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Biosynthesis of CMP-N,N’-Diacetyllegionaminic acid from UDP-N,N’-Diacetylbacillosamine in Legionella pneumophila

TitleBiosynthesis of CMP-N,N’-Diacetyllegionaminic acid from UDP-N,N’-Diacetylbacillosamine in Legionella pneumophila
Publication TypeJournal Article
Year of Publication2008
AuthorsGlaze, PA, Watson, DC, Young, NM, Tanner, ME
JournalBiochemistry
Volume47
Pagination3272-3282
Date PublishedMar
ISBN Number0006-2960
Abstract

Legionaminic acid is a nine-carbon (x-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.

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