Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi

• Published in: Molecular and biochemical parasitology Vol. 122; no. 2; pp. 189 - 200
• Main Authors: Takashima, Eizo, Inaoka, Daniel Ken, Osanai, Arihiro, Nara, Takeshi, Odaka, Masao, Aoki, Takashi, Inaka, Kozi, Harada, Shigeharu, Kita, Kiyoshi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-07-2002
• Subjects: Animals; Binding Sites; Cytosol - enzymology; Dihydroorotate dehydrogenase; Dihydroorotate Oxidase - genetics; Dihydroorotate Oxidase - isolation & purification; Dihydroorotate Oxidase - metabolism; Fumarate reductase; Fumarates; Kinetics; Methylviologen; Oxidation-Reduction; Paraquat - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Solubility; Succinate Dehydrogenase - genetics; Succinate Dehydrogenase - isolation & purification; Succinate Dehydrogenase - metabolism; Succinate production; Succinates - metabolism; Trypanosoma cruzi; Trypanosoma cruzi - enzymology; Trypanosoma cruzi - genetics; Trypanosoma cruzi - growth & development; Fumarate reductase; BA, barbiturate; FRD, fumarate reductase; QFR, quinol–fumarate reductase; SDH, succinate dehydrogenase; DHO, dihydroorotate; Succinate production; DHOD, dihydroorotate dehydrogenase; Trypanosoma cruzi; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DCIP, 2,6-dichlorophenol-indophenol; SQR, succinate–quinone reductase; MV, methylviologen; rDHOD, recombinant Trypanosoma cruzi DHOD; Methylviologen; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,4-tetrazolium bromide; Q 1, ubiquinone-1; Dihydroorotate dehydrogenase; TLC, thin-layer chromatography
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(02)00100-7

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.