Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity.

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.

Peroxisomal Delta3-cis-Delta2-trans-enoyl-CoA isomerase encoded by ECI1 is required for growth of the yeast Saccharomyces cerevisiae on unsaturated fatty acids.

We have identified the Saccharomyces cerevisiae gene ECI1 encoding Delta3-cis-Delta2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of (poly)unsaturated fatty acids. A mutant devoid of Eci1p was unable to grow on media containing unsaturated fatty acids such as oleic acid but was proficient for growth when a saturated fatty acid such as palmitic acid was the sole carbon source. Levels of ECI1 transcript were elevated in cells grown on oleic acid medium due to the presence in the ECI1 promoter of an oleate response element that bound the transcription factors Pip2p and Oaf1p. Eci1p was heterologously expressed in Escherichia coli and purified to homogeneity. It was found to be a hexameric protein with a subunit of molecular mass 32, 000 Da that converted 3-hexenoyl-CoA to trans-2-hexenoyl-CoA. Eci1p is the only known member of the hydratase/isomerase protein family with isomerase and/or 2-enoyl-CoA hydratase 1 activities that does not contain a conserved glutamate at its active site. Using a green fluorescent protein fusion, Eci1p was shown to be located in peroxisomes of wild-type yeast cells. Rat peroxisomal multifunctional enzyme type I containing Delta3-cis-Delta2-trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.

Delta3,5-delta2,4-dienoyl-CoA isomerase from rat liver. Molecular characterization.

rECH1, a recently identified rat cDNA (FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995) Genomics 27, 457-466) encodes a polypeptide belonging to the hydratase/isomerase superfamily. We modeled the structure of rECH1 based on rat mitochondrial 2-enoyl-CoA hydratase 1. The model predicts that rECH1p has the hydratase fold in the core domain and two domains for interaction with other subunits. When we incubated 3,5,8,11, 14-eicosapentaenoyl-CoA with purified rECH1p, the spectral data suggested a switching of the double bonds from the Delta3-Delta5 to the Delta2-Delta4 positions. This was confirmed by demonstrating that the product was a valid substrate for 2,4-dienoyl-CoA reductase. These results indicate that rECH1p is Delta3,5-Delta2,4-dienoyl-CoA isomerase. Subcellular fractionation and immunoelectron microscopy using antibodies to a synthetic polypeptide derived from the C terminus of rECH1p showed that rECH1p is located in the matrix of both mitochondria and peroxisomes in rat liver. Consistent with these observations, the 36,000-Da rECH1p has a potential N-terminal mitochondrial targeting signal as well as a C-terminal peroxisomal targeting signal type 1. Transport of the protein into the mitochondria with cleavage of the targeting signal results in a mature mitochondrial form with a molecular mass of 32,000 Da; transport to peroxisomes yields a protein of 36,000 Da.

The Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is encoded by the oleate-inducible gene SPS19.

beta-Oxidation is compartmentalized in mammals into both mitochondria and peroxisomes. Fatty acids with double bonds at even-numbered positions require for their degradation the auxiliary enzyme 2,4-dienoyl-CoA reductase, and at least three isoforms, two mitochondrial and one peroxisomal, exist in the rat. The Saccharomyces cerevisiae Sps19p is 34% similar to the human and rat mitochondrial reductases, and an SPS19 deleted strain was unable to utilize petroselineate (cis-C18:1(6)) as the sole carbon source, but remained viable on oleate (cis-C18:1(9)). Sps19p was purified to homogeneity from oleate-induced cells and the homodimeric enzyme (native molecular weight 69,000) converted 2,4-hexadienoyl-CoA into 3-hexenoyl-CoA in an NADPH-dependent manner and therefore contained 2,4-dienoyl-CoA reductase activity. Antibodies raised against Sps19p decorated the peroxisomal matrix of oleate-induced cells. SPS19 shares with the sporulation-specific SPS18 a common promoter region that contains an oleate response element. This element unidirectionally regulates transcription of the reductase and is sufficient for oleate induction of a promoterless CYC1-lacZ reporter gene. SPS19 is dispensable for growth and sporulation on solid acetate and oleate media, but is essential for these processes to occur on petroselineate.

Characterization and isolation of enzymes that hydrolyze short-chain acyl-CoA in rat-liver mitochondria.

In this study we investigated the presence of short-chain acyl-CoA hydrolases in rat liver mitochondria. One acetyl-CoA-hydrolyzing enzyme with a molecular mass of about 48 kDa was purified to apparent homogeneity as judged by SDS/PAGE. Immunoprecipitation experiments with antibodies raised to the purified protein showed that this enzyme corresponds to a minor portion of the total mitochondrial acetyl-CoA hydrolase activity, most (about 90%) of the total activity being due to an enzyme which was labile and required Triton X-100 for its stability. Neither of these acetyl-CoA-hydrolyzing enzymes appeared to be induced by treatment of rats with di(2-ethylhexyl)phthalate, a peroxisome proliferator which mediates induction of several cytosolic and mitochondrial long-chain acyl-CoA thioesterases. In addition, an enzyme that hydrolyzed acetoacetyl-CoA was partially purified; it was induced about 3.5-fold by di(2-ethylhexyl)phthalate treatment. In conclusion, these results demonstrate that rat liver mitochondria contain several enzymes capable of hydrolyzing short-chain acyl-CoA, indicating that regulation of the metabolism of short-chain acyl-CoAs and formation of ketone bodies, could be complex.