title: 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.affiliation: Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. tnara@juntendo.ac.jp
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nara
authors.forename: Muneaki
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hirawake
authors.forename: Chien-Wei
authors.initials: CW
authors.lastname: Liao
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Fukai
authors.forename: Shigeo
authors.initials: S
authors.lastname: Suzuki
authors.forename: Akiko
authors.initials: A
authors.lastname: Tsubouchi
authors.forename: Jorge
authors.initials: J
authors.lastname: Morales
authors.forename: Shinzaburo
authors.initials: S
authors.lastname: Takamiya
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Fujimura
authors.forename: Hikari
authors.initials: H
authors.lastname: Taka
authors.forename: Reiko
authors.initials: R
authors.lastname: Mineki
authors.forename: Chia-Kwung
authors.initials: CK
authors.lastname: Fan
authors.forename: Daniel Ken
authors.initials: DK
authors.lastname: Inaoka
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inoue
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanaka
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Harada
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
authors.forename: Takashi
authors.initials: T
authors.lastname: Aoki
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D004080:Dihydroorotase / Q000378:metabolism*; D047468:Immunoprecipitation; D011743:Pyrimidines / Q000096:biosynthesis*; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D011743:Pyrimidines; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); C030986:pyrimidine