Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein

metabolism, glycan, legionaminic acid, modeling, Flagellin, Campylobacter coli

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
5,0,5	Subst	17.9	167.2	30.4
5,0,7	Ac	175.1	22.5
5,0	bXLegp	?	100.6	41.3	67.9	58.4	74.4	52.6	69.8	19.5
5	P
	xXnucC


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
5,0,5	Subst	2.28	2.98
5,0,7	Ac	-	2.13
5,0	bXLegp	-	-	1.71-2.55	4.14	3.50	4.54	3.99	4.20	1.13
5	P
	xXnucC


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
5,0,5	Subst	NMR TSV error 2: unequal length of 13C and 1H datasets
5,0,7	Ac		22.5/2.13
5,0	bXLegp			41.3/1.71-2.55	67.9/4.14	58.4/3.50	74.4/4.54	52.6/3.99	69.8/4.20	19.5/1.13
5	P
	xXnucC

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

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CN=C(C)N[C@@H]1[C@@H](O)C[C@@](OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(N)nc3=O)[C@H](O)[C@@H]2O)(C(=O)O)O[C@H]1[C@H](NC(C)=O)[C@@H](C)O

652.551 g/mol (C₂₃H₃₇N₆O₁₄P)