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

Abstract

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.

• Authors: Takeshi Nara, Muneaki Hashimoto, Hiroko Hirawake, Chien-Wei Liao, Yoshihisa Fukai, Shigeo Suzuki, Akiko Tsubouchi, Jorge Morales, Shinzaburo Takamiya, Tsutomu Fujimura, Hikari Taka, Reiko Mineki, Chia-Kwung Fan, Daniel Ken Inaoka, Masayuki Inoue, Akiko Tanaka, Shigeharu Harada, Kiyoshi Kita, Takashi Aoki
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 418 Issue 1 Pg. 140-3 (Feb 03 2012) ISSN: 1090-2104 [Electronic] United States
• PMID: 22245425 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier Inc. All rights reserved.

Chemical References

• Pyrimidines
• Aspartate Carbamoyltransferase
• Dihydroorotase
• Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)
• pyrimidine

Topics

• Aspartate Carbamoyltransferase (metabolism)
• Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) (metabolism)
• Dihydroorotase (metabolism)
• Immunoprecipitation
• Pyrimidines (biosynthesis)
• Trypanosoma cruzi (enzymology)