Yeast Peroxisomal Multifunctional Enzyme: (3R)-Hydroxyacyl-CoA Dehydrogenase Domains A and B Are Required for Optimal Growth on Oleic Acid

Abstract

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...

The Multifunctional β-Oxidation Enzyme Is Required for Full Symptom Development by the Biotrophic Maize Pathogen Ustilago maydis

“…A separate hydratase domain (PF01575/IPR002539) was present in the C-terminal region. It is possible that the dehydrogenase domains may contribute to different substrate specificities; for example, the first domain is most active with longand medium-chain substrates, and the second domain with short-chain substrates in S. cerevisiae (60). The C-terminal domain encoding the 2-enoyl-CoA hydratase 2 is common to all multifunctional enzymes of the Mfe2 type (Fig.…”

Increasing the Carbon Flux toward Synthesis of Short-Chain-Length—Medium-Chain-Length Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae through Modification of the β-Oxidation Cycle

“…The chimeric genes were cloned into the yeast centromeric vector p416GPD, putting the genes under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter, resulting in the constructs p415GPD::PTS2-PHAC Re and p415GPD::PTS2-PHAC Ac (6). Mutants with mutations in the 3-hydroxyacyl-CoA dehydrogenase A and B domains of the S. cerevisae multifunctional enzyme (MFE-2) encoded by the FOX2 gene have been previously described by Qin et al (12). Briefly, the MFE-2(a⌬) mutant retains a broad activity towards short (C4)-, medium (C10)-, and long (C16)-chain R-3-hydroxyacyl-CoAs, while the MFE-2(b⌬) mutant shows highest activity with medium-and long-chain R-3-hydroxyacyl-CoAs and does not accept the short-chain R-3-hydroxybutyryl-CoA (12).…”

“…Mutants with mutations in the 3-hydroxyacyl-CoA dehydrogenase A and B domains of the S. cerevisae multifunctional enzyme (MFE-2) encoded by the FOX2 gene have been previously described by Qin et al (12). Briefly, the MFE-2(a⌬) mutant retains a broad activity towards short (C4)-, medium (C10)-, and long (C16)-chain R-3-hydroxyacyl-CoAs, while the MFE-2(b⌬) mutant shows highest activity with medium-and long-chain R-3-hydroxyacyl-CoAs and does not accept the short-chain R-3-hydroxybutyryl-CoA (12). The plasmid pYE352::ScMFE-2 containing the intact multifunctional gene from S. cerevisiae, as well as the plasmids pYE352::ScMFE-2(a⌬) and pYE352::ScMFE-2(b⌬) containing the mutated variants of the MFE-2 gene, have been previously described (12).…”

“…Briefly, the MFE-2(a⌬) mutant retains a broad activity towards short (C4)-, medium (C10)-, and long (C16)-chain R-3-hydroxyacyl-CoAs, while the MFE-2(b⌬) mutant shows highest activity with medium-and long-chain R-3-hydroxyacyl-CoAs and does not accept the short-chain R-3-hydroxybutyryl-CoA (12). The plasmid pYE352::ScMFE-2 containing the intact multifunctional gene from S. cerevisiae, as well as the plasmids pYE352::ScMFE-2(a⌬) and pYE352::ScMFE-2(b⌬) containing the mutated variants of the MFE-2 gene, have been previously described (12). All MFE-2 gene constructs were expressed in the vector pYE352, placing the genes under the control of the catalase A (CTA1) promoter and terminator (12).…”

“…The plasmid pYE352::ScMFE-2 containing the intact multifunctional gene from S. cerevisiae, as well as the plasmids pYE352::ScMFE-2(a⌬) and pYE352::ScMFE-2(b⌬) containing the mutated variants of the MFE-2 gene, have been previously described (12). All MFE-2 gene constructs were expressed in the vector pYE352, placing the genes under the control of the catalase A (CTA1) promoter and terminator (12). Plasmids harboring the various PHA synthases and MFE-2 constructs were transformed by the lithium acetate procedure (2) into the S. cerevisiae mutant fox2⌬0 (YKR009C:: kanMX4) in the BY4742 background (MAT␣ his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0) obtained from EUROSCARF (http://www.uni -frankfurt.de/fb15/mikro/euroscarf/index.html).…”