Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide - PubMed (original) (raw)

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

Hak Suk Chung et al. Proc Natl Acad Sci U S A. 2011.

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

A proposed Kdo dioxygenase that generates the Ko moiety of B. ambifaria and Y. pestis LPS. A. A possible Fe2+/α-ketoglutarate/O2-dependent dioxygenase, designated KdoO, might account for the origin of the Ko unit after Kdo2-lipid A formation, consistent with the fact that Ko-containing bacteria make CMP-Kdo and transfer two Kdo residues to lipid A in the same manner as bacteria lacking Ko (15). LpxO, which generates the secondary _S_-2-hydroxymyristate substituent of Salmonella lipid A, provides a precedent for an analogous Kdo2-lipid A hydroxylation (17). B. The bamb_0774 gene, located between waaC and waaA on the B. ambifaria chromosome [Copeland A, et al. (2006) Complete sequence of chromosomes 1, 2, and 3 and plasmid 1 of Burkholderia cenocepacia AMMD, submitted August 2006 to the European Molecular Biology Laboratory/GenBank/DNA Data Base in Japan databases] (35), was identified as a possible structural gene for the Kdo hydroxylase because it encodes a protein with the active site signature of a Fe2+/α-ketoglutarate/O2-dependent dioxygenase (18). KdoO otherwise displays no sequence similarity to LpxO. C. Alignment of Bamb_0774 from B. ambifaria and Y1812 from Y. pestis KIM. These proteins share 52% identity (shown in red) and 64% similarity, and both contain the potential iron-binding motif, HXDX_n_H (n > 40) (potential downstream His residues shown in blue). Schematic representations: Kdo, black boxes; Ko, orange box; glucosamine, blue ovals; acyl chains, green squiggles; phosphate group, P.

Fig. 2.

Fig. 2.

An altered form of Kdo2-lipid A in E. coli WBB06 expressing KdoO. Lipids were extracted (35) and separated by TLC, followed by charring with 10% sulfuric acid in ethanol. Cells harboring either pYpKdoO or pBaKdoO generated a slower migrating derivative of Kdo2-lipid A, consistent with Ko formation.

Fig. 3.

Fig. 3.

Kdo2-lipid A modification by KdoO involves hydroxylation and requires O2 A, B, and C. Cells expressing either YpKdoO or BaKdoO accumulate a Kdo2-lipid A derivative that is 16 amu larger than Kdo2-lipid A, consistent with the incorporation of a single oxygen atom and the generation of Ko-Kdo-lipid A (predicted [M-2H]2- at m/z 1,125.658). D and E. Growth in the absence of oxygen has no effect on the vector control but inhibits KdoO-dependent hydroxylation of Kdo2-lipid A. Schematic descriptions are the same as in Fig. 1.

Fig. 4.

Fig. 4.

18O from 18O2 is incorporated into the outer Kdo unit of Kdo2-lipid A by KdoO. A and B. KdoO expressing cells grown with 18O2 generate a Kdo2-lipid A derivative that is 1 m/z larger (as a doubly-charged species) than seen with 16O2-grown cells (blue vs. yellow spectra, respectively), confirming the incorporation of an O2-derived oxygen atom into the product. The asterisk* indicates the presence of the sodium adduct [M-3H + Na] ion of Kdo2-lipid A. C, D, and E. ESI-MS/MS analysis of Kdo2-lipid A, Ko-Kdo-lipid A, and [18O]Ko-Kdo-lipid A shows that YpKdoO modifies the outer Kdo unit, as evidenced in E by the [M-H]- ion of [18O]Ko at m/z 255.073, the [M-H216O-H]-ion of [18O]Ko at m/z 237.062, and the absence of corresponding [16O]Kdo fragments. Schematic descriptions are the same as in Fig. 1, except that [18O]Ko is indicated with a blue box.

Fig. 5.

Fig. 5.

In vitro assay for KdoO and its cofactor requirements. A. Time course of the conversion of Kdo2-[4′-32P]lipid A to Ko-Kdo-[4′-32P]lipid A by membrane-free lysates (0.05 mg/mL) of C41(DE3)/pYpKdoO.1. Abbreviation: NE, no enzyme. B. KdoO activity was insert dependent and required added Fe2+, α-ketoglutarate, and ascorbic acid. KdoO activity was inhibited by EDTA. Reactions were initiated by adding washed membranes of C41(DE3)/pYpKdoO.1 (0.02 mg/mL final concentration) and terminated by spotting 2 μL portions of the reactions onto silica TLC plates at various times. The plates were developed in chloroform∶methanol∶acetic acid∶H2O (25∶15∶3.5∶4 vol/vol) and quantified with a PhosphorImager screen.

Similar articles

Cited by

References

    1. Nikaido H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol R. 2003;67:593–656. - PMC - PubMed
    1. Raetz CRH, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635–700. - PMC - PubMed
    1. Raetz CRH, Reynolds CM, Trent MS, Bishop RE. Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem. 2007;76:295–329. - PMC - PubMed
    1. Miller SI, Ernst RK, Bader MW. LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol. 2005;3:36–46. - PubMed
    1. Park BS, et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature. 2009;458:1191–1195. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources