Purification and characterization of a plant peroxisomal Δ²,Δ³‐enoyl‐CoA isomerase acting on 3‐cis‐enoyl‐CoA and 3‐trans‐enoyl‐CoA

Abstract

A A3,AZ-enoyl-CoA isomerase was extracted from fat-degrading cotyledons of cucumber seedlings. The enzyme is required for the degradation of cis-unsaturated fatty acids, e.g. linoleic acid being present in the storage tissue of cucumber seedlings in high amounts. The A3,A2-enoyl-CoA isomerase was exclusively localized within peroxisomes. Its purification included chromatography on a hydrophobic matrix, a cation exchanger, and on hydroxylapatite. 17 000-fold purification yielded a protein of apparent homogeneity. The isomerase is a homodimer with a M , of 50000 and an isoelectric point of pH approximately 8.1. A3,A2-Enoyl-CoA isomerase reversibly catalyzes the conversion of both cis-3-enoyl-CoA and trans-3-enoyl-CoA into trans-2-enoyl-CoA. The isomerase exhibited optimal activity at pH 9 and was active with 3-enoyl-CoA species having chain lengths of C6 -CI2, with cis-hexenoyl-CoA being the most effective substrate. The purified enzyme did not show enoyl-CoA hydratase activity.Fatty acid degradation is an oxidative reaction sequence. As well as the chain-shortening and oxidative steps [l, 21, the metabolism of cis-unsaturated fatty acids requires the function of isomerases, both for the activity converting 3-cis-enoylCoA into 2-trans-enoyl-CoA and the activity converting 3-trans-enoyl-CoA into 2-trans-enoyl-CoA [3]. In the case of linoleic acid degradation, the respective substrate for these reactions are 3-cis-6-cis-dodecadienoyl-CoA, and 3-transdecenoyl-CoA originating from 2,4-decadienoyl-CoA by reduction [4, 51. It was therefore probable that both enzymes activities occur in fat-degrading tissue, one acting on 3-cis compound and the other one converting 3-trans derivatives.Isomerases responsible for bringing about 1,3-proton shifts have been described in the case of 3-oxosteroid isomerase [6] and isopentenyl diphosphate isomerase [7]. Isomerase activities acting on 3-enoyl-CoA species of fatty acids have been found in rat liver [8] and were characterized as 3-alkinyl thioester isomerase in hog liver [8]. The mitochondria1 isoenzyme in rat liver [9] is a homodimer with subunit M , 30000. In rat liver peroxisomes, the isomerase activity is part of the trifunctional protein possessing 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activities [lo]. In these cases, the isomerase activities were assayed with both 3-trans-enoyl-CoA and 3-cis-enoyl-CoA [8, 91. We describe the d3,A2-enoyl-CoA isomerase from a fatdegrading plant tissue, where it is exclusively confined to peroxisomes, but is not part of the trifunctional protein of fatty acid j-oxidation. MATERIALS AND METHODS Reagents and substratesCoA esters of alkenoic acids were synthesized via mixed anhydrides [ l l , 121 and were purified by HPLC on a CI8 column. 2-trans-Decenoic acid [13], 2-cis-decenoic acid [14], 3-cis-decenoyl-CoA [15, 161, 3-trans-hexenoic acid, 3-transoctenoic acid [I71 and 3-cis-hexenoic acid [15, 181 were synthesized according to established methods. 2-trans-4-transDecadienoic acid was prepared from decadienal [14]. The s...

Peroxisomal Δ3-cis-Δ2-trans-Enoyl-CoA Isomerase Encoded by ECI1 Is Required for Growth of the Yeast Saccharomyces cerevisiae on Unsaturated Fatty Acids

“…Enzymes-A search of the data bases for novel genes that contained in their promoters sequences resembling OREs with the consensus CGGN [15][16][17][18]23) and that ended with nucleotides similar to those coding for a peroxisomal targeting signal type 1 (Ref. 41) revealed a homologous pair of open reading frames YLR284c (Fig.…”

“…Human peroxisomal MFE type I and one of the two human mitochondrial isoforms have also been characterized (11)(12)(13). Other ⌬ 3 -cis-⌬ 2 -trans-enoyl-CoA isomerases that have been isolated include those from bovine and porcine mitochondria (14,15) as well as two contained in the glyoxysomes of cucumber seedlings (16,17). The isomerases whose amino acid sequences have been determined belong to the low homology hydratase/isomerase protein family that currently includes over 30 known members (18).…”

Evidence for Domain Structures of the Trifunctional Protein and the Tetrafunctional Protein Acting in Glyoxysomal Fatty Acid β‐Oxidation

“…The CoA esters of ~,~-3-hydroxydecanoic acid, D-3-hydroxydecanoic acid, 3-cis-hexenoic acid, and various 3-trans-alkenoic acids were synthesized as described [13,141. Sodium ~-3-hydroxybutyrate, ~-3-hydroxybutyryl-CoA, cro-tonoyl-CoA, acetoacetyl-CoA were purchased from Sigma.…”

“…6) have to be considered less positively charged, while peptides collected after the MFP should be highly positively charged. In more detail, we investigated the peptides in fractions [13][14][15][16][17][18] [17], and rat peroxisomal MFP [5]. The positions of the first amino acid residues within the MFP-a sequence are indicated by parentheses.…”

Molecular Identification and Characterization of the Arabidopsis Δ3,5,Δ2,4-Dienoyl-Coenzyme A Isomerase, a Peroxisomal Enzyme Participating in the β-Oxidation Cycle of Unsaturated Fatty Acids

“…The D 3 ,D 2 -enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase have been found to be essential in yeast (Saccharomyces cerevisiae) for the metabolism of fatty acids having cis-double bonds on even-chain carbons, such as petroselinic acid (6-cis-octadecenoic acid), catalyzing the stepwise conversion of the intermediate 2-trans,4-cisdienoyl-CoA to 3-trans-enoyl-CoA and 2-trans-enoylCoA, the latter intermediate being a normal substrate for the core b-oxidation cycle. Both D 3 ,D 2 -enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase activities have been detected in plants, but the corresponding genes have not been characterized (Behrends et al, 1988;Engeland and Kindl, 1991).…”