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Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide

Jason Lehrer,^1, Karen A. Vigeant,^1,, Laura D. Tatar,¹ and Miguel A. Valvano^1,2*

Infectious Diseases Research Group, Siebens-Drake Medical Research Institute, Departments of Microbiology and Immunology,¹ Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada²

Received 18 December 2006/ Accepted 15 January 2007

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 determined the steady-state kinetic parameters of the mutant proteins 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 in orienting 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 exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.

Published ahead of print on 19 January 2007.

J.L. and K.A.V. contributed equally to this work.

Present address: Bioniche Life Sciences Inc., Belleville, ON K8N 1E2, Canada.

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