The K17 capsular polysaccharide (CPS) produced by Acinetobacter baumannii G7, which carries the KL17 configuration at the capsule biosynthesis locus, was isolated and studied by chemical methods along with one- and two-dimensional 1H and 13C NMR spectroscopy. Selective cleavage of the glycosidic linkage of a 2,4-diacetamido-2,4,6-trideoxy-D-glucose (D-QuiNAc4NAc) residue by (i) trifluoroacetic acid solvolysis or (ii) alkaline β-elimination (NaOH-NaBH4) of the 4-linked D-alanine amide of a 2-acetamido-2-deoxy-D-galacturonic acid residue (d-GalNAcA6DAla) yielded trisaccharides that were isolated by Fractogel TSK HW-40 gel-permeation chromatography and identified by using NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The following structure was established for the trisaccharide repeat (K unit) of the CPS: →4)-α-D-GalpNAcA6DAla-(1→4)-α-D-GalpNAcA-(1→3)-β-D-QuipNAc4NAc-(1→. The presence of the itrA1 gene coding for the initial glycosylphosphotransferase in the KL17 gene cluster established the first sugar of the K unit as D-QuipNAc4NAc. KL17 includes genes for three transferases that had been annotated previously as glycosyltransferases (Gtrs). As only two Gtrs are required for the K17 structure and one D-GalpNAcA residue is modified by a D-alanine amide, these assignments were re-assessed. One transferase was found to belong to the ATPgrasp_TupA protein family that includes D-alanine-D-alanine ligases, and thus was renamed Alt1 (alanine transferase). Alt1 represents a novel family that amidate the carboxyl group of D-GalpNAcA or D-GalpA.

Acinetobacter baumannii, capsular polysaccharide, D-alanine, K locus, KL17

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,6	xDAla?	178.4	50.4	18.2
3,4,2	Ac	175.1-176.1	23.2-23.8
3,4	aDGalpNA	98.6-98.9	50.4	67.9	79.7-79.8	72.5	174.4
3,2	Ac	175.1-176.1	23.2-23.8
3	aDGalpNA	100.2	50.8	68.8	70.3	73.0	171.8
2	Ac	175.1-176.1	23.2-23.8
4	Ac	175.1-176.1	23.2-23.8
	bDQuipN4N	95.9	57.4	77.0	58.3-58.5	72.6	18.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,6	xDAla?	-	4.37	1.39
3,4,2	Ac	-	1.93-2.08
3,4	aDGalpNA	5.08	4.22	4.01	4.30	4.77	-
3,2	Ac	-	1.93-2.08
3	aDGalpNA	5.09	4.16	3.88	4.38	4.19	-
2	Ac	-	1.93-2.08
4	Ac	-	1.93-2.08
	bDQuipN4N	4.70	3.72	3.81	3.77	3.52	1.16


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,6	xDAla?		50.4/4.37	18.2/1.39
3,4,2	Ac		23.2-23.8/1.93-2.08
3,4	aDGalpNA	98.6-98.9/5.08	50.4/4.22	67.9/4.01	79.7-79.8/4.30	72.5/4.77	
3,2	Ac		23.2-23.8/1.93-2.08
3	aDGalpNA	100.2/5.09	50.8/4.16	68.8/3.88	70.3/4.38	73.0/4.19	
2	Ac		23.2-23.8/1.93-2.08
4	Ac		23.2-23.8/1.93-2.08
	bDQuipN4N	95.9/4.70	57.4/3.72	77.0/3.81	58.3-58.5/3.77	72.6/3.52	18.0/1.16

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@H]2O[C@H](C(=O)O)[C@H](O[C@H]3O[C@H](C(=O)N[C@H](C)C(=O)O)[C@H](O)[C@H](O)[C@H]3NC(C)=O)[C@H](O)[C@H]2NC(C)=O)[C@@H](NC(C)=O)[C@H](O)O[C@@H]1C

751.696 g/mol (C₂₉H₄₅N₅O₁₈)