Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly(16) and Gly(329) of the S. cerevisiae A and B domains, corresponding to Gly(16), which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Delta)) and mutational variants of the A and B domains (Ct MFE- 2(h2DeltaaDelta), Ct MFE- 2(h2DeltabDelta), and Ct MFE- 2(h2DeltaaDeltabDelta)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization.

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.

Enzymes converting D-3-hydroxyacyl-CoA to trans-2-enoyl-CoA. Microsomal and peroxisomal isoenzymes in rat liver.

Epiermization of 3-hydroxyacyl-CoA, which has been shown to occur as a two-step dehydration-hydration reaction (Hiltunen, J. K., Palosaari, P. M., and Kunau, W.-H. (1989) J. Biol. Chem. 264, 13536-13540; Smeland, E., Jianxun, L., Chu, C., Cuebas, D., and Schulz, H. (1989) Biochem. Biophys. Res. Commun. 160, 988-992) was studied in rat liver. Subcellular fractionations of rat liver on different density gradients revealed a dual distribution of activity, catalyzing dehydration of D-3-hydroxydecanoyl-CoA to trans-2-decenoyl-CoA (hydratase 2) in both peroxisomal and microsomal compartments. Both hydratase 2 activity peaks were separated by dye ligand chromatography from the extract of the combined heavy and light mitochondrial fractions. The activity eluted at low salt was identified as the microsomal isoform and was purified to apparent homogeneity. The M(r) of the native protein (subunit) was found to be 60,000 (31,500), indicating that it is homodimeric. The enzyme activity was inhibited by IgGs isolated from antisera raised against the denatured subunit. The activity eluted at high salt was tentatively identified to be peroxisomal of origin, and the M(r) of the native protein (subunit) was determined to be 62,000 (33,500). The peroxisomal enzyme was not recognized by the antibody to its microsomal counterpart. Analysis of the reaction products of microsomal enzyme activity by gas chromatography-mass spectrometry showed that the enzyme catalyzed reversibly hydration/dehydration between trans-2-enoyl-CoA and D-3-hydroxyacyl-CoA, but L-3-hydroxydecanoyl-CoA was not dehydrated to delta 2-enoyl-CoA compounds. Similar reaction characteristics were also determined for the peroxisomal hydratase by using stereospecific auxiliary enzymes. The present data demonstrate that rat liver contains microsomal and peroxisomal proteins possessing hydratase 2 activities. Although their kinetic properties are similar, immunological data, subunit sizes, and chromatographic evidence clearly indicate that they are different enzymes. Comparisons with other hydratases revealed that the microsomal and peroxisomal hydratase 2 described in the present work are proteins that have not been previously purified.