title: Genetic diversity and kinetic properties of Trypanosoma cruzi dihydroorotate dehydrogenase isoforms.
abstract: Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.
authors.affiliation: Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Idalia
authors.initials: I
authors.lastname: Sariego
authors.forename: Takeshi
authors.initials: T
authors.lastname: Annoura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nara
authors.forename: Muneaki
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Akiko
authors.initials: A
authors.lastname: Tsubouchi
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Iizumi
authors.forename: Takashi
authors.initials: T
authors.lastname: Makiuchi
authors.forename: Eri
authors.initials: E
authors.lastname: Murata
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
authors.forename: Takashi
authors.initials: T
authors.lastname: Aoki
mesh_terms: D000818:Animals; D001483:Base Sequence; D000090722:Dihydroorotate Dehydrogenase; D005650:Fumarates / Q000378:metabolism; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics*
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D005650:Fumarates; D007527:Isoenzymes; D011994:Recombinant Proteins; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; C032005:fumaric acid; D044925:Oxidoreductases Acting on CH-CH Group Donors