Biosynthesis of CMP-N,N'-diacetyllegionaminic acid from UDP-N,N'-diacetylbacillosamine in Legionella pneumophila

Biochemistry. 2008 Mar 11;47(10):3272-82. doi: 10.1021/bi702364s. Epub 2008 Feb 15.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / analogs & derivatives*
  • Acetylglucosamine / chemistry
  • Acetylglucosamine / metabolism
  • Cyclic CMP / chemistry*
  • Cyclic CMP / metabolism
  • Legionella pneumophila / metabolism*
  • Magnetic Resonance Spectroscopy
  • Models, Chemical
  • Molecular Structure
  • N-Acetylneuraminic Acid / chemistry
  • N-Acetylneuraminic Acid / metabolism
  • Sialic Acids / chemistry*
  • Sialic Acids / metabolism
  • Sugar Acids / chemistry
  • Sugar Acids / metabolism
  • Uridine Diphosphate / chemistry*
  • Uridine Diphosphate / metabolism

Substances

  • 2,4-diacetamido-2,4,6-trideoxyglucopyranose
  • 5,7-diacetamido-3,5,7,9-tetradeoxynonulosonic acid
  • 5,7-diacetamido-8-O-acetyl-3,5,7,9-tetradeoxy-glycero-talo-nonulosonic acid
  • Sialic Acids
  • Sugar Acids
  • Cyclic CMP
  • Uridine Diphosphate
  • N-Acetylneuraminic Acid
  • Acetylglucosamine