Cloning, Expression, and Purification of the Functional 2,4-Dienoyl-CoA Reductase from Rat Liver Mitochondria

Fillgrove, Kerry L.; Anderson, Vernon E.; Mizugaki, Michinao

doi:10.1006/prep.1999.1101

Cited by 3 publications

(4 citation statements)

References 18 publications

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“…Enzymes. The recombinant functional 24DCR was overexpressed in Escherichia coli and was purified by the previously described protocol (29). The purified enzyme exhibited a single band in Coomassie Blue stained SDS-PAGE and a molecular mass of 32 413 Da was determined by electrospray ionization mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

Orientation of Coenzyme A Substrates, Nicotinamide and Active Site Functional Groups in (Di)enoyl−coenzyme A Reductases

Fillgrove

Anderson

2000

Biochemistry

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The stereochemical course of reduction of dienoyl-coenzyme A (CoA) thiolesters catalyzed by the 2,4-dienoyl-CoA reductase from rat liver mitochondria was investigated. The configuration of the double bond in the 3-enoyl-CoA products was determined by (1)H NMR, and experiments to determine the stereochemical course of reduction at Calpha and Cdelta by use of 4-(2)H-labeled beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), were conducted in H(2)O and D(2)O. Defining the diastereoselectivity of the reaction, catalyzed by the Delta(3),Delta(2)-enoyl-CoA isomerase, facilitated the determination of the stereochemical course of reduction by 2, 4-dienoyl-CoA reductase. The absence of solvent exchange of the proton transferred during the Delta(3),Delta(2)-enoyl-CoA isomerase catalyzed equilibration of trans-2- and trans-3-enoyl-CoAs, coupled with the strong sequence homology to enoyl-CoA hydratase support the intramolecular suprafacial transfer of the pro-2R proton of trans-3-enoyl-CoA to the pro-4R position of trans-2-enoyl-CoA. The results indicate that the configuration of the double bond of the 3-enoyl-CoA product is trans and that a general acid-catalyzed addition of a solvent derived proton/deuteron occurs on the si face at Calpha of the dienoyl-CoA. The addition of the pro-4S hydrogen from NADPH occurs on the si face at Cdelta of trans-2, cis-4-dienoyl-CoA and on the re face at Cdelta of trans-2, trans-4-dienoyl-CoA. The stereochemical course of reduction of InhA, an enoyl-thiolester reductase from Mycobacterium tuberculosis, was also determined by use of ¿4-(2)HNADH in D(2)O. The reduction of trans-2-octenoyl-CoA catalyzed by InhA resulted in the syn addition of (2)H(2) across the double bond yielding (2R,3S)-¿2, 3-(2)H(2)ŏctanoyl-CoA. In the crystal structure of the InhA ternary complex, the residue donating the proton to Calpha could not be identified ¿Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589. The current results place further restrictions on the source of the proton and suggest the reduction is stepwise.

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Section: Methodsmentioning

confidence: 99%

Orientation of Coenzyme A Substrates, Nicotinamide and Active Site Functional Groups in (Di)enoyl−coenzyme A Reductases

Fillgrove

Anderson

2000

Biochemistry

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“…The concentrations of the trans-2,trans-4-dienoyl-CoA thiolesters were determined using an extinction coefficient at 296 nm of 23 000 M -1 cm -1 (32). The concentration of PPD-CoA was determined using an extinction coefficient at 340 nm of 40 400 M -1 cm -1 (Fillgrove and Anderson, unpublished results).…”

mentioning

confidence: 99%

“…Enzymes. The recombinant functional 24DCR was overexpressed in Escherichia coli and was purified by the previously described protocol (32). The purified enzyme exhibited a single band in Coomassie Blue stained SDS-PAGE and was used without any further manipulation.…”

mentioning

confidence: 99%

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

Fillgrove

Anderson

2001

Biochemistry

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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.

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Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase

Chu

Chen

et al. 2003

Protein Expression and Purification

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Cloning, Expression, and Purification of the Functional 2,4-Dienoyl-CoA Reductase from Rat Liver Mitochondria

Cited by 3 publications

References 18 publications

Orientation of Coenzyme A Substrates, Nicotinamide and Active Site Functional Groups in (Di)enoyl−coenzyme A Reductases

Orientation of Coenzyme A Substrates, Nicotinamide and Active Site Functional Groups in (Di)enoyl−coenzyme A Reductases

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

Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase

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