- Investigations on the Reaction Mechanism of Xenobiotic Reductase A (2010)
- Xenobiotic reductase A (XenA) from Pseudomonas putida 86 is a member of the Old Yellow Enzyme family of FMN containing enzymes. It catalyzes the NADH/NADPH dependent reduction of various substrates, including 2-cyclohexenone, coumarin, 7- and 8-hydroxycoumarin in a two-step mechanism consisting of a reductive and an oxidative half-reaction. The overall structure of the family members is similar but the active site residues show considerable variations. One distinct difference of XenA compared to other members is the presence of a cysteine residue (Cys25) in the active site, where most other members have a threonine. Further, the active site of XenA is lined up by two tyrosine (Tyr27 and Tyr183) and two tryptophan (Trp302 and Trp358) residues. To get a better understanding of the reaction mechanism of XenA we analyzed the enzyme using a combination of transient and steady-state kinetics, redox potentiometry and crystal structure analysis. Thermodynamic and kinetic investigations revealed a preference of XenA for NADPH over NADH. Furthermore, the reaction catalyzed by XenA follows a ping-pong mechanism in which both substrates are bound to the same position in the active site but interact with different amino acids. The crystal structures of XenA without and with coumarin bound to the active site were solved at true atomic resolution. The oxidized complex with coumarin showed a compressed active site geometry in which the isoalloxazine ring of FMN is sandwiched between coumarin and the protein backbone. The crystal structure of reduced XenA showed a distortion of the isoalloxazine ring and the movement of Trp302 into the active site. Furthermore, we analyzed the individual contributions of the five active site residues using site-directed mutagenesis. An exchange of Cys25 against serine shifted the reduction potential of the FMN/FMNH- couple by 82 mV, increased the limiting rate constant of the reductive and decreased the limiting rate constant of the oxidative half-reaction. Therefore we conclude that Cys25 modulates substrate binding and the reduction potential of FMN. Moreover, we revealed that Tyr27 contributes to the stabilization of the transition state during the reductive half-reaction by an interaction of its hydroxyl group with the transferred hydride ion. The exchange of Tyr183 resulted in a decreased affinity of XenA for NADPH and a considerable decrease of the rate of the oxidative half-reaction. These results are in agreement with its function as indispensable proton donor in the oxidative half-reaction. Exchanging Trp302 resulted in multiphasic kinetics for both half-reactions and a decreased affinity of XenA for NADPH. In combination with its movement between the reduced and oxidized state of XenA, we propose a redox dependent shaping of the active site by Trp302. Hence, this residue is responsible for the correct positioning of the substrates in both half-reactions, which is an essential part in the reaction mechanism. The results from the exchange of Trp358 indicated that this residue is involved in the orientation of the nicotinamide ring of NAD(P)H by spatial exclusion. Crystal structures of enzyme substrate complexes are usually determined from non-reactive states. The Y183F variant of XenA, lacking the proton donor of the oxidative half-reaction, allowed us to freeze-trap the true Michaelis complexes of reduced XenA in complex with four different substrates. For the first time we were able to observe 2-cyclohexenone in an active site. Finally, we prove that mode of substrate binding of XenA is redox dependent. In summary our results provide a more detailed description of the reaction mechanism of XenA and offer new insights on how substrates interact with flavoenzymes.