Three Highly Conserved Proteins Catalyze the Conversion of UDP-N-acetyl-d-glucosamine to Precursors for the Biosynthesis of O Antigen in Pseudomonas aeruginosaO11 and Capsule in Staphylococcus aureus Type 5

Abstract

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.

Solvent Stress Response of the Denitrifying Bacterium “ Aromatoleum aromaticum ” Strain EbN1

“…WbjB orthologs in Pseudomonas aeruginosa (WbjB) and Staphylococcus aureus (Cap5E) are involved in biosynthesis of N-acetyl-L-fucosamine (26), which is a constituent of surface polysaccharide structures (O antigens of lipopolysaccharide [LPS] and capsules, respectively). Differences in solvent tolerance have been correlated with LPS structure or composition in Pseudomonas strains; e.g., o-xylene-tolerant P. putida strain Idaho had rough LPS, while o-xylene-sensitive P. putida strain MW1200 had smooth LPS (32).…”

Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

“…The results of this study, in combination with recent studies in our laboratory, reinforce the notion that UDP-DGlcNAc is a fundamental precursor in bacterial polysaccharide biosynthesis. To date, our laboratory has been able to demonstrate that this sugar nucleotide is the precursor for: 2-acetamido-2,6-dideoxy-L-hexoses in the gluco, manno, talo (20,47), and galacto 2 configurations; for UDP-2-acetamido-2,6-dideoxy-D-xylo-4-hexulose, the proposed precursor for 2-acetamido-2,6-dideoxy-D-hexoses in the gluco and galacto configurations (10,11); and, for UDP-D-GlcNAcA, the proposed precursor for 2,3-diacetamido-2,3-dideoxy-hexuronic acids in the D-gluco, D-manno, and L-gulo configurations (present study). …”

Giant Virus Megavirus chilensis Encodes the Biosynthetic Pathway for Uncommon Acetamido Sugars

“…Moreover, it shared 37% identity with CapE from Staphylococcus aureus and WbjB from P. aeruginosa, both involved in UDP-FucNAc production (11), and with WbvB from V. cholerae, belonging to the UDP-L-QuiNAc pathway (10). Interestingly, whereas CapE and WbjB are characterized by motif MXXXK in the catalytic triad (11,27), the mg534 gene product contains the canonical sequon, YXXXK, typical of SDR enzymes, also found in PseB/FlaA1 (26,27). Mg534 lacks the so-called "latch" that has been identified in CapE (27), which is also absent in PseB/FlaA1 and related inverting 4,6-dehydratases.…”

“…1). Three enzymes from Pseudomonas aeruginosa O11 (WbjB, WbjC, and WbjD) and Staphylococcus aureus type 5 (CapE, CapF, and CapG) are required for the conversion of UDP-D-GlcNAc to UDP-N-acetyl-L-fucosamine (UDP-L-FucNAc) (11,12). For UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc), a parallel pathway is present in Vibrio cholera O37, including WbvB, WbvR, and WbvD (10).…”

“…Conflicting data have been reported in the literature about the identification of enzymes that catalyze the different steps of these pathways. Specifically, 3-epimerase activity was initially assigned to WbjB/CapE and WbvB, together with the 4,6-dehydratase and 5-epimerase activities (10,11). Hence, the following enzymes in the pathways, WbjC/CapF and WbvR, were proposed to only have a 4-reductase activity (10).…”