title: L-Mandelate dehydrogenase from Rhodotorula graminis: cloning, sequencing and kinetic characterization of the recombinant enzyme and its independently expressed flavin domain.
abstract: The l-mandelate dehydrogenase (L-MDH) from the yeast Rhodotorula graminis is a mitochondrial flavocytochrome b2 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 b2 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 b2, 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, kcat=225 s-1 at 25 degrees C) and artificial (ferricyanide, kcat=550 s-1 at 25 degrees C) electron acceptors. Kinetic isotope effects with [2-2H]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 b2 (L-lactate dehydrogenase) from S. cerevisiae.
authors.affiliation: Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland, U.K.
authors.forename: R M
authors.initials: RM
authors.lastname: Illias
authors.forename: R
authors.initials: R
authors.lastname: Sinclair
authors.forename: D
authors.initials: D
authors.lastname: Robertson
authors.forename: A
authors.initials: A
authors.lastname: Neu
authors.forename: S K
authors.initials: SK
authors.lastname: Chapman
authors.forename: G A
authors.initials: GA
authors.lastname: Reid
mesh_terms: D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005415:Flavins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008333:Mandelic Acids / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology*
chemical_list: D018076:DNA, Complementary; D005415:Flavins; D008333:Mandelic Acids; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C021538:L-mandelate dehydrogenase; C037938:mandelic acid