The Mechanism of Dienoyl-CoA Reduction by 2,4-Dienoyl-CoA Reductase Is Stepwise:  Observation of a Dienolate Intermediate

Abstract

The chemical mechanism of the 2,4-dienoyl-CoA reductase (EC 1.3.1.34) from rat liver mitochondria has been investigated. This enzyme catalyzes the NADPH-dependent reduction of 2,4-dienoyl-coenzyme A (CoA) thiolesters to the resulting trans-3-enoyl-CoA. Steady-state kinetic parameters for trans-2,trans-4-hexadienoyl-CoA and 5-phenyl-trans-2,trans-4-pentadienoyl-CoA were determined and demonstrated that the dienoyl-CoA and NADPH bind to the 2,4-dienoyl-CoA reductase via a sequential kinetic mechanism. Kinetic isotope effect studies and the transient kinetics of substrate binding support a random order of nucleotide and dienoyl-CoA addition. The large normal solvent isotope effects on V/K ((D)(2)(O)V/K) and V ((D)(2)(O)V) for trans-2,trans-4-hexadienoyl-CoA reduction indicate that a proton transfer step is rate limiting for this substrate. The stability gained by conjugating the phenyl ring to the diene in PPD-CoA results in the reversal of the rate-determining step, as evidenced by the normal isotope effects on V/K(CoA) ((D)V/K(CoA)) and V/K(NADPH) ((D)V/K(NADPH)). The reversal of the rate-determining step was supported by transient kinetics where a burst was observed for the reduction of trans-2,trans-4-hexadienoyl-CoA but not for 5-phenyl-trans-2,trans-4-pentadienoyl-CoA reduction. The chemical mechanism is stepwise where hydride transfer from NADPH occurs followed by protonation of the observable dienolate intermediate, which has an absorbance maximum at 286 nm. The exchange of the C alpha protons of trans-3-decenoyl-CoA, catalyzed by the 2,4-dienoyl-CoA reductase, in the presence of NADP(+) suggests that formation of the dienolate is catalyzed by the enzyme active site.

Genome streamlining and chemical defense in a coral reef symbiosis

“…action of PtzO, which lacks similarity to known trans-AT PKS accessory proteins, but is similar to characterized 2,4-dienoylCoA-reductases (43). Under this hypothesis, PtzO must act in trans with the PKS to synthesize the cis-double bond during chain elongation.…”

Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

“…The formation of a dienolate anion intermediate during the course of reaction has been shown by kinetic and spectrophotometric methods (48). Similarly, incubation of Ccr with crotonyl-CoA in the absence of CO 2 may also result in the formation of such an enolate anion, because the true electrophile CO 2 is missing, and the rate-limiting step is shifted to the addition of a solvent proton replacing that CO 2 molecule.…”

Structure and Reactivity of Human Mitochondrial 2,4-Dienoyl-CoA Reductase

“…The first part of the mechanism involves cofactor binding and orientation of the nicotinamide to create the floor of the catalytic site. Once the substrate is correctly positioned the pyridine nucleotide cofactor donates the C4 pro-4S hydride to C␦, the electrophilic carbon of the conjugated thiolester (24). The ternary complex structure places the hydride donor C4 3.5 Å from the substrate C␦, in the correct orientation to accept the pro-4S hydride (Fig.…”

“…DECR, though similar, is distinct from other enoyl-thiolester reductases, because it catalyzes an unusual addition onto a conjugated diene, whereas the other enzymes are generally involved in fatty acid elongation in which they reduce a 2,3 (or ␣,␤) double bond. A detailed, elegant biochemical and nuclear magnetic resonance study of rat liver mDECR by Fillgrove and Anderson (24) identified that the stereochemical course of the reduction is novel and that a specific lysine is essential for catalysis. In classic SDRs this lysine occurs in combination with a tyrosine in an active site YXXXK motif (where X is any amino acid) but in enoyl reductases the motif is YXXMXXXK.…”