The waaJ, waaT, and waaR genes encode a-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose:(galactosyl) LPS a-1,2-glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS a-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS a-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the enzyme's natural LPS acceptor. Although each enzyme could utilize the alternate donor epimer, activity was compromised due to significant decreases in k(cat) and corresponding increases in K(M(donor)). Finally, in vitro assays were performed to probe acceptor preference in the absence of cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His(6)-WaaT, His(6)-WaaJ showed a significantly decreased kcat and increased K(M(acceptor)). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for for glycoengineering applications

biosynthesis, Escherichia coli, core oligosaccharide, glycosyltransferases, lipopolysaccharide structure

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,2,2	aDGlcp	97.3	72.9	74.6	70.8	73.5	61.8
3,2	aDGlcp	92.8	76.5	72.9	71.3	73.6	62.5
3,3,2	Ac
3,3	aDGlcpN	93.9	54.9	72.5	71.6	73.6	62.1
3	aDGalp	95.5	69.5	72.2	65.9	71.5	62.2
	aDGlcp	101.9	71.7	76.2	72.1	73.7	61.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,2,2	aDGlcp	5.21	3.55	3.76	3.44	3.94	3.77-3.88
3,2	aDGlcp	5.52	3.75	3.89	3.47	3.84	3.75
3,3,2	Ac
3,3	aDGlcpN	5.14	3.98	3.82	3.54	4.00	3.83
3	aDGalp	5.91	4.21	4.24	4.26	4.27	3.77-3.95
	aDGlcp	5.28	3.69	4.14	3.84	3.83	3.76-3.89


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,2,2	aDGlcp	97.3/5.21	72.9/3.55	74.6/3.76	70.8/3.44	73.5/3.94	61.8/3.77-3.88
3,2	aDGlcp	92.8/5.52	76.5/3.75	72.9/3.89	71.3/3.47	73.6/3.84	62.5/3.75
3,3,2	Ac
3,3	aDGlcpN	93.9/5.14	54.9/3.98	72.5/3.82	71.6/3.54	73.6/4.00	62.1/3.83
3	aDGalp	95.5/5.91	69.5/4.21	72.2/4.24	65.9/4.26	71.5/4.27	62.2/3.77-3.95
	aDGlcp	101.9/5.28	71.7/3.69	76.2/4.14	72.1/3.84	73.7/3.83	61.3/3.76-3.89

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

869.773 g/mol (C₃₂H₅₅NO₂₆)