Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in...

Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of k_cat demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.