Biosynthesis of CMP-N,N'-diacetyllegionaminic acid from UDP-N,N'-diacetylbacillosamine in Legionella pneumophila.
Legionaminic acid is a nine-carbon alpha-keto acid that is similar in structure to other members of the sialic acid family that includes neuraminic acid and pseudaminic acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the O-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N, N'-diacetyllegionaminic acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic acid biosynthetic genes ( neuA,B,C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP- N, N'-diacetylbacillosamine 2-epimerase that converts UDP- N, N'-diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP- N-acetylglucosamine 2-epimerase (NeuC) of sialic acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GlcNAc as an alternate substrate. The NeuB homologue was found to be a N, N'-diacetyllegionaminic acid synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic acid and pseudaminic acid synthases. Finally, the NeuA homologue was shown to possess the CMP- N, N'-diacetyllegionaminic acid synthetase activity that generates the activated form of legionaminic acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative.
Glaze
Pavel A
PA
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
Watson
David C
DC
Young
N Martin
NM
Tanner
Martin E
ME
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003563:Cyclic CMP / Q000737:chemistry* / Q000378:metabolism; D016952:Legionella pneumophila / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D015394:Molecular Structure; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry* / Q000378:metabolism; D013400:Sugar Acids / Q000737:chemistry / Q000378:metabolism; D014530:Uridine Diphosphate / Q000737:chemistry* / Q000378:metabolism
C502751:2,4-diacetamido-2,4,6-trideoxyglucopyranose; C513444:5,7-diacetamido-3,5,7,9-tetradeoxynonulosonic acid; C110570:5,7-diacetamido-8-O-acetyl-3,5,7,9-tetradeoxy-glycero-talo-nonulosonic acid; D012794:Sialic Acids; D013400:Sugar Acids; D003563:Cyclic CMP; D014530:Uridine Diphosphate; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine