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

作者：Chung Hak Suk; Raetz Christian R H^*

来源：Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(2): 510-515.

DOI：10.1073/pnas.1016462108

• iron-alpha-ketoglutarate dependent dioxygenases
• Kdo-hydroxylase
• lipopolsaccharide
• D-glycero-D-talo-oct-2-ulosonic acid (Ko)

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 Kdo2-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 Kdo2-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 Kdo2-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.

• 出版日期2011-1-11