The outer membrane of gram-negative bacteria is stabilized by lipopolysaccharides (LPS). The O-antigenic polysaccharides of LPS are composed of repeating units that are exposed to and can interact with the environment. The glycosyltransferases that assemble these repeating units are encoded by the O-antigen gene cluster and utilize undecaprenol-phosphate-linked intermediates as natural acceptor substrates, and nucleotide sugars as donor substrates on the cytoplasmic face of the inner membrane. Many of the glycosyltransferase genes are known but the enzymatic functions of most of them remain to be identified. We describe here how the function of a recombinant glucosyltransferase WbdN from Escherichia coli O157 can be determined by NMR analysis of the enzyme product, using a synthetic acceptor substrate analog. A fluorescent acceptor substrate analog can be used in highly sensitive enzyme assays that allow the characterization of enzyme activity without the use of radioactive nucleotide sugar donor substrates.

NMR, glucosyltransferase, linkage analysis, WbdN, fluorescent acceptor substrate

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
0,0,3	bDGlcp	104.0	72.9	75.4	69.2	75.4	60.3
0,0,2	Ac
0,0	aDGalpN	94.6	48.3	77.4	68.6	71.6	60.9
0	P
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
0,0,3	bDGlcp	4.48	3.20	3.42	3.33	3.34	3.67-3.75
0,0,2	Ac
0,0	aDGalpN	5.53	4.31	3.98	4.19	4.13	3.65-3.70
0	P
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
0,0,3	bDGlcp	104.0/4.48	72.9/3.20	75.4/3.42	69.2/3.33	75.4/3.34	60.3/3.67-3.75
0,0,2	Ac
0,0	aDGalpN	94.6/5.53	48.3/4.31	77.4/3.98	68.6/4.19	71.6/4.13	60.9/3.65-3.70
0	P
	P

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

543.308 g/mol (C₁₄H₂₇NO₁₇P₂)