ArticleApplied microbiology and biotechnology2025
Enzyme engineering of cytochrome P450 RosC provides mechanistic insights into factors controlling iterative oxidation.
Article in Applied microbiology and biotechnology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Cytochrome P450 enzymes capable of performing iterative oxidation at the same substrate site contribute to compound diversification; however, reaction control is also necessary for the efficient production of the desired compound. RosC, a cytochrome P450 enzyme involved in the biosynthesis of the 16-membered ring macrolide antibiotic rosamicin, catalyzes stepwise oxidation of the ethyl group at C-20 via hydroxylation to an alcohol, followed by successive oxidation to the corresponding aldehyde and carboxylic acid. The P107S/L176Q mutant produces the hydroxylated intermediate in the first oxidation step, but the efficiency of the subsequent conversion to aldehyde and carboxylic acid is significantly reduced. To elucidate the factors responsible for the reduced efficiency of the second and subsequent oxidation steps in the P107S/L176Q mutant and to understand how RosC facilitates multistep oxidative modification, we compared the reaction time courses and substrate-binding affinities of RosC and the P107S/L176Q mutant. The mutant exhibited a reduced reaction rate for the initial hydroxylation and showed reduced substrate-binding affinities for both the hydroxylated and aldehydic intermediates. Furthermore, crystallographic analysis revealed that Leu-176 played a key role in binding to the desosamine moiety of the substrate, and its mutation resulted in the loss of this function. Ser-248 was presumed to play a role in re-anchoring the modification site to the active center through hydrogen bonding with the hydroxyl and aldehyde groups generated in the first and second reactions. These findings are expected to contribute to the multifunctionalization of cytochrome P450 enzymes and the regulation of their reactivity. KEY POINTS: • RosC-catalyzed three-step oxidation is limited to one step in the P107S/L176Q mutant. • P107S/L176Q mutations impair substrate binding by increasing structural fluctuations. • Leu-176 and Ser-248 position the substrate for RosC-catalyzed iterative oxidation.
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