Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide

  • Chung H
  • Raetz C
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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+), alpha-ketoglutarate, and ascorbic acid, confirming that KdoO is a Fe(2+)/alpha-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+)/alpha-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.

Author-supplied keywords

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Burkholderia/*enzymology
  • Hydrolysis
  • Lipids/chemistry
  • Lipopolysaccharides/*chemistry
  • Mass Spectrometry/methods
  • Mixed Function Oxygenases/chemistry
  • Models, Chemical
  • Molecular Sequence Data
  • Oxygen/chemistry
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Electrospray Ionization/method
  • Sugar Acids/*chemistry
  • Yersinia pestis/*enzymology

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Authors

  • H S Chung

  • C R Raetz

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