3WVS

Crystal Structure of Cytochrome P450revI

Summary for 3WVS

• Entry DOI: 10.2210/pdb3wvs/pdb
• Descriptor: Putative monooxygenase, PROTOPORPHYRIN IX CONTAINING FE, (2E,4S,5S,6E,8E)-10-{(2R,3S,6S,8R,9S)-9-butyl-8-[(1E,3E)-4-carboxy-3-methylbuta-1,3-dien-1-yl]-3-methyl-1,7-dioxaspiro[5.5]undec-2-yl}-5-hydroxy-4,8-dimethyldeca-2,6,8-trienoic acid, ... (6 entities in total)
• Functional Keywords: monooxygenase, oxidoreductase
• Biological source: Streptomyces
• Total number of polymer chains: 1
• Total formula weight: 46523.76

Authors

Nagano, S.,Takahashi, S.,Osada, H.,Shiro, Y. (deposition date: 2014-06-06, release date: 2014-10-01, Last modification date: 2024-05-29)

Primary citation

Takahashi, S.,Nagano, S.,Nogawa, T.,Kanoh, N.,Uramoto, M.,Kawatani, M.,Shimizu, T.,Miyazawa, T.,Shiro, Y.,Osada, H.
Structure-function analyses of cytochrome P450revI involved in reveromycin A biosynthesis and evaluation of the biological activity of its substrate, reveromycin T.
J.Biol.Chem., 289:32446-32458, 2014

PubMed Abstract: 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. R190A and R81A 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 Arg(190) 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.

PubMed: 25258320
DOI: 10.1074/jbc.M114.598391

Experimental method

X-RAY DIFFRACTION (1.4 Å)