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Characterization of the SgcF Epoxide Hydrolase Supporting an ( R )-Vicinal Diol Intermediate for Enediyne Antitumor Antibiotic C-1027 Biosynthesis

2009; American Chemical Society; Volume: 131; Issue: 45 Linguagem: Inglês

10.1021/ja901242s

1943-2984

Shuangjun Lin, Geoff P. Horsman, Yihua Chen, Wenli Li, Ben Shen,

Microbial Natural Products and Biosynthesis

C-1027 is a chromoprotein antitumor antibiotic consisting of an apoprotein and the C-1027 chromophore. The C-1027 chromophore possesses four distinct structural moieties—an enediyne core, a deoxy aminosugar, a benzoxazolinate, and an (S)-3-chloro-5-hydroxy-β-tyrosine—the latter two of which are proposed to be appended to the enediyne core via a convergent biosynthetic strategy. Here we report the in vitro characterization of SgcF, an epoxide hydrolase from the C-1027 biosynthetic gene cluster that catalyzes regio- and stereospecific hydrolysis of styrene oxide, serving as an enediyne core epoxide intermediate mimic, to form a vicinal diol. Abolishment of C-1027 production in the ΔsgcF mutant strain Streptomyces globisporus SB1010 unambiguously establishes that sgcF plays an indispensable role in C-1027 biosynthesis. SgcF efficiently hydrolyzes (S)-styrene oxide, displaying an apparent Km of 0.6 ± 0.1 mM and kcat of 48 ± 1 min−1, via attack at the α-position to exclusively generate the (R)-phenyl vicinal diol, consistent with the stereochemistry of the C-1027 chromophore. These findings support the role of SgcF in the proposed convergent pathway for C-1027 biosynthesis, unveiling an (R)-vicinal diol as a key intermediate. Interestingly, SgcF can also hydrolyze (R)-styrene oxide to afford preferentially the (R)-phenyl vicinal diol via attack at the β-position, albeit with significantly reduced efficiency (apparent Km of 2.0 ± 0.4 mM and kcat = 4.3 ± 0.3 min−1). Although the latter activity unlikely contributes to C-1027 biosynthesis in vivo, such enantioconvergence arising from complementary regioselective hydrolysis of a racemic substrate could be exploited to engineer epoxide hydrolases with improved regio- and/or enantiospecificity.