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 prese...

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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
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Summary:	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.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0166-6851 1872-9428
DOI:	10.1016/S0166-6851(02)00100-7