Peroxisomal Trifunctional Enzyme Research Articles

Molecular mechanisms of fatty acid beta-oxidation enzyme catalysis.

The -oxidation of fatty acids is an important metabolic process that takes place in various organisms ranging from E. coli to Homo sapiens. In animal cells there are different -oxidation systems in mitochondria and peroxisomes. Mitochondrial -oxidation of fatty acids provides a significant part of the energy in some organs. For example, free fatty acids are the preferred substrate for energy production in heart. On the other hand, the peroxisomal -oxidation system degrades very long-chain fatty acids and other uncommon carboxylic acids but does not provide useful energy.1 More complexity was found in plant cells where glyoxysomes—which correspond to peroxisomes—have a unique -oxidation system.2 Fungi possess peroxisomal bifunctional -oxidation enzymes employing D-specific substrates but not L-isomers. The stereochemical specificity of the latter enzymes is the opposite of that for the animal peroxisomal trifunctional enzyme. During prolonged fasting or long-lasting exercise, mitochondrial -oxidation supplies energy to various tissues, producing acetyl-CoA for liver ketogenesis to supply circulating ketone bodies as an important alternative fuel in extrahepatic tissues. In addition to the mitochondrial matrix -oxidation enzymes, a long-chain-specific -oxidation enzyme system with a very long-chain acyl-CoA dehydrogenase and a trifunctional oxidation complex has been discovered on the mitochondrial inner membrane.5 Although the fatty acid -oxidation spiral comprises only four reactions, understanding of the complexity of fatty acid degradation has dramatically increased in recent years due to the discovery of a variety of new -oxidation enzymes. This article will discuss the enzymes that catalyze the second and third steps of the -oxidation pathway, an area of recent and substantial progress.

Structures of the human cDNA and gene encoding the 78 kDa gastrin-binding protein and of a related pseudogene

The nucleotide sequence encoding the human 78 kDa gastrin binding protein (GBP) has been deduced from overlapping fragments generated by the polymerase chain reaction with oligonucleotides based on the sequence of the porcine GBP (Mantamadiotis, T. et al. (1993) Biochim. Biophys. Acta 1170, 211–215) and cDNA from the colonic carcinoma cell line LIM 1215 as template. The mature human GBP is 90% identical to the porcine GBP. Clones encoding the human GBP gene, which contains 19 exons, have been isolated from human genomic libraries. The positions of the exon/intron junctions are completely different from the junctions in the gene encoding the related peroxisomal trifunctional enzyme. Clones encoding a related pseudogene have also been isolated and sequenced.

Nucleotide sequence encoding a novel member of the hydratase/dehydrogenase family

The nucleotide sequence encoding a novel member of the family of fatty acid oxidation enzymes has been determined. Clones were generated from porcine gastric corpus cDNA by application of the polymerase chain reaction (PCR) using oligodeoxyri-bonucleotides based on the amino acid sequence of a 78 kDa gastrin-binding protein isolated from porcine gastric mucosal membranes (Baldwin et al., J. Biol. Chem. 261 (1986) 12252–12257). Clones encoding the 3' and 5' ends of the cDNA were then isolated by conventional screening of a porcine liver cDNA library, and by application of anchored PCR, respectively. The composite cDNA of 2744 nucleotides encoded a protein of 763 amino acids, the sequence of which was related to a rat peroxisomal trifunctional enzyme, Δ 3, Δ 2-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and to other members of the same enzyme family. Northern blots indicated that the 3-kb mRNA encoding the novel protein was abundant in gastric corpus and liver, with lower amounts also present in gastric antrum and forestomach.

Partial Structure of the Gene Encoding the 78-kDa Gastrin Binding Protein Excludes a Close Relationship with the Peroxisomal Trifunctional Enzyme

A 10.9 kb porcine gerlomic clone encoding nucleotides 124-732 of the cDNA for the porcine 78 kDa gastrin-binding protein has been isolated and characterized. The coding sequence is interrupted by 7 introns, which vary in length from 93 to 3000bp. The positions of the intron/exon junctions are different from the junctions in the gene encoding the rat peroxisomal trifunctional enzyme. Despite 33% amino acid sequence identity between the two proteins it is concluded that the porcine gastrin binding protein is not closely related to the rat trifunctional enzyme.

NADPH-dependent beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms.

The mitochondrial metabolism of 5-enoyl-CoAs, which are formed during the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms, was studied with mitochondrial extracts and purified enzymes of beta-oxidation. Metabolites were identified spectrophotometrically and by high performance liquid chromatography. 5-cis-Octenoyl-CoA, a putative metabolite of linolenic acid, was efficiently dehydrogenated by medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis-octadienoyl-CoA, which was isomerized to 3,5-octadienoyl-CoA either by mitochondrial delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) or by peroxisomal trifunctional enzyme. Further isomerization of 3,5-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA in the presence of soluble extracts of either rat liver or rat heart mitochondria was observed and attributed to a delta 3,5,delta 2,4-dienoyl-CoA isomerase. Qualitatively similar results were obtained with 2-trans-5-trans-octadienoyl-CoA formed by dehydrogenation of 5-trans-octenoyl-CoA. 2-trans-4-trans-Octadienoyl-CoA was a substrate for NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34). A soluble extract of rat liver mitochondria catalyzed the isomerization of 2-trans-5-cis-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA, which upon addition of NADPH, NAD+, and CoA was chain-shortened to hexanoyl-CoA, butyryl-CoA, and acetyl-CoA. Thus we conclude that odd-numbered double bonds, like even-numbered double bonds, can be reductively removed during the beta-oxidation of polyunsaturated fatty acids.

