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

Kimberly A. White, Igor A. Kaltashov, Robert J. Cotter, Christian R.H. Raetz

Research output: Contribution to journalArticle

Abstract

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 IV(A). E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IV(A) 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 IV(A) is 0.51 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IV(A) 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 IV(A), Kdo-lipid IV(A) was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IV(A) was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IV(A) 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.

Original languageEnglish (US)
Pages (from-to)16555-16563
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number26
DOIs
StatePublished - Jun 27 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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