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pubmed:abstractText |
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 Ala(245) by a threonine enables the oxidation of alternative substrates using either H(2)O(2) or O(2)/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 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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