L-MANDELATE DEHYDROGENASE FROM RHODOTORULA-GRAMINIS - CLONING, SEQUENCING AND KINETIC CHARACTERIZATION OF THE RECOMBINANT ENZYME AND ITS INDEPENDENTLY EXPRESSED FLAVIN DOMAIN

The L-mandelate dehydrogenase (L-MDH) from the yeast Rhodotorula grami nis is a mitochondrial flavocytochrome b(2) which catalyses the oxidat ion of mandelate to phenylglyoxylate coupled with the reduction of cyt ochrome c. We have used the N-terminal sequence of the enzyme to isola te the gene encoding this enzyme using the PCR. Comparison of the geno mic sequence with the sequence of cDNA prepared by reverse transcripti on PCR revealed the presence of 11 introns in the coding region. The p redicted amino acid sequence indicates a close relationship with the f lavocytochromes b(2) from Saccharomyces cerevisiae and Hansenula anoma la, with about 40%,, identity to each. The sequence shows that a key r esidue 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 specifi cities of the two enzymes. We have developed an expression system and purification protocol for recombinant L-MDH. In addition, we have expr essed 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 f lavin domain have been carried out. These indicate that L-MDH is effic ient with both physiological (cytochrome c, k(cat) = 225 s(-1) at 25 d egrees C) and artificial (ferricyanide, k(cat) = 550 s(-1) at 25 degre es C) electron accepters. Kinetic isotope effects with [2-H-2]mandelat e indicate that H-C-2 bond cleavage contributes somewhat to rate-limit ation. However, the value of the isotope effect erodes significantly a s the catalytic cycle proceeds. Reduction potentials at 25 degrees C w ere measured as - 120 mV for the 2-electron reduction of the flavin an d -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) fr om S. cerevisiae.