title: Structure-function analyses of cytochrome P450revI involved in reveromycin A biosynthesis and evaluation of the biological activity of its substrate, reveromycin T.
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.
authors.affiliation: From the Chemical Biology Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan, the Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan, shunjitaka@riken.jp.
authors.forename: Shunji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: the Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori 680-8582, Japan, the Biometal Science Laboratory, RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan, and snagano@bio.tottori-u.ac.jp.
authors.forename: Shingo
authors.initials: S
authors.lastname: Nagano
authors.affiliation: From the Chemical Biology Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Nogawa
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 980-8578, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kanoh
authors.affiliation: the Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Uramoto
authors.affiliation: the Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kawatani
authors.affiliation: the Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: the Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyazawa
authors.affiliation: the Biometal Science Laboratory, RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan, and.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Shiro
authors.affiliation: From the Chemical Biology Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan, the Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
mesh_terms: D000818:Animals; D055162:Biocatalysis; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D006900:Hydroxylation; D007533:Isoleucine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism; D020014:K562 Cells; D007700:Kinetics; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011714:Pyrans / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013141:Spiro Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D011714:Pyrans; D013141:Spiro Compounds; C068098:reveromycin A; D003577:Cytochrome P-450 Enzyme System; C000595190:cytochrome P-450 107E6 protein, human; D007533:Isoleucine-tRNA Ligase
keywords: Biosynthesis; Crystallography; Cytochrome P450; Enzyme Catalysis; Polyketide; Reveromycin; Secondary Metabolite; Streptomyces