Estudos estruturais das enzimas histidina amônio liase, imidazolona propionase de Trypanosoma cruzi e gliceraldeído-3-fosfato desidrogenase de Candida albicans

Abstract

The study of the three dimensional (3D) structure of proteins helps us to understand the relations among amino acid sequence, structure and function, what is essential for a better understanding of the pathogenic mechanisms of organisms and for the development of new drugs. In this context, the present work aims to determine the three dimensional structures of enzymes Histidine Ammonia-Lyase and Imidazolone Propionase from Trypanosoma cruzi (TcHAL, TcIP) and Glyceraldehyde-3-phosphate dehydrogenase from Candida albicans (CaGAPDH). After TcHAL structure refinement, one could observe the abscence of the so called protection domain of the active site, what is different to previously known 3D structures of Phenylalanine Ammonia-Liases from plants and fungi, therefore, it is the first solved structure of an eukaryotic ammonia lyase that does not present this domain. The TcIP enzyme was even expressed in E. coli (DE3) using the vector pET-28a(+), however, the purification tests by affinity chromatography with different metal ions were not sufficient to obtain it in an adequate purity for the crystallization trials; therefore, its structure was hypothesized by homology modeling. The CaGAPDH enzyme was expressed in E. coli (DE3) using the vector pET-28a(+) and purified by Ni+2 affinity chromatography. Crystals from CaGAPDH appeared in 3 days after the drops were assembled and the best one diffracted to a maximum resolution of 2.98 Å. Indexing and processing showed that the crystal belongs to the space group P21 and presents translational non-crystallographic symmetry. A study after structure refinement showed that CaGAPDH has two conjugated mutations, Glu296 on place of a common Ala and Ala258 on place of common Lys or Asn, face to face residues, which possibly maintain structural properties. Thus, the results presented here provide support for further investigations to explain the participation of the enzymes HAL and IP in the metabolism of T. cruzi, as well as for GAPDH in C. albicans. In addition, the structural information presented here may contribute to the development of new chemotherapeutic agents for Chagas disease and also for candidiasis further evidence of CaGAPDH as a molecular target.