| Sujet | ANTIGEN; Antigens; bacterial; Bacterial Proteins; Base Sequence; biosynthesis; Canada; capillary; capsular polysaccharide; capsule; Catalysis; deficiency; DISEASE; DNA; DNA Primers; Electrophoresis; Electrophoresis,Capillary; ENZYME; enzymes; GENE; Genes; immunology; INTERMEDIATE; Kinetics; LIPOPOLYSACCHARIDE; MAGNETIC; Magnetic Resonance Spectroscopy; MAGNETIC-RESONANCE; metabolism; Microbiology; Mixture; MS; MUTANT; MUTANTS; N-acetylglucosamine; NUCLEAR; Nuclear Magnetic Resonance; nuclear magnetic resonance spectroscopy; Nuclear Magnetic Resonance,Biomolecular; NUCLEAR-MAGNETIC-RESONANCE; O antigen; O Antigens; O-antigen; PATHWAY; Phenotype; POLYSACCHARIDE; polysaccharide structure; polysaccharide structures; protein; Proteins; Pseudomonas; Pseudomonas aeruginosa; PSEUDOMONAS-AERUGINOSA; RESONANCE; SEROTYPE; SPECTROSCOPY; Spectrum Analysis,Mass; Staphylococcus aureus; STRAIN; structure; sugar; SUGARS; Support,Non-U.S.Gov't; Support,U.S.Gov't,P.H.S.; surface; Uridine Diphosphate; Uridine Diphosphate N-Acetylglucosamine; Uridine Diphosphate Sugars; yield |
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| Résumé | N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis of N-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype |
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