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...
-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.
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