WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-D-manno-oct-2- ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX 5GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA.
CITATION STYLE
Schmidt, H., Hansen, G., Singh, S., Hanuszkiewicz, A., Lindner, B., Fukase, K., … Mesters, J. R. (2012). Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis. Proceedings of the National Academy of Sciences of the United States of America, 109(16), 6253–6258. https://doi.org/10.1073/pnas.1119894109
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