Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

• Published in: Biochemical and biophysical research communications 2012-02, Vol.418 (1), p.140-143
• Main Authors: Nara, Takeshi, Hashimoto, Muneaki, Hirawake, Hiroko, Liao, Chien-Wei, Fukai, Yoshihisa, Suzuki, Shigeo, Tsubouchi, Akiko, Morales, Jorge, Takamiya, Shinzaburo, Fujimura, Tsutomu, Taka, Hikari, Mineki, Reiko, Fan, Chia-Kwung, Inaoka, Daniel Ken, Inoue, Masayuki, Tanaka, Akiko, Harada, Shigeharu, Kita, Kiyoshi, Aoki, Takashi
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; ANTIBODIES; Aspartate Carbamoyltransferase - metabolism; Aspartate transcarbamoylase; BIOSYNTHESIS; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - metabolism; Carbamoyl-phosphate synthetase II; COPRECIPITATION; De novo pyrimidine biosynthetic pathway; Dihydroorotase; Dihydroorotase - metabolism; DRUGS; ESCHERICHIA COLI; FUNGI; Immunoprecipitation; LIGASES; PHOSPHATES; Pyrimidines - biosynthesis; TRYPANOSOMA; Trypanosoma cruzi; Trypanosoma cruzi - enzymology; UMP; URIDINE
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2011.12.148

► An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. ► Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. ► CPSII bound with both ATC and DHO. ► ATC bound with both CPSII and DHO. ► A functional tri-enzyme complex might precede the establishment of the fused enzyme. The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded—and led to—gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.