Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 05 Jan 2019 07:32:01 Microbiology (2002), 148, 571–582 Printed in Great Britain Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli Amal O. Amer 1 and Miguel A. Valvano 1,2 Author for correspondence : Miguel A. Valvano. Tel : 1 519 661 3996. Fax: 1 519 661 3499. e-mail : mvalvanouwo.ca Departments of Microbiology and Immunology 1 and Medicine 2 , University of Western Ontario, London, Ontario N6A 5C1, Canada The integral membrane protein WecA mediates the transfer of N- acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner- membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme. Keywords : undecaprenol, N-acetylglucosamine, O-antigen biosynthesis, membrane protein, phosphodiester bond INTRODUCTION Lipopolysaccharide (LPS) is a major component of the outer membrane in Gram-negative bacteria. It consists of lipid A, core oligosaccharide and the O-specific polysaccharide chain or O antigen (Whitfield, 1995). The biosynthesis of LPS is a complex process requiring many enzymes, encoded by a large number of genes, which catalyse the independent formation of a lipid A-core oligosaccharide complex and a polyisoprenyl- linked O antigen. The biosynthesis and assembly of O antigen involves a characteristic transmembrane export of polyisoprenyl-linked saccharides that are synthesized ................................................................................................................................................. Abbreviations : GlcNAc, N-acetylglucosamine ; GPT, UDP-GlcNAc : dolichol phosphate GlcNAc-1-phosphate transferase ; TMHMM, Transmembrane Hidden Markov Model ; Und-P, undecaprenyl phosphate. on the cytoplasmic face of the plasma membrane. Depending on the components that may be required for this process, the assembly of O antigen can be dis- tinguished into wzy-dependent and wzy-independent pathways (Whitfield, 1995). In wzy-dependent systems, the translocation of individual polyisoprenyl-linked O- repeating oligosaccharide subunits to the periplasmic face of the plasma membrane is mediated by the Wzx protein (Feldman et al., 1999). Once on the periplasmic side, the individual subunits are polymerized by the Wzy O-antigen polymerase (Whitfield, 1995). In wzy-inde- pendent systems, polymerized O antigens are trans- ported to the periplasmic face of the plasma membrane by an ATP-binding cassette transporter-dependent path- way (Bronner et al., 1994). Regardless of the specific export system used, polymerized O antigens are finally ligated onto the lipid A-core oligosaccharide acceptor 0002-5151  2002 SGM 571