Cloning and characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat).
In this study, we cloned the Pseudomonas aeruginosa zwf gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD+- or NADP+-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted zwf gene product is 490 residues, which could form a tetramer with a molecular mass of approximately 220 kDa. G6PDH activity and zwf transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and zwf transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be approximately 220 kDa by size exclusion chromatography. Estimated Km values of purified G6PDH acting on glucose-6-phosphate, NADP+, and NAD+ were 530, 57, and 333 microM, respectively. The specific activities with NAD+ and NADP+ were calculated to be 176 and 69 micromol/min/mg. An isogenic zwf mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.
Ma
J F
JF
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524, USA.
Hager
P W
PW
Howell
M L
ML
Phibbs
P V
PV
Hassett
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010269:Paraquat / Q000633:toxicity*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic
D005954:Glucosephosphate Dehydrogenase; D010269:Paraquat