Ma J F, Hager P W, Howell M L, Phibbs P V, Hassett D J
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524, USA.
J Bacteriol. 1998 Apr;180(7):1741-9. doi: 10.1128/JB.180.7.1741-1749.1998.
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.
在本研究中，我们克隆了铜绿假单胞菌的zwf基因，该基因编码葡萄糖-6-磷酸脱氢酶（G6PDH），这是一种催化葡萄糖-6-磷酸以NAD⁺或NADP⁺依赖性方式转化为6-磷酸葡萄糖酸的酶。预测的zwf基因产物有490个氨基酸残基，可形成分子量约为220 kDa的四聚体。当甘油、葡萄糖或葡萄糖酸盐等诱导底物丰富时，G6PDH活性和zwf转录在对数早期阶段达到最大值。相反，当细菌接近稳定期、诱导底物有限或生物体在含有柠檬酸盐、琥珀酸盐或乙酸盐的基础盐培养基中生长时，G6PDH活性和zwf转录均急剧下降。G6PDH被纯化至同质，通过尺寸排阻色谱法估计其分子量约为220 kDa。纯化的G6PDH作用于葡萄糖-6-磷酸、NADP⁺和NAD⁺的估计Km值分别为530、57和333 μM。用NAD⁺和NADP⁺计算的比活性分别为176和69 μmol/min/mg。一个同基因的zwf突变体无法在补充有甘露醇的基本培养基上生长。该突变体还表现出对氧化还原活性超氧化物生成剂甲基紫精（百草枯）的敏感性增加。由于G6PDH活性的一种副产物是NADPH，后一数据表明该辅因子对于防御百草枯毒性至关重要的酶的活性必不可少。
