The Catalytic and Kinetic Mechanisms of NADPH-dependent Alkenal/one Oxidoreductase

Abstract

NADPH-dependent alkenal/one oxidoreductase (AOR) from the rat is a phase 2/antioxidative enzyme that is known to catalyze the reduction of the carbon-carbon double bond of ␣,␤-unsaturated aldehydes and ketones. It is also known for its leukotriene B 4 12-hydroxydehydrogenase activity. In order to begin to understand these dual catalytic activities and validate its classification as a reductase of the medium-chain dehydrogenase/ reductase family, an investigation of the mechanism of its NADPH-dependent activity was undertaken. Recombinant AOR and a 3-nonen-2-one substrate were used to perform steady-state initial velocity, product inhibition, and dead end inhibition experiments, which elucidated an ordered Theorell-Chance kinetic mechanism with NADPH binding first and NADP ؉ leaving last. A nearly 20-fold preference for NADPH over NADH was also observed. The dependence of kinetic parameters V and V/K on pH suggests the involvement of a general acid with a pK of 9.2. NADPH isomers stereospecifically labeled with deuterium at the 4-position were used to determine that AOR catalyzes the transfer of the pro-R hydride to the ␤-carbon of an ␣,␤-unsaturated ketone, illudin M. Two-dimensional nuclear Overhauser effect NMR spectra demonstrate that this atom becomes the R-hydrogen at this position on the metabolite. Using [4R-2 H]NADPH, small primary kinetic isotope effects of 1.16 and 1.73 for V and V/K, respectively, were observed and suggest that hydride transfer is not rate-limiting. Atomic absorption spectroscopy indicated an absence of Zn 2؉ from active preparations of AOR. Thus, AOR fits predictions made for medium-chain reductases and bears similar characteristics to well known medium-chain alcohol dehydrogenases. NAD(P)H-dependent alkenal/one oxidoreductase (AOR)1 is an enzyme that reduces the carbon-carbon double bond of a variety of ␣,␤-unsaturated aldehydes and ketones (1). It is coregulated in the rat with a variety of phase 2/antioxidative enzymes, including NAD(P)H:quinone reductase, glutathione S-transferases, and UDP-glucuronosyltransferases, through the Keap1/Nrf2 signaling pathway (2). ␣,␤-Unsaturated aldehydes and ketones are electrophilic and capable of reacting, via a Michael addition mechanism, with important cellular nucleophiles, which in turn leads to macromolecular (protein, DNA) dysfunction and cell death. Because saturated carbonyls lack this reactive moiety, they are often far less toxic. Thus, hydrogenation of the ␣,␤-double bond by AOR conceptually results in detoxication (1, 3).Of the AOR substrates identified, several are common environmental pollutants (methyl vinyl ketone and acrolein) or products of lipid peroxidation (4, 5). The latter process involves reaction of oxygen free radicals with polyunsaturated fatty acids to form aliphatic ␣,␤-unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE), 2-hexenal, and 2,4-decadienal (5). These reactive molecules probably mediate many of the detrimental effects of oxidative stress. 4HNE is extremely cytotoxic, an abundant product of lipid perox...

Modulation of Nitro-fatty Acid Signaling

“…This is the first report of a mammalian enzyme capable of catalyzing nitroalkene reduction via conjugate hydride addition to the electrophilic -bond. The similar electron-withdrawing character of nitro and keto groups also suggests that the mechanism of nitroalkene reduction by PtGR-1 is similar to that reported for ␣,␤-unsaturated carbonyls (62). PtGR-1 is the product of the DIG-1 (dithiolethione-inducible gene-1) gene and is identical to the leukotriene-B 4 dehydrogenase that catalyzes the oxidation of leukotriene B 4 (LTB 4 ) to 12-oxo-LTB 4 in humans and rats (37).…”

FaQR, Required for the Biosynthesis of the Strawberry Flavor Compound 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, Encodes an Enone Oxidoreductase

“…The physiological function of plant quinone oxidoreductases in developmental processes has been little studied, and to date no specific function for this class of proteins has been discovered except for an alkenal/one oxidoreductase (Mano et al, 2002;Dick and Kensler, 2004). Here, we present the isolation and biochemical and molecular characterization of a strawberry fruit ripening-related protein and its corresponding gene, exhibiting identity with plant quinone oxidoreductase enzymes and genes, respectively.…”

Mechanistic and Structural Studies of Apoform, Binary, and Ternary Complexes of the Arabidopsis Alkenal Double Bond Reductase At5g16970

“…For guinea pig 12-HD/PGR and rat liver AOR, a ketoreductase reaction mechanism has been proposed with a conjugated enolate as the reaction intermediate (29,55). Apparently, the latter enzyme is also able to reduce one of the phenylpropanoids, cinnamyl aldehyde (17) (30), whereas AtDBR1 does not reduce it.…”

“…Considering the fact that one side wall of the substratebinding site is made up of a nicotinamide ring of the tightly bound cofactor, the binding of either p-coumaryl aldehyde (6) or 4-HNE (15) can presumably be facilitated by cofactor binding. Therefore, it is reasonable to speculate that NADPH binds first and NADP ϩ leaves last, indicating that the kinetic mechanism of AtDBR could be an ordered Bi-Bi or Theorell-Chance mechanism, as in the cases of 12-HD/PGR and AOR (55,57). The dissociation of the NADP ϩ could, however, be ratelimiting.…”

“…when compared with the very well established Zn 2ϩ -containing MDRs such as alcohol dehydrogenase. Indeed, a majority of the enzymes provisionally belonging to this group are still depicted as putative gene products (55).…”