L(+)-Mandelate dehydrogenase from Rhodotorula graminis: purification, partial characterization and identification as a flavocytochrome b.
L(+)-Mandelate dehydrogenase was purified to homogeneity from the yeast Rhodotorula graminis KGX 39 by a combination of (NH4)2SO4 fractionation, ion-exchange and hydrophobic-interaction chromatography and gel filtration. The amino-acid composition and the N-terminal sequence of the enzyme were determined. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50172 (4 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9. The enzyme is a tetramer as judged by comparison of its subunit M(r) value of 59,100 and native M(r) of 239,900, estimated by SDS/PAGE and gel filtration respectively. There is one molecule of haem and approx. one molecule of non-covalently bound FMN per subunit. 2,6-Dichloroindophenol, cytochrome c and ferricyanide can all serve as electron acceptors. L(+)-Mandelate dehydrogenase is stereospecific for its substrate. D(-)-Mandelate and L(+)-hexahydromandelate are competitive inhibitors. The enzyme has maximum activity at pH 7.9 and it has a pI value of 4.4. HgCl2 and 4-chloromercuribenzoate are potent inhibitors, but there is no evidence that the enzyme is subject to feedback inhibition by potential metabolic effectors. The evidence suggests that L(+)-mandelate dehydrogenase from R. graminis is a flavocytochrome b which is very similar to, and probably (at least so far as the haem domain is concerned) homologous with, certain well-characterized yeast L(+)-lactate dehydrogenases, and that the chief difference between them is their mutually exclusive substrate specificities.
Yasin
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Department of Biochemistry, University of Glasgow, U.K.
Fewson
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CA
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003573:Cytochrome b Group / Q000378:metabolism*; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012248:Rhodotorula / Q000201:enzymology*; D012492:Salts; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents
D000596:Amino Acids; D003573:Cytochrome b Group; D005415:Flavins; D012492:Salts; D013439:Sulfhydryl Reagents; D006418:Heme; D000429:Alcohol Oxidoreductases; C021538:L-mandelate dehydrogenase