title: The incorporation of glucosamine into enterobacterial core lipopolysaccharide: two enzymatic steps are required.
abstract: The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to ld-HeppII.
authors.affiliation: Departamento de Microbiología y Parasitología Sanitarias, Facultad de Farmacia, Universidad de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Regué
authors.forename: Luis
authors.initials: L
authors.lastname: Izquierdo
authors.forename: Sandra
authors.initials: S
authors.lastname: Fresno
authors.forename: Natalia
authors.initials: N
authors.lastname: Jimenez
authors.forename: Nuria
authors.initials: N
authors.lastname: Piqué
authors.forename: M Michela
authors.initials: MM
authors.lastname: Corsaro
authors.forename: Michelangelo
authors.initials: M
authors.lastname: Parrilli
authors.forename: Teresa
authors.initials: T
authors.lastname: Naldi
authors.forename: Susana
authors.initials: S
authors.lastname: Merino
authors.forename: Juan M
authors.initials: JM
authors.lastname: Tomás
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D005944:Glucosamine / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000254:growth & development / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011134:Polysaccharides / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D011134:Polysaccharides; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D005944:Glucosamine