Triose Phosphate Isomerase from Boophilus microplus Embryos
Jorge Moraes1; Nallely Cabrera2; Daniela Freitas3; Horacio Reyes-Vivas4; J. Lenin Domínguez Ramírez2; Aoi Masuda3; Adela Rodríguez-Romero5; Rodrigo Arreola2; Itabajara Vaz jr.3; Marietta Tuena de Gómez-Puyou2; Ruy Pérez-Montfort2; Armando Gómez-Puyou2 and Carlos Logullo1.
1) Laboratório de Química e Função de Proteínas e Peptídeos – CBB – UENF; 2) Instituto de Fisiologıa Celular, Universidad Nacional Autonoma de México, DF México; 3) Centro de Biotecnologia – UFRGS; 4) Instituto Nacional de Pediatria, Laboratório de Bioquímica-Genética, DF, México; 5) Instituto de Química, Universidad Nacional Autonoma de México, DF, México.
Triose phosphate isomerase (TIM) catalyzes the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. TIMs are usually described as a homodimer with monomers formed by eight central β strands that are surrounded by eight α-helices; the strands and helices are connected by loops.
In this work B. microplus embryos TIM-Bm gene was cloned by RT-PCR and expressed in pET system. The recombinant TIM-Bm was purified and some of its structural and kinetic features were determined and compared to T. cruzi TIM. TIM-Bm's kinetic properties of Km and Vmax were measured with glyceraldehyde 3-phosphate as substrate showing 0.47 mM and 6031 μmols.min-1.mg ptn-1, respectively.
Structurally, TIM-Bm circular dichroism spectrum was almost identical to T. cruzi TIM, but their intrinsic fluorescence at an excitation wavelength of 280 nm has different intensities. TIM-Bm deduced amino acid sequence presents more cystein residues (9) for each monomer than the previously described TIMs. It can be a useful tool for inhibition studies with this enzyme, once such mechanisms targets non-conserved cysteine residues between different species. A homology model was generated based on that deduced sequence and it suggests a possible disulfide bound between cys 47 and 51 residues. Purified TIM-Bm was also submitted to crystallization systems (Crystal Screen I and II). Crystals were observed after 24 hours in Crystal Screen I containing 0.1 M HEPES-Na, with 1.4 M tri-Sodium citrate dehydrate, pH 7.5, and diffracted a maximum resolution of 2.4 Å. Supported by CAPES PRONEX, FAPERJ and CNPq.
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