Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase.
In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of L- and D-lactate to pyruvate catalysed by L-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and D-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate- pyruvate+ mutants in a cyb2 genetic background. Two of them were devoid of D-LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on D-, L-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein L-gulono-gamma-lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of L-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.
Lodi
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Institute of Genetics, University of Parma, Italy.
Ferrero
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003580:Cytochromes / Q000096:biosynthesis; D005800:Genes, Fungal; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D045022:Lactate Dehydrogenases; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D003580:Cytochromes; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C081234:D-lactate dehydrogenase (cytochrome)