Publications

• Candida tropicalis expresses two mitochondrial 2-enoyl thioester reductases that are able to form both homodimers and heterodimers.

  Torkko J.M., Koivuranta K.T., Kastaniotis A.J., Airenne T.T., Glumoff T., Ilves M., Hartig A., Gurvitz A., Hiltunen J.K.

  Here we report on the cloning of a Candida tropicalis gene, ETR2, that is closely related to ETR1. Both genes encode enzymatically active 2-enoyl thioester reductases involved in mitochondrial synthesis of fatty acids (fatty acid synthesis type II) and respiratory competence. The 5'- and 3'-flanki ... >> More

  Here we report on the cloning of a Candida tropicalis gene, ETR2, that is closely related to ETR1. Both genes encode enzymatically active 2-enoyl thioester reductases involved in mitochondrial synthesis of fatty acids (fatty acid synthesis type II) and respiratory competence. The 5'- and 3'-flanking (coding) regions of ETR2 and ETR1 are about 90% (97%) identical, indicating that the genes have evolved via gene duplication. The gene products differ in three amino acid residues: Ile67 (Val), Ala92 (Thr), and Lys251 (Arg) in Etr2p (Etr1p). Quantitative PCR analysis and reverse transcriptase-PCR indicated that both genes were expressed about equally in fermenting and ETR1 predominantly respiring yeast cells. Like the situation with ETR1, expression of ETR2 in respiration-deficient Saccharomyces cerevisiae mutant cells devoid of Ybr026p/Etr1p was able to restore growth on glycerol. Triclosan that is used as an antibacterial agent against fatty acid synthesis type II 2-enoyl thioester reductases inhibited growth of FabI overexpressing mutant yeast cells but was not able to inhibit respiratory growth of the ETR2- or ETR1-complemented mutant yeast cells. Resolving of crystal structures obtained via Etr2p and Etr1p co-crystallization indicated that all possible dimer variants occur in the same asymmetric unit, suggesting that similar dimer formation also takes place in vivo. << Less

  J. Biol. Chem. 278:41213-41220(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Studies on the metabolism of unsaturated fatty acids. IX. Stereochemical studies of the reaction catalyzed by trans-2-enoyl-coenzyme A reductase of Escherichia coli.

  Mizugaki M., Nishimaki T., Shiraishi T., Kawaguchi A., Okuda S., Yamanaka H.

  The steric course of the reaction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase from Escherichia coli was investigated. trans-2-[6,7,8-2H7]Octenoyl-CoA was synthesized as a substrate and incubated with partially purified trans-2-enoyl-CoA reductase in the presence of 4R- or 4S-[4-2H1]NA ... >> More

  The steric course of the reaction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase from Escherichia coli was investigated. trans-2-[6,7,8-2H7]Octenoyl-CoA was synthesized as a substrate and incubated with partially purified trans-2-enoyl-CoA reductase in the presence of 4R- or 4S-[4-2H1]NADPH. The deuterium-labeled octenoyl-CoA was also incubated with the reductase in the presence of NADPH in 2H2O. Aliquots of octanoic acids formed were analyzed, after esterification, by gas chromatography-mass spectrometry (GC-MS) to demonstrate that the pro-4R hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA and that hydrogen from water was introduced into the C-2 position of octenoyl-CoA. The remaining portions of octanoic acids isolated from the incubation mixtures were converted to their CoA esters by the action of acyl-CoA synthetase, and they were dehydrogenated by treatment with acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2R and pro-3R hydrogen atoms of acyl-CoA. The deuterium contents of the products were also analyzed by GC-MS, and the results indicated that the reduction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase occurred by an anti-addition of hydrogen via a 2-Re, 3-Re attack on the trans-double bond of the substrate. << Less

  J Biochem 92:1649-1654(1982) [PubMed] [EuropePMC]

• Structural enzymological studies of 2-enoyl thioester reductase of the human mitochondrial FAS II pathway: new insights into its substrate recognition properties.

  Chen Z.J., Pudas R., Sharma S., Smart O.S., Juffer A.H., Hiltunen J.K., Wierenga R.K., Haapalainen A.M.

  Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the bi ... >> More

  Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway. << Less

  J. Mol. Biol. 379:830-844(2008) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Molecular cloning and expression of mammalian peroxisomal trans-2-enoyl-coenzyme A reductase cDNAs.

