Dihydroorotate dehydrogenase from Saccharomyces cerevisiae: spectroscopic investigations with the recombinant enzyme throw light on catalytic properties and metabolism of fumarate analogues.
In all organisms the fourth catalytic step of the pyrimidine biosynthesis is driven by the flavoenzyme dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11). Cytosolic DHODH of the established model organism Saccharomyces cerevisiae catalyses the oxidation of dihydroorotate to orotate and the reduction of fumarate to succinate. Here, we investigate the structure and mechanism of DHODH from S. cerevisiae and show that the recombinant ScDHODH exists as a homodimeric enzyme in vitro. Inhibition of ScDHODH by the reaction product was observed and kinetic studies disclosed affinity for orotate (K(ic)=7.7 microM; K(ic) is the competitive inhibition constant). The binding constant for orotate was measured through comparison of UV-visible spectra of the bound and unbound recombinant enzyme. The midpoint reduction potential of DHODH-bound flavine mononucleotide determined from analysis of spectral changes was -242 mV (vs. NHE) under anaerobic conditions. A search for alternative electron acceptors revealed that homologues such as mesaconate can be used as electron acceptors.
Zameitat
Elke
E
Institute of Physiological Chemistry, Philipps-University Marburg, Marburg, Germany.
Pierik
Antonio J
AJ
Zocher
Kathleen
K
Löffler
Monika
M
D002384:Catalysis; D000090722:Dihydroorotate Dehydrogenase; D005650:Fumarates / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008958:Models, Molecular; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013057:Spectrum Analysis
D000090722:Dihydroorotate Dehydrogenase; D005650:Fumarates; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors