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. 2017 Jan 1;8(1):444-451.
doi: 10.1039/c6sc01826b. Epub 2016 Aug 19.

Construction of an octosyl acid backbone catalyzed by a radical S-adenosylmethionine enzyme and a phosphatase in the biosynthesis of high-carbon sugar nucleoside antibiotics

Nisha He  1   2   3 , Pan Wu  1 , Yongxing Lei  2   3 , Baofu Xu  2   3 , Xiaochen Zhu  2 , Gudan Xu  1 , Yaojie Gao  1 , Jianzhao Qi  1 , Zixin Deng  1 , Gongli Tang  4 , Wenqing Chen  1 , Youli Xiao  2
Affiliations

Construction of an octosyl acid backbone catalyzed by a radical S-adenosylmethionine enzyme and a phosphatase in the biosynthesis of high-carbon sugar nucleoside antibiotics

Nisha He et al. Chem Sci. .

Abstract

Unique bicyclic octosyl uronic acid nucleosides include ezomycin, malayamycin, and octosyl acid (OA). They are structurally characterized by OA, an unusual 8-carbon furanosyl nucleoside core proposed to be the precursor to polyoxin and nikkomycin. Despite the well-known bioactivity of these nucleoside antibiotics, the biosynthesis of OA has not been elucidated yet. Here we report the two pivotal enzymatic steps in the polyoxin biosynthetic pathway leading to the identification of OA as a key intermediate. Our data suggest that this intermediate is formed via a free radical reaction catalyzed by the radical S-adenosylmethionine (SAM) enzyme, PolH, and using 3'-enolpyruvyl uridine 5'-monophosphate (3'-EUMP) as a substrate. Subsequent dephosphorylation catalyzed by phosphatase PolJ converts the resulting octosyl acid 5'-phosphate (OAP) to OA. These results provide, for the first time, significant in vitro evidence for the biosynthetic origins of the C8 backbone of OA.

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Figures

Fig. 1
Fig. 1. Selected examples of nucleoside antibiotics containing or originating from the octosyl acid (OA) scaffold.
Scheme 1
Scheme 1. Proposed or characterized biosynthetic pathway for building the high-carbon sugar nucleoside skeleton.
Fig. 2
Fig. 2. Biochemical characterization of PolH. (A) SDS-PAGE of the anaerobically purified PolH; (B) UV-Vis spectra of the as-isolated (blue), reconstituted (red), and partially reduced (green) PolH; (C) EPR spectrum of reconstituted PolH reduced by sodium dithionite.
Fig. 3
Fig. 3. Characterization of the reaction of 3′-EUMP catalyzed by reconstituted PolH. (A) HPLC analysis of the activity assay (monitoring at 260 nm). (1) PolH with 3′-EUMP, SAM and dithionite; (2) without SAM; (3) with denatured PolH; (4) 3′-EUMP standard; (5) 5′-dA standard; (6) SAM standard; (7) purified PolH reaction product (OAP); (8) co-injection of OAP with 3′-EUMP; (B) ESI-MS/MS (78.957, 96.967, 111.018, 295.056, 350.028, and 393.034 (from left to right)); (C) 1H-NMR of the purified product (OAP) from the PolH reaction; (D) the reaction scheme of PolH. *The impurities from background reactions in the HPLC assay or contamination from glycerol in the 1H-NMR sample.
Fig. 4
Fig. 4. Characterization of the reaction of 3′-EUMP catalyzed by reconstituted PolH in 60% D2O containing buffer. (A) Reaction scheme; (B) LC-MS; (C) ESI-MS/MS (78.957, 96.967, 111.018, 295.057, 296.062, 350.028, 351.033, 393.034, and 394.039 (from left to right)).
Scheme 2
Scheme 2. The proposed reaction mechanism catalyzed by PolH.
Scheme 3
Scheme 3. The proposed biosynthetic pathway for polyoxin. R = H, CH3, CH2OH or COOH.
Fig. 5
Fig. 5. Target-directed genome mining of the potential nucleoside antibiotics containing AHA using PolH and PolJ as probes. The genome sequence data was obtained from GenBank, and the related ORFs associated with aminohexuronic acid (uracil polyoxin C) are linked together and probably encode potential nucleoside antibiotics related to polyoxin and nikkomycin.
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