CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-D-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP+ and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD+-dependent and has distinct UDP-6-deoxy-D-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4?-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.
pseudaminic acid, Bacillus, aminotransferase, CMP-pseudaminic acid, flagellin glycosylation, dehydratase, enzyme catalysis, bacterial metabolism, Bacillus thuringiensis, carbohydrate biosynthesis, glycobiology, UDP-GlcNAc
NCBI PubMed ID: 25414257Publication DOI: 10.1074/jbc.M114.612747Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: peled@ccrc.uga.edu
Institutions: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA, From the Department of Plant Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
Methods: 13C NMR, 1H NMR, gel filtration, NMR-2D, PCR, SDS-PAGE, DNA techniques, kinetics assays, NMR-1D, genetic methods, biochemical methods, HPLC, UV, enzymatic analysis, LC-ESI-MS/MS, HILIC-HPLC-UV, HILIC-ESI-MS
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