期刊
BIOCHEMISTRY
卷 45, 期 16, 页码 5343-5350出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi0602056
关键词
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资金
- NIGMS NIH HHS [GM65699, GM039334] Funding Source: Medline
Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-alpha 1,4-GaINAc-alpha 1,4-(Glc beta 1,3)-GalNAc-alpha 1,4-GalNAc-alpha 1,4-GaINAc-alpha 1,3-Bac-alpha 1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PgIC, the glycosyl-I-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked heptasaccharide donor. In addition, we report the first synthetic route to uridine 5'diphosphobacillosamine. Using the uridine 5'-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PgIC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PgIC does not accept uridine 5'-diphospho-N-acetylglucosamine or uridine 5'-diphospho-N-acetylgalactosamine as substrates but will accept uridine 5'-diphospho-6-hydroxybacillosamine, an analogue of bacillosamine that retains the C-6 hydroxyl functionality from the biosynthetic precursor. The in vitro characterization of PgIC as a bacillosamine 1-phosphoryl transferase provides direct evidence for the early steps in the C. jejuni N-linked glycosylation pathway, and the coupling of PgIC with the latter glycosyltransferases (PgIA, PgIJ, PgIH, and PglI) allows for the one-pot chernoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor.
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