With an increase in antibiotic-resistant strains, the nosocomial pathogen Acinetobacter baumannii has become a serious threat to global health. Glycoconjugate vaccines containing fragments of bacterial exopolysaccharide (EPS) are an emerging therapeutic to combat bacterial infection. Herein, we characterize the bacteriophage PhiAB6 tailspike protein (TSP), which specifically hydrolyzed the EPS of A. baumannii strain 54149 (Ab-54149). Ab-54149 EPS exhibited the same chemical structure as two antibiotic-resistant A. baumannii strains. The PhiAB6 TSP-digested products comprised oligosaccharides of two repeat units, typically with stoichiometric pseudaminic acid (Pse). The 1.48-1.89-A resolution crystal structures of an N-terminally-truncated PhiAB6 TSP and its complexes with the semi-hydrolyzed products revealed a trimeric beta-helix architecture that bears intersubunit carbohydrate-binding grooves, with some features unusual to the TSP family. The structures suggest that Pse in the substrate is an important recognition site for PhiAB6 TSP. A region in the carbohydrate-binding groove is identified as the determinant of product specificity. The structures also elucidated a retaining mechanism, for which the catalytic residues were verified by site-directed mutagenesis. Our findings provide a structural basis for engineering the enzyme to produce desired oligosaccharides, which is useful for the development of glycoconjugate vaccines against A. baumannii infection.

Acinetobacter baumannii, pseudaminic acid, crystal structure, bacteriophage, glycoconjugate vaccine, Tailspike

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
3,3,3,6,6,5	Ac
3,3,3,6,6,7	Ac
3,3,3,6,6	bXPsep	?	?	35.4	64.9	48.7	70.0	53.5	67.0	15.7
3,3,3,6	bDGlcp	104.0	73.2	75.3	70.2	74.6	61.1
3,3,3	bDGalp
3,3,2	Ac
3,3	bDGalpN	102.9	51.4	79.6	68.0	74.8	61.1
3,6	bDGlcp	104.2	73.3	75.3	69.4	73.9	61.2
3	bDGalp	104.7	69.3	81.7	68.5	73.7	70.8
2	Ac
	?DGalpN


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
3,3,3,6,6,5	Ac
3,3,3,6,6,7	Ac
3,3,3,6,6	bXPsep	-	-	1.53-2.08	4.19	4.17	3.80	4.13	4.15	1.10
3,3,3,6	bDGlcp	4.41	3.23	3.42	3.50	3.64	3.60-3.72
3,3,3	bDGalp
3,3,2	Ac
3,3	bDGalpN	4.63	3.98	3.83	4.09	3.63	3.71
3,6	bDGlcp	4.39	3.21	3.41	3.38	3.46	3.48-3.62
3	bDGalp	4.46	3.52	3.66	4.10	3.81	3.83-3.93
2	Ac
	?DGalpN


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
3,3,3,6,6,5	Ac
3,3,3,6,6,7	Ac
3,3,3,6,6	bXPsep			35.4/1.53-2.08	64.9/4.19	48.7/4.17	70.0/3.80	53.5/4.13	67.0/4.15	15.7/1.10
3,3,3,6	bDGlcp	104.0/4.41	73.2/3.23	75.3/3.42	70.2/3.50	74.6/3.64	61.1/3.60-3.72
3,3,3	bDGalp
3,3,2	Ac
3,3	bDGalpN	102.9/4.63	51.4/3.98	79.6/3.83	68.0/4.09	74.8/3.63	61.1/3.71
3,6	bDGlcp	104.2/4.39	73.3/3.21	75.3/3.41	69.4/3.38	73.9/3.46	61.2/3.48-3.62
3	bDGalp	104.7/4.46	69.3/3.52	81.7/3.66	68.5/4.10	73.7/3.81	70.8/3.83-3.93
2	Ac
	?DGalpN

The spectrum also has 2 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]([C@H](C)O)[C@@H]1O[C@@](OC[C@H]2O[C@@H](OC[C@H]3O[C@@H](O[C@@H]4[C@@H](NC(C)=O)[C@H](O[C@H]5[C@@H](O)[C@@H](CO[C@@H]6O[C@H](CO)[C@@H](O)[C@H](O)[C@H]6O)O[C@@H](O[C@@H]6[C@@H](NC(C)=O)C(O)O[C@H](CO)[C@@H]6O)[C@@H]5O)O[C@H](CO)[C@@H]4O)[C@H](O)[C@@H](O)[C@H]3O)[C@H](O)[C@@H](O)[C@@H]2O)(C(=O)O)C[C@H](O)[C@@H]1NC(C)=O

1389.277 g/mol (C₅₃H₈₈N₄O₃₈)