A mono-functional 3-deoxy-D-manno-octulosonic acid (Kdo) transferase and a Kdo kinase in extracts of Haemophilus influenzae

被引:48
作者
White, KA
Kaltashov, IA
Cotter, RJ
Raetz, CRH
机构
[1] DUKE UNIV, MED CTR, DEPT BIOCHEM, DURHAM, NC 27710 USA
[2] JOHNS HOPKINS UNIV, DEPT PHARMACOL, MIDDLE ATLANTIC MASS SPECTROMETRY LAB, BALTIMORE, MD 21205 USA
关键词
D O I
10.1074/jbc.272.26.16555
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. coli Kdo transferase and demonstrated that homogeneous preparations of KdtA polypeptide catalyzed the attachment of both Kdo groups to the precursor, lipid IVA. E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IVA in extracts of H. influenzae. The mono-functional Kdo transferase of H. influenzae is membrane-bound, and the reaction is dependent upon a CMP-Kdo-generating system, as in E. coli. The specific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IVA were prepared for analysis. Matrix-assisted laser desorption/ionization mass spectrometry revealed a parent ion (M - H)(-) at m/z 1626.0, consistent with the addition of a single Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IVA with a parent ion (M - H)(-) at m/z 1703.9. The identification of the monofunctional H. influenzae Kdo transferase, which is encoded by a KdtA homologue that displays 50% identity to its E. coli counterpart, should facilitate the mechanistic dissection of more complex multi-functional Kdo transferases, like those of E. coli and Chlamydia trachomatis.
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页码:16555 / 16563
页数:9
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