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.

Calmodulin (CaM) is a ubiquitous Ca2+‐binding protein that can regulate a wide variety of cellular events. The protein contains 9 Met out of a total of 148 amino acid residues. The binding of Ca2+ to CaM induces conformational changes and exposes two Met‐rich hydrophobic surfaces which provide the main protein‐protein contact areas when CaM interacts with its target enzymes. Two‐dimensional (1H,13C)‐heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy was used to study selectively 13C‐isotope labelled Met methyl groups in apo‐CaM, Ca2+‐CaM and a complex of CaM with the CaM‐binding domain of skeletal muscle Myosin Light Chain Kinase (MLCK). The resonance assignment of the Met methyl groups in these three functionally different states were obtained by site‐directed mutagenesis (Met→Leu). Chemical shift changes indicate that the methyl groups of the Met residues are in different environments in apo‐, calcium‐, and MLCK‐bound‐CaM. The T 1 relaxation rates of the individual Met methyl carbons in the three forms of CaM indicate that those in Ca2+‐CaM have the highest mobility. Our results also suggest that the methyl groups of the unbranched Met sidechains in general are more flexible than those of aliphatic amino acid residues such as Leu and Ile.

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(a⌬) and pYE352::ScMFE-2(b⌬) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(a⌬b⌬) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE-2(h2⌬)) and mutational variants of the A and B domains (Ct MFE-2(h2⌬a⌬), Ct MFE-2(h2⌬b⌬), and Ct MFE-2(h2⌬a⌬b⌬)) 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)-hydroxyacylCoA 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.The degradation of fatty acids in yeast was observed to be confined to the peroxisome that contains a complete fatty acid ␤-oxidation system, an acyl-CoA oxidase, a multifunctional enzyme (MFE) 1 type 2 possessing 2-enoyl-CoA hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase (HADH) activities and a 3-ketoacyl-CoA thiolase (1, 2). Because multifunctional enzyme type 1 (MFE-1), which metabolizes trans-2-acyl-CoA to 3-ketometabolites via (3S)-hydroxyacyl-CoA, is missing in yeast peroxisomes, the ␤-oxidation proceeds via the (3R)-hydroxayacylCoA intermediate. In contrast to yeast, both MFE-1 and MFE-2 are present in mammalian peroxisomes (3, 4). MFE-1 is proposed to be responsible for metabolism of straight chain fatty acyl-CoA esters and MFE-2 mainly for metabolism of ␣-methyl branched acyl-CoA esters (5). Among several reported mutations leading to MFE-2 deficiency in humans is a G16S mutation in the nucleotide-binding site (6).Yeast MFE-2 has been cloned from Candida tropicalis (7) and characterized from Saccharomyces cerevisiae (2). The amino acid sequence comparison of MFE-2(s) reveals that yeast enzymes contain the two domains A and B belonging to the short chain alcohol dehydrogenase/reductase superfamily (8), whereas the mammalian MFE-2 has only one. The (3R)-HADH activities of MFE-2 have been assigned to the short chain alcohol dehydrogenase/reductase domains in both the yeast (2) and mammalian enzymes (Refs. 9 and 10 and Fig. 1). An interesting question arises from what the physiological functions of the two domains are or even whether both of them show en...

Peroxisomes are capable of oxidizing a variety of substrates including (poly)unsaturated enoyl-CoA esters. The beta-oxidation of unsaturated enoyl-CoA esters in peroxisomes, and also in mitochondria, is not just chain-shortening but also involves the metabolizing of pre-existing carbon-to-carbon double bonds. In addition to the enzymes of the beta-oxidation spiral itself, this metabolism requires the participation of auxiliary enzymes: delta 3, delta 2-enoyl-CoA isomerase; 2,4-dienoyl-CoA reductase; 2-enoyl-CoA hydratase 2 or 3-hydroxyacyl-CoA epimerase; and delta 3,5 delta 2,4-dienoyl-CoA isomerase. Many of these enzymes are present as isoforms, and can be found located in multiple subcellular compartments, for example, peroxisomes, mitochondria or the endoplasmic reticulum, while some of the activities are integral parts of multifunctional enzymes of beta-oxidation systems.

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