Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-α-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-β-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-α-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose.

Campylobacter, NMR spectroscopy, pseudaminic acid, Helicobacter pylori, Substrate Specificity, glycosylation, bacillosamine, Flagellin

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
5,0,0,2	Ac	?	22.9
5,0,0	aDGlcpN	95.1	54.5	71.7	70.3	73.8	61.2
5,0	P
5	P
	xXnucU	89.2	74.6	70.5	83.9	65.6	167.2	152.6	103.4	142.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
5,0,0,2	Ac	-	2.08
5,0,0	aDGlcpN	5.51	3.99	3.81	3.55	3.92	3.82-3.86
5,0	P
5	P
	xXnucU	5.97	4.37	4.36	4.28	4.18-4.23	-	-	5.97	7.95


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
5,0,0,2	Ac		22.9/2.08
5,0,0	aDGlcpN	95.1/5.51	54.5/3.99	71.7/3.81	70.3/3.55	73.8/3.92	61.2/3.82-3.86
5,0	P
5	P
	xXnucU	89.2/5.97	74.6/4.37	70.5/4.36	83.9/4.28	65.6/4.18-4.23			103.4/5.97	142.4/7.95

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O

607.355 g/mol (C₁₇H₂₇N₃O₁₇P₂)