Kinetic Characterization of Xenobiotic Reductase A from Pseudomonas putida 86

Abstract

Xenobiotic reductase A (XenA) from Pseudomonas putida is a member of the old-yellow-enzyme family of flavin-containing enzymes and catalyzes the NADH/NADPH-dependent reduction of various substrates, including 8-hydroxycoumarin and 2-cyclohexenone. Here we present a kinetic and thermodynamic analysis of XenA. In the reductive half-reaction, complexes of oxidized XenA with NADH or NADPH form charge-transfer (CT) intermediates with increased absorption around 520-560 nm, which occurs with a second-order rate constant of 9.4 x 10(5) M(-1) s(-1) with NADH and 6.4 x 10(5) M(-1) s(-1) with NADPH, while its disappearance is controlled by a rate constant of 210-250 s(-1) with both substrates. Transfer of hydride from NADPH proceeds 24 times more rapidly than from NADH. This modest kinetic preference of XenA for NADPH is unlike the typical discrimination between NADH and NADPH by binding affinity. Docking studies combined with electrostatic energy calculations indicate that the 2'-phosphate group attached to the adenine moiety of NADPH is responsible for this difference. The reductions of 2-cyclohexenone and coumarin in the oxidative half-reaction are both concentration-dependent under the assay conditions and reveal a more than 50-fold larger limiting rate constant for the reduction of 2-cyclohexenone compared to that of coumarin. Our work corroborates the link between XenA and other members of the old-yellow-enzyme family but demonstrates several differences in the reactivity of these enzymes.

Cysteine as a Modulator Residue in the Active Site of Xenobiotic Reductase A: A Structural, Thermodynamic and Kinetic Study

“…The rate constants for reductive and oxidative half-reactions are dependent on the reduction potential of the flavin cofactors. 13 The reduction potential of the FMN/FMNH -couple in XenA was previously determined to be -263 mV, 13 which is considerably lower than the values found for other members of the OYE family. [14][15][16] XenA shows several structural variations that can be responsible for the difference in the reduction potential.…”

“…We therefore assume that, as in XenA-wt, the reduction potential of the FMN/FMNH -couple is substantially lower than the FMN/FMNH -couple and that either the enzyme or the reaction conditions allowed for faster equilibration than observed in the experiment with XenA-wt. 13 The inability to form semiquinone species has been observed also for the related flavoenzymes morphinone reductase, 20 PETN reductase 14 and YqjM. 21 To investigate the influence of Cys25 on the relative stability of the oxidized and reduced state of XenA, we determined the reduction potentials of the FMN/FMNH -redox couple of both variants.…”

“…This method was used to ensure single electron transfer and allow the formation of semiquinone species. Unlike XenAwt, which shows the initial formation of an anionic semiquinone, 13 only quinone and hydroquinone species of FMN are observed during photoreduction of XenA-C25S and XenA-C25A (Fig. 2a).…”

“…12 The exchange of Cys26 (C26D and C26G) altered the reactivity of YqjM and changed its enantioselectivity. 18 The recent structural 6 and kinetic 13 studies of XenA did not reveal the contributions of individual active site residues. To gain insight into the role of Cys25 we studied its effect on the structure, the stabilization of substrate complexes and the kinetics of the catalytic cycle.…”

Old Yellow Enzyme-Catalysed Asymmetric Hydrogenation: Linking Family Roots with Improved Catalysis

“…In agreement with a bi-bi ping-pong mechanism, great similarity exists among OYEs in the binding mode of the nicotinamide moiety of NADPH and phenolic inhibitors such as para-hydroxybenzaldehyde (p-HBA) ( Figure 5) or para-nitrophenol (p-NP). Both aromatic rings are oriented through π-π stacking with the FMN isoalloxazine ring and hydrogen bonding with His167/Asn194 and His164/His191 (YqjM/OYE1) [115]. The crystal structure of OYE1 (class II) with an NADP + analogue showed that the oxygen of the amide on the pyridinium ring is (hydrogen) bonding with the two conserved histidine residues, thus positioning the C4 atom close to the N5 atom of FMN for the hydride transfer [83].…”