In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system.
In Campylobacter jejuni 2,4-diacetamido-2,4,6-trideoxy-alpha-d-glucopyranose, termed N,N'-diacetylbacillosamine (Bac2,4diNAc), is the first carbohydrate in the glycoprotein N-linked heptasaccharide. With uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a starting point, two enzymes of the general protein glycosylation (Pgl) pathway in C. jejuni (PglF and PglE) have recently been shown to modify this sugar nucleotide to form UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-d-glycopyranose (UDP-4-amino-sugar) [Schoenhofen, I. C., et al. (2006) J. Biol. Chem. 281, 723-732]. PglD has been proposed to catalyze the final step in N,N'-diacetylbacillosamine synthesis by N-acetylation of the UDP-4-amino-sugar at the C4 position. We have cloned, overexpressed, and purified PglD from the pgl locus of C. jejuni NCTC 11168 and identified it as the acetyltransferase that modifies the UDP-4-amino-sugar to form UDP-N,N'-diacetylbacillosamine, utilizing acetyl-coenzyme A as the acetyl group donor. The UDP-N,N'-diacetylbacillosamine product was purified from the reaction by reverse phase C18 HPLC and the structure determined by NMR analysis. Additionally, the full-length PglF was overexpressed and purified in the presence of detergent as a GST fusion protein, allowing for derivation of kinetic parameters. We found that the UDP-4-amino-sugar was readily synthesized from UDP-GlcNAc in a coupled reaction using PglF and PglE. We also demonstrate the in vitro biosynthesis of the complete heptasaccharide lipid-linked donor by coupling the action of eight enzymes (PglF, PglE, PglD, PglC, PglA, PglJ, PglH, and PglI) in the Pgl pathway in a single reaction vessel.
Olivier
Nelson B
NB
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Chen
Mark M
MM
Behr
Jonathan R
JR
Imperiali
Barbara
B
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000123:Acetyltransferases / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000096:biosynthesis; D021241:Spectrometry, Mass, Electrospray Ionization
C502751:2,4-diacetamido-2,4,6-trideoxyglucopyranose; D011134:Polysaccharides; D000123:Acetyltransferases; D000117:Acetylglucosamine