Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.

Abstract

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate-growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDHI gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.Two isozymes of malate dehydrogenase in mammalian cells catalyze the interconversion of malate plus NAD+ and oxaloacetate plus NADH. The mitochondrial isozyme functions in the tricarboxylic acid cycle, and the cytosolic isozyme catalyzes a step in gluconeogenesis from pyruvate. Also, as components of the malate/aspartate shuttle cycle, the malate dehydrogenase isozymes control the exchange of metabolic intermediates and reducing equivalents across the mitochondrial membrane. Analyses of amino acid sequences for the isozymes from porcine heart (4, 5) and of nucleotide sequences for the porcine and murine cDNAs (25,26) (MATo leu2 his3-AJ trpl gcrl-J-J; 34) was used for isolation of the nonmitochondrial isozyme of malate dehydrogenase. Yeast strains S173-6B (MATa leu2-3,112 his3-AJ ura3-57 trpl-289; 7) and SAMDH1, a derivative of S173-6B containing a chromosomal disruption of the MDH1 gene (47), were used for transformation, growth studies, and cell fractionation. The parental S. cerevisiae strain MMY011 (MATa ade2-1 his3-11,15 leu2-3,112 trpl-l ura3-1 canl-100) and 370 on April 28, 2019 by guest

TCA cycle‐independent acetate metabolism via the glyoxylate cycle in Saccharomyces cerevisiae

“…It thus appears that the succinate dehydrogenase complex (SDHC), a tetramer of Sdh1, Sdh2, Sdh3 and Sdh4 (Lombardo et al, 1990;Chapman et al, 1992;Bullis and Lemire, 1994;Daignan-Fornier et al, 1994), is the only TCA cycle-specific enzyme required for acetate utilization on YNBA. Our results also show that the enzymes involved in the glyoxylate cycle operation, such as ACO (Aco1) (Gangloff et al, 1990), malate synthase (Mls1) (Hartig et al, 1992;Kunze et al, 2002), isocitrate lyase (Icl1) (Fernandez et al, 1992;Taylor et al, 1996) and Mdh2 (Minard and McAlister-Henn, 1991), are indispensable for the growth of yeast cells with acetate on YNBA, regardless of their involvement in the TCA cycle. Considering that both the cytoplasmic and mitochondrial isoforms of fumarase (FUM) are encoded by a single gene, FUM1 (Wu and Tzagoloff, 1987), and that operation of the TCA cycle is dispensable on YNBA, as shown above, it is suggested that conversion of fumarate to malate Figure 6.…”

“…In previous studies, the enzyme isoforms of the TCA or glyoxylate cycle have been studied mainly in terms of the correlation between their subcellular distributions and metabolic functions. Three MDH genes, MDH1 (McAlister-Henn and Thompson, 1987), MDH2 (Minard and McAlister-Henn, 1991) and MDH3 (Steffan and McAlister-Henn, 1992), encoding mitochondrial, cytosolic and peroxisomal variants, have been identified in S. cerevisiae. The cytosolic isozyme Mdh2, but not the peroxisomal enzyme Mdh3, functions in the glyoxylate cycle (Minard and McAlister-Henn, 1991;Van Roermund et al, 1995), whereas the mitochondrial isoform Mdh1 participates in the TCA cycle (McAlister-Henn and Thompson, 1987).…”

“…Three MDH genes, MDH1 (McAlister-Henn and Thompson, 1987), MDH2 (Minard and McAlister-Henn, 1991) and MDH3 (Steffan and McAlister-Henn, 1992), encoding mitochondrial, cytosolic and peroxisomal variants, have been identified in S. cerevisiae. The cytosolic isozyme Mdh2, but not the peroxisomal enzyme Mdh3, functions in the glyoxylate cycle (Minard and McAlister-Henn, 1991;Van Roermund et al, 1995), whereas the mitochondrial isoform Mdh1 participates in the TCA cycle (McAlister-Henn and Thompson, 1987). Two isoenzymes of ACO encoded by a single gene, ACO1, are distributed between two distinct subcellular compartments, the mitochondria and the cytosol, and participate in the TCA cycle and the glyoxylate cycle, respectively (Gangloff et al, 1990;Regev-Rudzki et al, 2005).…”

Malate dehydrogenase: A model for structure, evolution, and catalysis

“…A phylogenetic tree was constructed by using the program PILEUP. The following complete MDH amino acid sequences were available: maize (Metzler et al, 1989); sorghum (Luchetta et al, 1991); pig (c) (Birktoft et al, 1989b); mouse (c) (Setoyama et al, 1988); T. fluvus (Nishiyama et al, 1986); S. typhimurium (Lu & Abdelal, 1993); E. coli (McAlister-Henn et al, MDHs (Grant et al, 1987); pig (m) (Birktoft & Banaszak, 1987); mouse (m) (Takeshima et al, 1988); rat (m) 1984); watermelon (Gietl et al, 1990); yeast (Minard & McAlister-Henn, 1991); H. marismortui (Cendrin et al, 1993). The LDH sequences used were rabbit chain" (Sass et al, 1989) and dogfish chain" (Eventoff et al ., 1977) and B. stearothermophilus LDH (Barstow et al, 1986 …”

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH

“…The sequences of both the mouse and yeast c-and mMDH genes have been reported (Minard and McAlister-Henn, 1991;Setoyama et al, 1988;Takeshima et al, 1988;Thompson et al, 1988). MDH genes have also been isolated from several bacterial sources (McAlister-Henn et al, 1987;Nicholls et al, 1990;Nishiyama et al, 1986).…”