Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway.
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-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-alpha1-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 PglC, the glycosyl-1-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 PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC 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 PglC 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 PglC with the latter glycosyltransferases (PglA, PglJ, PglH, and PglI) allows for the "one-pot" chemoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor.
Glover
Kerney Jebrell
KJ
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Weerapana
Eranthie
E
Chen
Mark M
MM
Imperiali
Barbara
B
D016123:Campylobacter jejuni / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002384:Catalysis / Q000187:drug effects; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D015870:Gene Expression; D006031:Glycosylation; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000737:chemistry / Q000378:metabolism*
D003902:Detergents; D011134:Polysaccharides; C510546:UDP-bacillosamine; D014539:Uridine Diphosphate Sugars; D010770:Phosphotransferases