Cloning, Purification, and Biochemical Characterization of PA1225, a Novel FAD Containing NADPH:Quinone Reductase from Pseudomonas aeruginosa PAO1

The gene product of pa1225 found in the opportunistic bacterium Pseudomonas aeruginosa strain PAO1 is currently annotated as a putative NAD(P)H dehydrogenase. Prior investigation of LysR (PA4203), which regulates the transcription of detoxifying enzymes, revealed PA1225 to be the most upregulated gene in the LysR deletion mutant in PAO1. A combination of steady-state and rapid kinetics was used to investigate the enzymatic properties of PA1225, which may play a potential role in the viability of P. aeruginosa PAO1 in toxic environments. In this study, it was experimentally determined that PA1225 catalyzes the oxidation of NADPH and NADH to reduce one- and two-ring quinones. PA1225 has a clear preference for NADPH as the reducing substrate as determined by a 50-fold difference in kcat/Km with respect to NADH at pH 6.0. The effect of pH was also investigated with NADPH and NADH to elucidate changes in kred and Kd