The O antigens of outer membrane-bound lipopolysaccharides (LPS) in Gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-β 1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid (GlcNAc-PP-PhU) was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by HPLC and analyzed by NMR. The spectra showed the expected Glc-β 1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require divalent cation (Mn(2+) or Mg(2+)) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

O-antigen, gene cluster, Glucosyltransferases, enzymatic syntheses, Escherichia coli O56, Escherichia coli O152

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
0,0,3	bDGlcp	104.7	74.5	77.5	71.0	77.8	62.1
0,0,2	Ac
0,0	?DGlcpN	95.8	53.8	82.9	70.4	74.8	62.2
0	P
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
0,0,3	bDGlcp	4.41	3.18	3.37	3.33	3.33	3.67-3.89
0,0,2	Ac
0,0	?DGlcpN	5.54	4.14	3.86	3.42	4.00	3.66
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	104.7/4.41	74.5/3.18	77.5/3.37	71.0/3.33	77.8/3.33	62.1/3.67-3.89
0,0,2	Ac
0,0	?DGlcpN	95.8/5.54	53.8/4.14	82.9/3.86	70.4/3.42	74.8/4.00	62.2/3.66
0	P
	P

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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