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Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruzi.

ABSTRACT: Aspartate transcarbamoylase (ATCase), the second enzyme of the de novo pyrimidine-biosynthetic pathway, catalyzes the production of carbamoyl aspartate from carbamoyl phosphate and L-aspartate. In contrast to Escherichia coli ATCase and eukaryotic CAD multifunctional fusion enzymes, Trypanosoma cruzi ATCase lacks regulatory subunits and is not part of the multifunctional fusion enzyme. Recombinant T. cruzi ATCase expressed in E. coli was purified and crystallized in a ligand-free form and in a complex with carbamoyl phosphate at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. Ligand-free crystals (space group P1, unit-cell parameters a = 78.42, b = 79.28, c = 92.02 A, alpha = 69.56, beta = 82.90, gamma = 63.25 degrees) diffracted X-rays to 2.8 A resolution, while those cocrystallized with carbamoyl phosphate (space group P2(1), unit-cell parameters a = 88.41, b = 158.38, c = 89.00 A, beta = 119.66 degrees) diffracted to 1.6 A resolution. The presence of two homotrimers in the asymmetric unit (38 kDa x 6) gives V(M) values of 2.3 and 2.5 A(3) Da(-1) for the P1 and P2(1) crystal forms, respectively.

SUBMITTER: Matoba K

PROVIDER: S-EPMC2795605 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruzi.

Matoba Kazuaki K Nara Takeshi T Aoki Takashi T Honma Teruki T Tanaka Akiko A Inoue Masayuki M Matsuoka Shigeru S Inaoka Daniel Ken DK Kita Kiyoshi K Harada Shigeharu S

Acta crystallographica. Section F, Structural biology and crystallization communications 20090826 Pt 9

Aspartate transcarbamoylase (ATCase), the second enzyme of the de novo pyrimidine-biosynthetic pathway, catalyzes the production of carbamoyl aspartate from carbamoyl phosphate and L-aspartate. In contrast to Escherichia coli ATCase and eukaryotic CAD multifunctional fusion enzymes, Trypanosoma cruzi ATCase lacks regulatory subunits and is not part of the multifunctional fusion enzyme. Recombinant T. cruzi ATCase expressed in E. coli was purified and crystallized in a ligand-free form and in a c ...[more]

PMID: 19724137

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Project description:Genetic exchange has been described in several kinetoplastid parasites, but the most well-studied mating system is that of Trypanosoma brucei, the causative organism of African sleeping sickness. Sexual reproduction takes place in the salivary glands (SG) of the tsetse vector and involves meiosis and production of haploid gametes. Few genetic crosses have been carried out to date and consequently there is little information about the mating compatibility of different trypanosomes. In other single-celled eukaryotes, mating compatibility is typically determined by a system of two or more mating types (MT). Here we investigated the MT system in T. brucei.We analysed a large series of F1, F2 and back crosses by pairwise co-transmission of red and green fluorescent cloned cell lines through experimental tsetse flies. To analyse each cross, trypanosomes were cloned from fly SG containing a mixture of both parents, and genotyped by microsatellites and molecular karyotype. To investigate mating compatibility at the level of individual cells, we directly observed the behaviour of SG-derived gametes in intra- or interclonal mixtures of red and green fluorescent trypanosomes ex vivo.Hybrid progeny were found in all F1 and F2 crosses and most of the back crosses. The success of individual crosses was highly variable as judged by the number of hybrid clones produced, suggesting a range of mating compatibilities among F1 progeny. As well as hybrids, large numbers of recombinant genotypes resulting from intraclonal mating (selfers) were found in some crosses. In ex vivo mixtures, red and green fluorescent trypanosome gametes were observed to pair up and interact via their flagella in both inter- and intraclonal combinations. While yellow hybrid trypanosomes were frequently observed in interclonal mixtures, such evidence of cytoplasmic exchange was rare in the intraclonal mixtures.The outcomes of individual crosses, particularly back crosses, were variable in numbers of both hybrid and selfer clones produced, and do not readily fit a simple two MT model. From comparison of the behaviour of trypanosome gametes in inter- and intraclonal mixtures, we infer that mating compatibility is controlled at the level of gamete fusion.

Dataset Information

Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruzi.

Publications

Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruzi.

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