title: Structure of the Escherichia coli heptosyltransferase WaaC: binary complexes with ADP and ADP-2-deoxy-2-fluoro heptose.
abstract: Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-negative bacteria and are therefore essential for cell growth and viability. The heptosyltransferase WaaC is a glycosyltransferase (GT) involved in the synthesis of the inner core region of LPS. It catalyzes the addition of the first L-glycero-D-manno-heptose (heptose) molecule to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue of the Kdo2-lipid A molecule. Heptose is an essential component of the LPS core domain; its absence results in a truncated lipopolysaccharide associated with the deep-rough phenotype causing a greater susceptibility to antibiotic and an attenuated virulence for pathogenic Gram-negative bacteria. Thus, WaaC represents a promising target in antibacterial drug design. Here, we report the structure of WaaC from the Escherichia coli pathogenic strain RS218 alone at 1.9 A resolution, and in complex with either ADP or the non-cleavable analog ADP-2-deoxy-2-fluoro-heptose of the sugar donor at 2.4 A resolution. WaaC adopts the GT-B fold in two domains, characteristic of one glycosyltransferase structural superfamily. The comparison of the three different structures shows that WaaC does not undergo a domain rotation, characteristic of the GT-B family, upon substrate binding, but allows the substrate analog and the reaction product to adopt remarkably distinct conformations inside the active site. In addition, both binary complexes offer a close view of the donor subsite and, together with results from site-directed mutagenesis studies, provide evidence for a model of the catalytic mechanism.
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Université Paris Descartes, Faculté de Pharmacie, 4, Avenue de l'Observatoire, F-75270 Paris cedex 06, France.
authors.forename: Sylvestre
authors.initials: S
authors.lastname: Grizot
authors.forename: Michèle
authors.initials: M
authors.lastname: Salem
authors.forename: Vanida
authors.initials: V
authors.lastname: Vongsouthi
authors.forename: Lionel
authors.initials: L
authors.lastname: Durand
authors.forename: François
authors.initials: F
authors.lastname: Moreau
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Dohi
authors.forename: Stéphane
authors.initials: S
authors.lastname: Vincent
authors.forename: Sonia
authors.initials: S
authors.lastname: Escaich
authors.forename: Arnaud
authors.initials: A
authors.lastname: Ducruix
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017611:Fluorine Compounds / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006539:Heptoses / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins; D017611:Fluorine Compounds; D006539:Heptoses; D000244:Adenosine Diphosphate; D016695:Glycosyltransferases; C080934:lipooligosaccharide 1,5-heptosyltransferase