Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi.
Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.
Takashima
Eizo
E
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Japan.
Inaoka
Daniel Ken
DK
Osanai
Arihiro
A
Nara
Takeshi
T
Odaka
Masao
M
Aoki
Takashi
T
Inaka
Kozi
K
Harada
Shigeharu
S
Kita
Kiyoshi
K
D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D004081:Dihydroorotate Oxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005650:Fumarates; D007700:Kinetics; D010084:Oxidation-Reduction; D010269:Paraquat / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013385:Succinate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013386:Succinates / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D005650:Fumarates; D011994:Recombinant Proteins; D013386:Succinates; C032005:fumaric acid; D004081:Dihydroorotate Oxidase; D013385:Succinate Dehydrogenase; D010269:Paraquat