Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan , and Effects of the NADP Molecule on Enzyme Stability.

This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan (). The glucose-6-phosphate dehydrogenase () gene from . was isolated by PCR and the sequence of the product showed that is fused with gene. The fused Tv:: gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP were performed. These results revealed that the protein becomes more stable in the presence of the NADP. In addition, we determined the dissociation constant for the binding () of NADP in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.