L-Mandelate dehydrogenase from Rhodotorula graminis: cloning, sequencing and kinetic characterization of the recombinant enzyme and its independently expressed flavin domain

R M Illias, R Sinclair, Douglas Robertson, Achim Neu, Stephen K Chapman, Graeme A Reid

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    Abstract

    The L-mandelate dehydrogenase (L-MDH) from the yeast Rhodotorula graminis is a mitochondrial flavocytochrome b(2) which catalyses the oxidation of mandelate to phenylglyoxylate coupled with the reduction of cytochrome c. We have used the N-terminal sequence of the enzyme to isolate the gene encoding this enzyme using the PCR. Comparison of the genomic sequence with the sequence of cDNA prepared by reverse transcription PCR revealed the presence of 11 introns in the coding region. The predicted amino acid sequence indicates a close relationship with the flavocytochromes b(2) from Saccharomyces cerevisiae and Hansenula anomala, with about 40%,, identity to each. The sequence shows that a key residue for substrate specificity in S. cerevisiae flavocytochrome b(2), Leu-230, is replaced by Gly in L-MDH. This substitution is likely to play an important part in determining the different substrate specificities of the two enzymes. We have developed an expression system and purification protocol for recombinant L-MDH. In addition, we have expressed and purified the flavin-containing domain of L-MDH independently of its cytochrome domain. Detailed steady-state and pre-steady-state kinetic investigations of both L-MDH and its independently expressed flavin domain have been carried out. These indicate that L-MDH is efficient with both physiological (cytochrome c, k(cat) = 225 s(-1) at 25 degrees C) and artificial (ferricyanide, k(cat) = 550 s(-1) at 25 degrees C) electron accepters. Kinetic isotope effects with [2-H-2]mandelate indicate that H-C-2 bond cleavage contributes somewhat to rate-limitation. However, the value of the isotope effect erodes significantly as the catalytic cycle proceeds. Reduction potentials at 25 degrees C were measured as - 120 mV for the 2-electron reduction of the flavin and -10 mV for the 1-electron reduction of the haem. The general trends seen in the kinetic studies show marked similarities to those observed previously with the flavocytochrome b(2) (L-lactate dehydrogenase) from S. cerevisiae.

    Original languageEnglish
    Pages (from-to)107-115
    Number of pages9
    JournalBiochemical Journal
    Volume333
    Issue number1
    Publication statusPublished - 1 Jul 1998

    Keywords

    • NUCLEOTIDE-SEQUENCE
    • ESCHERICHIA-COLI
    • FLAVOCYTOCHROME B(2)
    • CYTOCHROME-C PEROXIDASE
    • INTERMEMBRANE SPACE
    • MOLECULAR-CLONING
    • YEAST FLAVOCYTOCHROME-B2
    • L-LACTATE
    • SPINACH GLYCOLATE OXIDASE
    • SACCHAROMYCES-CEREVISIAE

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