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

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 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, Mg 2؉ , and Mn 2؉ suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg 2؉ , 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.

Bioinformatic insights into the biosynthesis of the Group B carbohydrate in Streptococcus agalactiae

“…SAG0140 is also highly homologous to RgpG, which carries out the comparable reaction initiating rhamnose-glucose polysaccharide (RGP) synthesis in Streptococcus mutans (Yamashita et al, 1999;Shibata et al, 2002) and WecA, which initiates LPS and enterobacterial common antigen biosynthesis in Escherichia coli (Lehrer et al, 2007). Indeed, the predicted topology of SAG0140 matches that determined for WecA (Lehrer et al, 2007) and key amino acid sequence motifs are conserved. Thus we propose that SAG0140 initiates GBC biosynthesis on a polyisoprenol-phosphate lipid carrier.…”

“…However, we were unable to identify an appropriate NAG transferase in the GBC locus, but instead noted that SAG0140 is significantly homologous to TagO [135/324 (41 %) amino acid identity], which carries out the identical reaction in teichoic acid synthesis in Bacillus subtilis (Soldo et al, 2002;Bhavsar & Brown, 2006;D'Elia et al, 2006). SAG0140 is also highly homologous to RgpG, which carries out the comparable reaction initiating rhamnose-glucose polysaccharide (RGP) synthesis in Streptococcus mutans (Yamashita et al, 1999;Shibata et al, 2002) and WecA, which initiates LPS and enterobacterial common antigen biosynthesis in Escherichia coli (Lehrer et al, 2007). Indeed, the predicted topology of SAG0140 matches that determined for WecA (Lehrer et al, 2007) and key amino acid sequence motifs are conserved.…”

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

“…Hence we suggest that His-324/His-325 may sequester the phosphate monoanion of undecaprenyl phosphate, and activate it for nucleophilic attack on the phospho-enzyme intermediate. MraY and also an integral membrane protein [13]. The reaction catalysed by WecA is the reversible reaction of UDPGlcNAc with undecaprenyl phosphate to give undecaprenyldiphospho-GlcNAc and UMP.…”

“…The reaction catalysed by WecA is the reversible reaction of UDPGlcNAc with undecaprenyl phosphate to give undecaprenyldiphospho-GlcNAc and UMP. WecA also contains conserved aspartic acid residues that are important for catalysis: replacement of Asp-156 by Asn in E. coli WecA gives inactive enzyme, whereas replacement of Asp-90 or Asp-91 by Asn gives mutant enzymes with residual activity [13], though replacement of both residues gives inactive enzyme [14].…”

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

“…The challenge was to introduce a pathway which would lead to an AsnGlcNAc linkage, then couple this with the mammalian glycosylation pathway which would generate an acceptable sugar moiety, but this was achieved using metabolic engineering ( [123] [124] [125]. The E. coli was then further engineered to synthesis a mannose3-Nacetylglucosamine2 (Man3GlcNAc2) glycan chain, a common core structure shared in all eukaryotes [126].…”