  Das A.K., Uhler M.D., Hajra A.K.

  Chain elongation of fatty acids is an important cellular process and is believed to occur in the endoplasmic reticulum of all eukaroytic cells. Herein we describe the cloning and characterization of a peroxisomal NADPH-specific trans-2-enoyl-CoA reductase, the key enzyme for a proposed peroxisomal ... >> More

  Chain elongation of fatty acids is an important cellular process and is believed to occur in the endoplasmic reticulum of all eukaroytic cells. Herein we describe the cloning and characterization of a peroxisomal NADPH-specific trans-2-enoyl-CoA reductase, the key enzyme for a proposed peroxisomal chain elongation pathway. The reductase was solubilized and partially purified from guinea pig liver peroxisomes by affinity chromatography. On SDS-polyacrylamide gel electrophoresis, a 40-kDa band was identified as the enzyme, and its partial amino acid sequence (27 amino acids) was determined. A full-length cDNA for the reductase was cloned from a guinea pig liver cDNA library. The open reading frame of this nucleotide sequence encodes a 302-amino acid polypeptide with a calculated molecular mass of 32.5 kDa. Full-length mouse and human cDNA clones encoding homologous proteins have also been isolated. All of these translated polypeptides have the type I peroxisomal targeting signal, AKL, at the carboxyl terminus. The identity of the cloned enoyl-CoA reductase cDNAs was confirmed by expressing the guinea pig and human cDNAs in Escherichia coli. The His-tagged recombinant enzymes were found to have very high NADPH-specific 2-enoyl-CoA reductase activity with similar properties and specificity as the liver peroxisomal reductase. Both the natural and the recombinant enzyme catalyze the reduction of trans-2-enoyl-CoAs of varying chain lengths from 6:1 to 16:1, having maximum activity with 10:1 CoA. Northern blot analysis demonstrated that a single transcript of 1.3 kilobases is present in most mouse tissues, with particularly high concentrations in liver and kidney. << Less

  J. Biol. Chem. 275:24333-24340(2000) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Characterization of 2-enoyl thioester reductase from mammals: an ortholog of Ybr026p/Mrf1'p of the yeast mitochondrial fatty acid synthesis type II.

  Miinalainen I.J., Chen Z.-J., Torkko J.M., Pirilae P.L., Sormunen R.T., Bergmann U., Qin Y.-M., Hiltunen J.K.

  A data base search with YBR026c/MRF1', which encodes trans-2-enoyl thioester reductase of the intramitochondrial fatty acid synthesis (FAS) type II in yeast (Torkko, J. M., Koivuranta, K. T., Miinalainen, I. J., Yagi, A. I., Schmitz, W., Kastaniotis, A. J., Airenne, T. T., Gurvitz, A., and Hiltune ... >> More

  A data base search with YBR026c/MRF1', which encodes trans-2-enoyl thioester reductase of the intramitochondrial fatty acid synthesis (FAS) type II in yeast (Torkko, J. M., Koivuranta, K. T., Miinalainen, I. J., Yagi, A. I., Schmitz, W., Kastaniotis, A. J., Airenne, T. T., Gurvitz, A., and Hiltunen, K. J. (2001) Mol. Cell. Biol. 21, 6243-6253), revealed the clone AA393871 (HsNrbf-1, nuclear receptor binding factor 1) in human EST data bank. Expression of HsNrbf-1, tagged C-terminally with green fluorescent protein, in HeLa cells, resulted in a punctated fluorescence signal, superimposable with the MitoTracker Red dye. Wild-type polypeptide was immunoisolated from the extract of bovine heart mitochondria. Recombinant HsNrbf-1p reduces trans-2-enoyl-CoA to acyl-CoA with chain length from C6 to C16 in an NADPH-dependent manner with preference to medium chain length substrate. Furthermore, expression of HsNRBF-1 in the ybr026cDelta yeast strain restored mitochondrial respiratory function allowing growth on glycerol. These findings provide evidence that Nrbf-1ps act as a mitochondrial 2-enoyl thioester reductase, and mammalian cells may possess bacterial type fatty acid synthetase (FAS type II) in mitochondria, in addition to FAS type I in the cytoplasm. << Less

  J. Biol. Chem. 278:20154-20161(2003) [PubMed] [EuropePMC]