title: Pen and Pal are nucleotide-sugar dehydratases that convert UDP-GlcNAc to UDP-6-deoxy-D-GlcNAc-5,6-ene and then to UDP-4-keto-6-deoxy-L-AltNAc for CMP-pseudaminic acid synthesis in Bacillus thuringiensis.
abstract: 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.
authors.affiliation: From the Department of Plant Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Zi
authors.initials: Z
authors.lastname: Li
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Soyoun
authors.initials: S
authors.lastname: Hwang
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ericson
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Kyle
authors.initials: K
authors.lastname: Bowler
authors.affiliation: From the Department of Plant Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 peled@ccrc.uga.edu.
authors.forename: Maor
authors.initials: M
authors.lastname: Bar-Peled
mesh_terms: D001413:Bacillus thuringiensis / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D001426:Bacterial Proteins / Q000235:genetics*; D003568:Cytidine Monophosphate / Q000378:metabolism; D005408:Flagellin / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D006031:Glycosylation; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D013400:Sugar Acids / Q000737:chemistry / Q000378:metabolism*
chemical_list: C513444:5,7-diacetamido-3,5,7,9-tetradeoxynonulosonic acid; D001426:Bacterial Proteins; D013400:Sugar Acids; D005408:Flagellin; C026712:UDP-glucosamine; D006836:Hydro-Lyases; D003568:Cytidine Monophosphate; D005944:Glucosamine
keywords: Aminotransferase; Bacillus; Bacterial Metabolism; CMP-Pseudaminic Acid; Carbohydrate Biosynthesis; Dehydratase; Enzyme Catalysis; Glycobiology; UDP-GlcNAc