Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis

• Published in: Proceedings of the National Academy of Sciences - PNAS 2012-04, Vol.109 (16), p.6253-6258
• Main Authors: Schmidt, Helgo, Hansen, Guido, Singh, Sonia, Hanuszkiewicz, Anna, Lindner, Buko, Fukase, Koichi, Woodard, Ronald W, Holst, Otto, Hilgenfeld, Rolf, Mamat, Uwe, Mesters, Jeroen R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Aquifex; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding sites; Binding Sites - genetics; Biocatalysis; Biological Sciences; Biosynthesis; Crystal structure; Crystallography, X-Ray; Glutamic Acid - chemistry; Glutamic Acid - genetics; Glutamic Acid - metabolism; Glycine - chemistry; Glycine - genetics; Glycine - metabolism; Glycosyltransferases - chemistry; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; Gram-negative bacteria; Gram-Negative Bacteria - enzymology; Gram-Negative Bacteria - genetics; Gram-Negative Bacteria - metabolism; Lipid A - biosynthesis; Lipids; lipopolysaccharides; Lipopolysaccharides - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Interaction Domains and Motifs; Protein Structure, Tertiary; Sequence Homology, Amino Acid; site-directed mutagenesis; Spectrometry, Fluorescence; Transferases - chemistry; Transferases - genetics; Transferases - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1119894109

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/TX5GXNXLE) 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.