title: Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.
abstract: 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(5)GXNXLE) 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.
authors.affiliation: Institute of Biochemistry, Center for Structural and Cell Biology in Medicine,  University of Lübeck, 23538 Lübeck,  Germany.
authors.forename: Helgo
authors.initials: H
authors.lastname: Schmidt
authors.forename: Guido
authors.initials: G
authors.lastname: Hansen
authors.forename: Sonia
authors.initials: S
authors.lastname: Singh
authors.forename: Anna
authors.initials: A
authors.lastname: Hanuszkiewicz
authors.forename: Buko
authors.initials: B
authors.lastname: Lindner
authors.forename: Koichi
authors.initials: K
authors.lastname: Fukase
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Woodard
authors.forename: Otto
authors.initials: O
authors.lastname: Holst
authors.forename: Rolf
authors.initials: R
authors.lastname: Hilgenfeld
authors.forename: Uwe
authors.initials: U
authors.lastname: Mamat
authors.forename: Jeroen R
authors.initials: JR
authors.lastname: Mesters
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008050:Lipid A / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008050:Lipid A; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D018698:Glutamic Acid; D014166:Transferases; D016695:Glycosyltransferases; C019917:3-deoxy-D-manno-octulosonate transferase; D005998:Glycine