Abstract
UDP-glucose dehydrogenase (UGDH) catalyzes the NAD+-dependent twofold oxidation of UDP-glucose to yield UDP-glucuronic acid, a substrate for polysaccharide biosynthesis in many strains of pathogenic bacteria. Although the functions and kinetic properties of UGDH have been studied extensively in organisms ranging from bovine to bacteria; the biochemical properties of UGDH in Pseudomonas aeruginosa PAO1, an important pathogen, is still unknown. In this study, we have cloned, expressed, and affinity-purified the product of two open reading frame, PA2022 and PA3559, which have been annotated as nucleotide sugar dehydrogenase and determined their kinetic parameters, substrate specificities, and oligomeric states. The Km of PA2022 and PA3559 for UDP-glucose are 0.061 mM and 0.646 mM, while the Km of these two enzymes for NAD+ is 0.379 mM and 1.003 mM, respectively. Besides, the substrate specificity of PA2022 is quite different from that of PA3559 that is more specific to UDP-glucose. PA2022 can also utilize TDP-glucose and UDP-N-acetylglucosamine with the one-third velocity of UDP-glucose. The gel filtration data showed that both the UGDH are active as dimer in solution. Comparison between the wild type PAO1 and mutants revealed that the possible functions of the UGDHs in P. aeruginosa included the participation in antibiotic resistance and biofilm formation. The PA2022-PA3559 double mutant RJ103 was more susceptible than wild type PAO1 to chloramphenicol, cefotaxime, and ampicillin. Moreover, its twitching motility was impaired and it produced substantially thinner biofilm than wild type PAO1. The data suggests that the possible biological role of UGDH, which syntheses UDP-glucuronic acid as a precursor for hyaluronic acid, involves in the biosynthesis of critical exopolysaccharide components and may regulate P. aeruginosa pathogenesis.