Kinetic Characterization of PA1225 from Pseudomonas aeruginosa PAO1 Reveals a New NADPH:Quinone Reductase.

ABSTRACT

The pa1225 gene of Pseudomonas aeruginosa strain PAO1 was cloned, and the resulting enzyme (PA1225) was purified and revealed to be an NADPHquinone reductase. By using kinetics, fluorescence, and mass spectrometric analyses, PA1225 was shown to utilize FAD to transfer a hydride ion from NADPH to quinones. The enzyme could also use NADH, but with an efficiency that was 40-fold lower than that of NADPH as suggested by the kcat/ Km values at pH 6.0. Similar initial rates of reaction were determined with 1,4-benzoquinone and 2,6-dimethoxy-1,4-benzoquinone in the range between 25 and 200 µM, suggesting a low Km value for the quinone-oxidizing substrate. The lack of inhibition by NADP+ versus NADPH at saturating concentrations of 1,4-benzoquinone was consistent with a ping-pong bi-bi mechanism. The reductive half-reaction at pH 6.0 had Kd values of 0.07 mM with NADPH and 1.8 mM with NADH; the kred for flavin reduction was independent of pH with values of ∼10 s-1 with NADPH and ∼5 s-1 with NADH. Thus, the enzyme specificity for the reducing substrate arises primarily from a tighter binding of NADPH than of NADH. At pH 6.0, the kcat value with NADPH and 1,4-benzoquinone was 10.1 s-1, consistent with the hydride transfer from NADPH to FAD being fully rate limiting for the overall turnover of the enzyme. The enzyme showed negligible NADPH oxidase and azoreductase activities. This study enables annotation of the pa1225 gene as NADPHquinone reductase, elucidates the enzymatic function of PA1225 in P. aeruginosa PAO1, and establishes that PA1225 is not an azoreductase as previously proposed.