Characterization of 2-Enoyl Thioester Reductase from Mammals

Abstract

Fatty acid synthesis (FAS)1 can apparently take place in various subcellular organelles in eukaryotes. The eukaryotic extracytosolic FASs, like the fungal mitochondrial (1, 2), plant mitochondrial (3), or plastid (4) systems, are similar to the prokaryotic type of FAS (type II) in terms of having a set of separate monofunctional enzymes (5) in contrast to the eukaryotic cytosolic FAS (type I), which consists of multifunctional polypeptide(s) (6, 7). Fatty acid chain elongation in the mammalian and fungal endoplasmic reticulum also resembles FAS type II, except for the facts that it is membrane-associated (8) and has CoA as an acyl group carrier instead of acyl carrier protein (ACP).The properties of the eukaryotic FASs and their role in metabolism appear to vary depending on the organism and the subcellular organelle; this is analogous to the ␤-oxidation of fatty acids. Although both mitochondrial and peroxisomal ␤-oxidation perform chain shortening of acyl-CoA substrates, the kinetic properties of the enzymes differ, and they clearly do not represent a metabolic redundancy, as exemplified by inborn errors of fatty acid oxidation (for review see Ref. 9). The disruption of any of the mitochondrial FAS type II genes in yeast results in a respiration-deficient phenotype (1, 2, 10), although the cytosolic FAS is still operational. Also the mitochondrial FAS in the yeast cannot compensate for a defective cytosolic pathway, and the inactivation of the cytosolic FAS is lethal, unless the cells are supplemented with fatty acids in the culture media (11).We have recently identified a novel type of 2-enoyl thioester reductase from Candida tropicalis (Etr1p) and Saccharomyces cerevisiae (Ybr026p) that is involved in mitochondrial FAS (12). The ybr026c⌬ strain exhibits reduced mitochondrial cytochrome contents and an inability to grow on nonfermentable carbon sources (13). The yeast FAS type II 2-enoyl thioester reductases are homodimeric, NADPH-dependent, and are not sequence-related to the homotetrameric reductases of bacterial FAS type II.Rat nuclear receptor binding factor-1 (RnNrbf-1p), which was described as homolog of the Ybr026p/Mrf1Јp by protein data base searches, was observed previously to interact with peroxisome proliferator-activated receptor ␣ as well as with a number of other nuclear receptors in the yeast two-hybrid assay (14). In the current work open reading frames (ORF) encoding the human homolog (HsNrbf-1p) of the rat Nrbf-1p was identified in human EST data bank, and the cDNA encoding the bovine homolog (BtNrbf-1p) was cloned. The data demonstrated that Nrbf-1ps are mitochondrial proteins that catalyze an NADPH-dependent reduction of trans-2-enoyl thioesters to the corresponding saturated acyl thioesters. As the expression of human NRBF-1 in the ybr026c⌬ yeast strain rescues the respiration-deficient phenotype, it is concluded that Nrbf-1ps are the orthologs of the yeast 2-enoyl thioester reductases, enzymes previously unidentified in mammals.

Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

“…Knockdown efficiencies of ϳ75% for malonyltransferase and enoyl reductase were insufficient to compromise the lipoylation status of any of the mitochondrial proteins. 3 The metabolism of malonate, the ACSF3 substrate, has not been studied extensively in animals. However, malonate is known to be present in substantial concentrations, and levels as high as 200 nmol/g have been measured in normal brain (31).…”

“…The mitochondrial systems are composed of an acyl carrier protein (ACP) 2 and malonyltransferase (which together generate the malonyl-ACP substrate used for chain extension by ␤-ketoacyl synthase) and a trio of ␤-carbon-processing enzymes (␤-ketoacyl reductase, dehydrase, and enoyl reductase) that completely saturate the acyl chain prior to the following round of chain extension. All of these enzymes have been characterized in mammalian mitochondria (1)(2)(3)(4)(5), and recent evidence indicates that one of the major functions of the pathway is to generate the octanoyl precursor required for formation of the lipoyl moieties that are essential for post-translational modification of several mitochondrial proteins (6,7). The source of malonyl-CoA as the substrate for a mitochondrial fatty acid synthase system is unknown (8).…”

“…To evaluate the effect of ACSF3 knockdown on mitochondrial function, we assessed the ability of mitochondria to utilize [2][3][4][5][6][7][8][9][10][11][12][13][14] C]malonate for fatty acid synthesis. The presence in these mitochondrial preparations of substantial thioesterase activity against malonyl-CoA precluded estimation of the amount of this metabolite formed (7).…”

Cloning, Expression, and Characterization of the Human Mitochondrial β-Ketoacyl Synthase

“…The presence of a similar mitochondrial FAS system in animals has been suspected for some time, based initially on the discovery of an ACP-like protein in animal mitochondria (8,9). However, only recently have other components of a putative mitochondrial FAS been identified, cloned, and characterized; they include the human ACP and malonyl transferase (10) and enoyl reductase (11). In continuation of the search for other components, we have now identified and characterized a single candidate for a type II human mitochondrial ␤-ketoacyl synthase, the critical enzyme required for catalysis of the chain-elongating condensation reaction.…”

“…Synthase Activity Using Acetyl-CoA as Primer-Recombinant mitochondrial ␤-ketoacyl synthase was assayed for condensing activity using [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetyl-CoA as the primer and malonyl-ACP mit as the chain extender. The malonyl-ACP mit was first generated in situ from malonyl-CoA and holoACP mit using human mitochondrial malonyl transferase (10).…”