Functional characterization and membrane topology of Escherichia coli WecA, a glycosyltransferase initiating the biosynthesis of enterobacterial common antigen and O antigen lipopolysaccharide

Infectious Diseases Research Group, Siebens-Drake Medical Research Institute, Department of Microbiology and Immunology, and Medicine, University of Western Ontario, London, ON, N6A 5C1, Canada

^* To whom correspondence should be addressed. Email: mvalvano{at}uwo.ca.

	Abstract

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O antigen LPS synthesis in a wecA-deficient strain and also their steady-state kinetic parameters in an in vitro transfer assay. Apparent K_M and V_MAX values for UDP-GlcNAc, Mg²⁺, and Mn²⁺ suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg²⁺, possibly playing a role to orient the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156, and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C-terminally fused with the green fluorescent protein displayed a punctate distribution of fluorescence along the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.