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Isolation and characterization of cDNA for human 120 kDa mitochondrial 2,4-dienoyl-coenzyme A reductase.

ABSTRACT: 2,4-Dienoyl-CoA reductase (EC 1.3.1.34) participates in beta-oxidation of (poly)unsaturated enoyl-CoAs and it appears in mammalian mitochondria as two isoforms with molecular masses of 120 and 60 kDa [Hakkola and Hiltunen (1993) Eur. J. Biochem. 215, 199-204]. The 120 kDa isomer is a homotetrameric enzyme, and here we report cDNA cloning of its subunit from human. cDNA clones were isolated by reverse transcriptase-PCR from a fibrosarcoma cell line and by screening from a human liver lambda gt11 cDNA library. The 1128 bp clone contained an open reading frame of 1008 bp encoding a polypeptide of 335 amino acid residues with a predicted molecular mass of 36066 Da. This polypeptide represents the immature monomer of the 120 kDa enzyme, and it contains a predicted N-terminal mitochondrial targeting signal. The amino acid (nucleotide) sequence of human 2,4-dienoyl-CoA reductase shows 82.7% (81.7%) similarity (identity) to the corresponding sequence from the rat. Northern-blot analysis gave a single mRNA species of 1.2 kb in several human tissues, the amounts present in the tissues tested ranking as follows: heart approximately liver approximately pancreas > kidney >> skeletal muscle approximately lung. Immunoblotting of human and rat liver samples with an antibody to the subunit of the rat 120 kDa isoform indicates that the mature human enzyme is larger than its counterpart in the rat. The comparison of amino acid sequences for rat and human enzymes proposes that the difference in the size is 10 amino acid residues. The results show that the rat and human reductases are similar in many characteristics and that the reductase is expressed in human tissues capable of beta-oxidation of fatty acids.

SUBMITTER: Koivuranta KT

PROVIDER: S-EPMC1137403 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Project description:Leishmania spp. are protozoan parasites that cause a spectrum of important diseases in humans. These parasites develop as extracellular promastigotes in the digestive tract of their insect vectors and as obligate intracellular amastigotes that infect macrophages and other phagocytic cells in their vertebrate hosts. Promastigote-to-amastigote differentiation is associated with marked changes in metabolism, including the upregulation of enzymes involved in fatty acid ?-oxidation, which may reflect adaptation to the intracellular niche. Here, we have investigated the function of one of these enzymes, a putative 2,4-dienoyl-coenzyme A (CoA) reductase (DECR), which is specifically required for the ?-oxidation of polyunsaturated fatty acids. The Leishmania DECR shows close homology to bacterial DECR proteins, suggesting that it was acquired by lateral gene transfer. It is present in other trypanosomatids that have obligate intracellular stages (i.e., Trypanosoma cruzi and Angomonas) but is absent from dixenous parasites with an exclusively extracellular lifestyle (i.e., Trypanosoma brucei). A DECR-green fluorescent protein (GFP) fusion protein was localized to the mitochondrion in both promastigote and amastigote stages, and the levels of expression increased in the latter stages. A Leishmania major ?decr null mutant was unable to catabolize unsaturated fatty acids and accumulated the intermediate 2,4-decadienoyl-CoA, confirming DECR's role in ?-oxidation. Strikingly, the L. major ?decr mutant was unable to survive in macrophages and was avirulent in BALB/c mice. These findings suggest that ?-oxidation of polyunsaturated fatty acids is essential for intracellular parasite survival and that the bacterial origin of key enzymes in this pathway could be exploited in developing new therapies.IMPORTANCE The Trypanosomatidae are protozoan parasites that infect insects, plants, and animals and have evolved complex monoxenous (single host) and dixenous (two hosts) lifestyles. A number of species of Trypanosomatidae, including Leishmania spp., have evolved the capacity to survive within intracellular niches in vertebrate hosts. The adaptations, metabolic and other, that are associated with development of intracellular lifestyles remain poorly defined. We show that genomes of Leishmania and Trypanosomatidae that can survive intracellularly encode a 2,4-dienoyl-CoA reductase that is involved in catabolism of a subclass of fatty acids. The trypanosomatid enzyme shows closest similarity to the corresponding bacterial enzymes and is located in the mitochondrion and essential for intracellular growth of Leishmania The findings suggest that acquisition of this gene by lateral gene transfer from bacteria by ancestral monoxenous Trypanosomatidae likely contributed to the development of a dixenous lifestyle of these parasites.

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Isolation and characterization of cDNA for human 120 kDa mitochondrial 2,4-dienoyl-coenzyme A reductase.

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