_eryF (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 P450_eryF 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-, 11-, 12-, and 16-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 P450_eryF-androstenedione complex. P450_eryF 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 P450_eryF mutants are also attractive templates for the engineering of novel P450 activities.

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