Kinetic characterization of xenobiotic reductase A from Pseudomonas putida 86.
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.
Bioinorganic Chemistry, University of Bayreuth, 95447 Bayreuth, Germany.
Olivia
O
Spiegelhauer
Frank
F
Dickert
Sophia
S
Mende
Dimitri
D
Niks
Russ
R
Hille
Matthias
M
Ullmann
Holger
H
Dobbek
D000465:Algorithms; D001665:Binding Sites; D002384:Catalysis; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D003512:Cyclohexanones / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity; D015262:Xenobiotics / Q000378:metabolism*
D003374:Coumarins; D003512:Cyclohexanones; D015262:Xenobiotics; D009243:NAD; C049536:8-hydroxycoumarin; C028215:2-cyclohexen-1-one; D009249:NADP; D010088:Oxidoreductases