Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans.
Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.
Zameitat
Elke
E
Institute for Physiological Chemistry, Philipps-University, Marburg, Germany. zameitat@gmx.de
Gojković
Zoran
Z
Knecht
Wolfgang
W
Piskur
Jure
J
Löffler
Monika
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000611:Aminobiphenyl Compounds / Q000378:metabolism / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000378:metabolism / Q000494:pharmacology; D002176:Candida albicans / Q000201:enzymology* / Q000472:pathogenicity*; D017124:Conserved Sequence; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D011798:Quinaldines
D000611:Aminobiphenyl Compounds; D001713:Biphenyl Compounds; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011993:Recombinant Fusion Proteins; C093361:2,2'-((3,3'-dimethoxy(1,1'-biphenyl)-4,4'-diyl)diimino)bis-benzoic acid; C046943:brequinar; D011798:Quinaldines