Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase.

摘要: In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded CYB2 gene, (D-LCR) 1.1.2.4), respectively. We selected several lactate− pyruvate+ mutants in a cyb2 genetic background. Two them were devoid D -LCR activity (dld mutants, belonging same complementation group). The mutation mapped structural gene. This was demonstrated gene dosage effect thermosensitivity enzyme thermosensitive revertants. DLD cloned for growth on d-, strain WWF18-3D, carrying both disruption dld mutation. minimal complete complementing sequence localized subcloning experiments. From analysis an open reading frame (ORF) identified that could encode polypeptide 576 amino-acids, corresponding calculated molecular weight 64000 Da. deduced protein showed significant homology with previously described microsomal flavoprotein l-gulono-γ-lactone oxidase isolated from Rattus norvegicus, which catalyses terminal step l-ascorbic acid biosynthesis. These results are discussed together role L-LCR D-LCR metabolism S. cerevisiae.