Predictive model for epoxide hydrolase-generated stereochemistry in the biosynthesis of nine-membered enediyne antitumor antibiotics

TitlePredictive model for epoxide hydrolase-generated stereochemistry in the biosynthesis of nine-membered enediyne antitumor antibiotics
Publication TypeJournal Article
Year of Publication2013
AuthorsHorsman G.P, Lechner A., Ohnish Y., Moore BS, Shen B
JournalBiochemistry
Volume52
Pagination5217-5224
Date Published2013/08
Type of ArticleArticle
ISBN Number0006-2960
Accession NumberWOS:000323013700010
Keywordsc-1027; chemistry; chemotherapy; core; divergence; gene-cluster; insights; sequence; synthase
Abstract

Nine-membered enediyne antitumor antibiotics C-1027, neocarzinostatin (NCS), and kedarcidin (KED) possess enediyne cores to which activity-modulating peripheral moieties are attached via (R)- or (S)-vicinal diols. We have previously shown that this stereochemical difference arises from hydrolysis of epoxide precursors by epoxide hydrolases (EHs) with different regioselectivities. The inverting EHs, such as SgcF, hydrolyze an (S)-epoxide substrate to yield an (R)-diol in C-1027 biosynthesis, whereas the retaining EHs, such as NcsF2 and KedF, hydrolyze an (S)-epoxide substrate to yield an (S)-diol in NCS and KED biosynthesis. We now report the characterization of a series of EH mutants and provide a predictive model for EH regioselectivity in the biosynthesis of the nine-membered enediyne antitumor antibiotics. A W236Y mutation in SgcF increased the retaining activity toward (S)-styrene oxide by 3-fold, and a W236Y/Q237M double mutation in SgcF, mimicking NcsF2 and KedF, resulted in a 20-fold increase in the retaining activity. To test the predictive utility of these mutations, two putative enediyne biosynthesis-associated EHs were identified by genome mining and confirmed as inverting enzymes, SpoF from Salinospora tropica CNB-440 and SgrF (SGR_625) from Streptomyces griseus IFO 13350. Finally, phylogenetic analysis of EHs revealed a familial classification according to inverting versus retaining activity. Taken together, these results provide a predictive model for vicinal diol stereochemistry in enediyne biosynthesis and set the stage for further elucidating the origins of EH regioselectivity.

DOI10.1021/bi400572a
Short TitleBiochemistry
Integrated Research Themes: 
Student Publication: 
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