Structure-Function Analyses of Cytochrome P450revI Involved in Reveromycin A Biosynthesis and Evaluation of the Biological Activity of Its Substrate, Reveromycin T [Enzymology]

September 25th, 2014 by Takahashi, S., Nagano, S., Nogawa, T., Kanoh, N., Uramoto, M., Kawatani, M., Shimizu, T., Miyazawa, T., Shiro, Y., Osada, H.

Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4-Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. Arg190Ala and Arg81Ala mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg190 and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM-derivatives with anti-osteoclast activity.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Structure-Function Analyses of Cytochrome P450revI Involved in Reveromycin A Biosynthesis and Evaluation of the Biological Activity of Its Substrate, Reveromycin T [Enzymology]