title: Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide.
abstract: Several gram-negative pathogens, including Yersinia pestis, Burkholderia cepacia, and Acinetobacter haemolyticus, synthesize an isosteric analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), known as D-glycero-D-talo-oct-2-ulosonic acid (Ko), in which the axial hydrogen atom at the Kdo 3-position is replaced with OH. Here we report a unique Kdo 3-hydroxylase (KdoO) from Burkholderia ambifaria and Yersinia pestis, encoded by the bamb_0774 (BakdoO) and the y1812 (YpkdoO) genes, respectively. When expressed in heptosyl transferase-deficient Escherichia coli, these genes result in conversion of the outer Kdo unit of Kdo(2)-lipid A to Ko in an O(2)-dependent manner. KdoO contains the putative iron-binding motif, HXDX(n>40)H. Reconstitution of KdoO activity in vitro with Kdo(2)-lipid A as the substrate required addition of Fe(2+), α-ketoglutarate, and ascorbic acid, confirming that KdoO is a Fe(2+)/α-ketoglutarate/O(2)-dependent dioxygenase. Conversion of Kdo to Ko in Kdo(2)-lipid A conferred reduced susceptibility to mild acid hydrolysis. Although two enzymes that catalyze Fe(2+)/α-ketoglutarate/O(2)-dependent hydroxylation of deoxyuridine in fungal extracts have been reported previously, kdoO is the first example of a gene encoding a deoxy-sugar hydroxylase. Homologues of KdoO are found exclusively in gram-negative bacteria, including the human pathogens Burkholderia mallei, Yersinia pestis, Klebsiella pneumoniae, Legionella longbeachae, and Coxiella burnetii, as well as the plant pathogen Ralstonia solanacearum.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, P. O. Box 3711, Durham, NC 27710, USA.
authors.forename: Hak Suk
authors.initials: HS
authors.lastname: Chung
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019117:Burkholderia / Q000201:enzymology*; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013400:Sugar Acids / Q000737:chemistry*; D015010:Yersinia pestis / Q000201:enzymology*
chemical_list: D008055:Lipids; D008070:Lipopolysaccharides; D013400:Sugar Acids; D006899:Mixed Function Oxygenases; D010100:Oxygen; C120356:3-deoxy-manno-oct-2-ulopyranosonic acid