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

Abstract

Fatty acid catabolism by ␤-oxidation mainly occurs in mitochondria and to a lesser degree in peroxisomes. Polyunsaturated fatty acids are problematic for ␤-oxidation, because the enzymes directly involved are unable to process all the different double bond conformations and combinations that occur naturally. In mammals, three accessory proteins circumvent this problem by catalyzing specific isomerization and reduction reactions. Central to this process is the NADPH-dependent 2,4-dienoyl-CoA reductase. We present high resolution crystal structures of human mitochondrial 2,4-dienoylCoA reductase in binary complex with cofactor, and the ternary complex with NADP ؉ and substrate trans-2,trans-4-dienoyl-CoA at 2.1 and 1.75 Å resolution, respectively. The enzyme, a homotetramer, is a shortchain dehydrogenase/reductase with a distinctive catalytic center. Close structural similarity between the binary and ternary complexes suggests an absence of large conformational changes during binding and processing of substrate. The site of catalysis is relatively open and placed beside a flexible loop thereby allowing the enzyme to accommodate and process a wide range of fatty acids. Seven single mutants were constructed, by site-directed mutagenesis, to investigate the function of selected residues in the active site thought likely to either contribute to the architecture of the active site or to catalysis. The mutant proteins were overexpressed, purified to homogeneity, and then characterized. The structural and kinetic data are consistent and support a mechanism that derives one reducing equivalent from the cofactor, and one from solvent. Key to the acquisition of a solvent-derived proton is the orientation of substrate and stabilization of a dienolate intermediate by Tyr-199, Asn-148, and the oxidized nicotinamide.Essential fatty acids and derivatives that mammals acquire from exogenous sources or by endogenous biosynthesis fulfill critical functions in numerous metabolic pathways, in endocrine, and signaling processes (1, 2). Fatty acids also provide much of the cells energy following degradation in a sequence of four enzyme-catalyzed reactions termed the ␤-oxidation cycle (1, 3, 4), a highly exergonic metabolic process so named because oxidation occurs at C␤ of a fatty acyl-coenzyme A (CoA) 1 derivative preceding cleavage of the C␣-C␤ bond. The initial step leading into ␤-oxidation is fatty acid activation by formation of a thiolester bond with CoA in a reaction catalyzed by acyl-CoA synthetase. The oxidation of the C␣-C␤ bond produces an olefin, then hydration and oxidation produce a carbonyl group at C␤. The fourth step is cleavage of the ␤-keto ester in a reverse Claisen condensation resulting in a new acyl-CoA derivative truncated by two C atoms, which are used to produce acetyl-CoA. These reactions proceed until eventually only acetyl-CoA is produced. Extensive studies have afforded a very clear picture of structure, mechanism, and specificity of the four enzymes (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-L-hydrox...

The Crystal Structure of Progesterone 5β-Reductase from Digitalis lanata Defines a Novel Class of Short Chain Dehydrogenases/Reductases

“…As a result, the tyrosine moves by the distance of one complete helical turn along the helix from which it is displayed. In dienoyl-reductases, which catalyze a 1-6 hydrogen addition, the tyrosine residue is moved to a different framework segment altogether (50). Why in 5␤-POR yet another solution for the active site has been adopted when compared with other enoyl-reductases is again not obvious.…”

Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

“…1C). This is in contrast to the tetramer observed for mDCR packed in the crystal lattice (20). Such a pure (Ͼ95% purity as judged by SDS-PAGE analysis) but heterogeneous pDCR was active against the substrate HXC when tested for activity (Table 2).…”

“…An invariant lysine, a tyrosine, a serine, and an asparagine have been shown to participate in reactions catalyzed by enzymes belonging to the SDR superfamily, including mDCR (20). Analysis of the primary amino acid sequence of the pDCR by aligning it with that of its counterpart from mitochondrion revealed that pDCR does not contain two of the four residues important for catalysis (Fig.…”

“…Activity Assay and Kinetic Studies-Reductase activity was determined kinetically by following the substrate-dependent decrease in absorbance of NADPH at 340 nm at room temperature (20). The assay mixture contained 50 mM PBS, pH 7.4, 100 M EDTA, 125 M NADPH, and 10 g of enzyme in a final volume of 1.0 ml.…”

“…Although mitochondrial DCRs (mDCR) and their counterparts in the peroxisome (pDCR) catalyze similar reactions, they share only 35% sequence identity. More important, two of the residues (tyrosine and serine) of the catalytic tetrad, a hallmark of the SDR family of enzymes, including mDCR (20), are missing in the pDCR (see Fig. 1A).…”