Abstract
This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl 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 (Kd) 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.
Highlights
Trichomonas vaginalis is a unicellular and anaerobic pathogenic protozoon facultative that reproduces by binary fusion [1]
Morales-Luna et al [23] showed the fusion of 6pgl with the g6pd gene occurs in the protozoan parasite Giardia lamblia (G. lamblia), it is located at the 3 end, similar to the results deposited for the genome [20] and found in our results for T. vaginalis
The purified protein showed a molecular mass of 81.6 KDa per monomer, but we found this protein in the form of a tetramer; this differs from the other reported fused proteins, such as the glucose-6-phosphate dehydrogenase (G6PD)::6PGL of Giardia lamblia, which are phylogenetically similar and feature observable dimeric and tetrameric forms
Summary
Trichomonas vaginalis is a unicellular and anaerobic pathogenic protozoon facultative that reproduces by binary fusion [1]. The metabolic pathways of this parasite share similar characteristics with eukaryotes and anaerobic prokaryotes, but T. vaginalis does not have the ability to synthesize macromolecules de novo since it lacks the enzymes necessary for the synthesis of purine, pyrimidine, and some lipids as cholesterol; which are acquired from vaginal secretions or through the phagocytosis of host and bacterial cells [13,14] This pathogen belongs to the parabasal lineage of microaerophilic eukaryotes that do not have mitochondria or peroxisomes but do possess organelles called hydrogenosomes, a structure where molecular hydrogen and adenosine triphosphate (ATP) are produced. This is the first study on the characterization of a protein that participates in the PPP of T. vaginalis to propose it as a therapeutic target for the analysis (or development) of specific drugs and that serves to control this pathogen that is of clinical relevance to humans
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