Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1.
UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.
Hung
Ruei-Jiun
RJ
Institute of Molecular Medicine, National Tsing Hua University, Hsin Chu 300, Taiwan.
Chien
Han-Sheng
HS
Lin
Ruei-Zeng
RZ
Lin
Ching-Ting
CT
Vatsyayan
Jaya
J
Peng
Hwei-Ling
HL
Chang
Hwan-You
HY
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000502:physiology; D016415:Sequence Alignment; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism; D014574:Uronic Acids / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D007527:Isoenzymes; D014574:Uronic Acids; D014535:Uridine Diphosphate Glucuronic Acid; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014532:Uridine Diphosphate Glucose