The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme to yeast peroxisomes.

The gene encoding Candida tropicalis peroxisomal trifunctional enzyme, hydratase-dehydrogenase-epimerase (HDE), was expressed in both Candida albicans and Saccharomyces cerevisiae. The cellular location of HDE was determined by subcellular fractionation followed by Western blot analysis of peroxisomal and cytosolic fractions using antiserum specific for HDE. HDE was found to be exclusively targeted to and imported into peroxisomes in both heterologous expression systems. Deletion and mutational analyses were used to determine the regions within HDE which are essential for its targeting to peroxisomes. Deletion of a carboxyl-terminal tripeptide Ala-Lys-Ile completely abolished targeting of HDE to peroxisomes, whereas large internal deletions of HDE (amino acids 38-353 or 395-731) had no effect on HDE targeting to peroxisomes in either yeast. This tripeptide is similar to, but distinct from, other tripeptide peroxisomal targeting sequences (PTSs) as identified in peroxisomal firefly luciferase and four mammalian peroxisomal proteins. Substitutions within the carboxyl-terminal tripeptide (Ala----Gly and Lys----Gln) supported targeting of HDE to peroxisomes of C. albicans but not of S. cerevisiae. This is the first detailed analysis of the peroxisomal targeting signal in a yeast peroxisomal protein.

Sequence of the gene encoding Candida tropicalis peroxisomal trifunctional enzyme

The gene ( HDE) encoding the peroxisomal trifunctional enzyme, hydratase-dehydrogenase-epimerase, of the yeast Candida tropicalis, was cloned and its nucleotide sequence is reported.

Glucose-responsive and oleic acid-responsive elements in the gene encoding the peroxisomal trifunctional enzyme of Candida tropicalis

We have investigated the regulation of expression of the gene ( HDE), encoding the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase (HDE), of the diploid yeast Candida tropicalis. Heterologous expression in Saccharomyces cerevisiae of constructs containing deletions in the upstream region of the HDE gene has allowed for determination of regions responsible for the control of expression of the HDE gene. Expression was monitored by immunoblot analysis of yeast lysates with anti-HDE serum. Regions have been identified that are responsible for both repression by glucose and induction by oleic acid. A glucose-responsive region lies between nucleotides (nt) −526 and −393, An oleic acid-responsive region lies between ntt-393 and −341. An additional region controlling derepression by nonfermentable carbon sources is located downstream from nt −341. Comparison of the nt sequences of these regions to upstream regions of other oleic acid-responsive genes of C. tropicalis has identified possible consensus nt sequences for glucose- and oleic acid-responsive upstream elements in these genes. The regulation of the HDE gene in S. cerevisiae closely resembles that found in C. tropicalis, suggesting that similar mechanisms of transcriptional control operate in both yeasts.

Occurrence of a long-chain Δ3,Δ2-enoyl-CoA isomerase in rat liver

Isoproteins of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8), an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids having double bonds at odd-numbered positions, were studied in livers of control and clofibrate-treated rats. When liver extracts were applied to a hydroxyapatite column at pH 7.0, the previously characterized peroxisomal trifunctional hydratase-dehydrogenase-isomerase enzyme and the mitochondrial isomerase, which shows a preference for short-chain substrates, were eluted almost in parallel. In addition to these activities, a separate isomerase was observed to elute at a lower potassium phosphate concentration in the gradient. Experiments with extracts of purified mitochondria and peroxisomes demonstrated the mitochondrial origin of this third activity. Studies on the kinetic properties of the third isomerase showed that it has a preference for C10-C12 substrates. An Mr of 200,000 was obtained for the native protein by gel-filtration chromatography. Antibodies to mitochondrial short-chain isomerase and peroxisomal trifunctional enzyme did not recognize this novel mitochondrial isoenzyme. The immunological non-cross-reactivity can be interpreted as suggesting that the different isomerases are not closely related at the level of the primary structure of the polypeptide chain. The present data demonstrate that, similar to many other enzymes of beta-oxidation, delta 3,delta 2-enoyl-CoA isomerase has at least three isoenzymes in rat liver: mitochondrial short- and long-chain isomerases and an additional peroxisomal isoenzyme, which in this case is a part of a multifunctional protein.

CDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional β-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A) + RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in λgt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA ( HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The M r of the protein is 99 350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels.