A MONO-FUNCTIONAL 3-DEOXY-D-MANNO-OCTULOSONIC ACID (KDO) TRANSFERASE AND A KDO KINASE IN EXTRACTS OF HAEMOPHILUS-INFLUENZAE

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 homogeneo us preparations of KdtA polypeptide catalyzed the attachment of both K do groups to the precursor, lipid IVA. E. coli KdtA produced only trac es of mono-glycosylated product. We now show that a single Kdo is tran sferred 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 spec ific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, m illigram quantities of Kdo-lipid IVA were prepared for analysis. Matri x-assisted laser desorption/ionization mass spectrometry revealed a pa rent ion (M - H)(-) at m/z 1626.0, consistent with the addition of a s ingle Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent subst rate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphoryla ted in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid I VA with a parent ion (M - H)(-) at m/z 1703.9. The identification of t he 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-fu nctional Kdo transferases, like those of E. coli and Chlamydia trachom atis.