Recombinant expression and nucleotide hydrolysis activity of NTPDase 4 from Trichomonas vaginalis

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the non-viral sexually transmitted infection most prevalent in world. It is important to investigate biochemical aspects of the parasite that contribute to our understanding of the biology and applications in the treatment and diagnosis of the infection. The nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme that hydrolyses extracellular adenine and guanine nucleotides, forming nucleosides adenosine and guanosine. This is important for parasite survival through the purine salvage pathway, since adenosine is the precursor for the entire purine nucleotides pool in T. vaginalis. Herein we expressed TvNTPDase4 in the bacterial system Escherichia coli. Our data demonstrate that the enzyme is active, being able to hydrolyze ATP, ADP and AMP at a concentration of 10 μg of purified protein/reaction. The inhibitors gadolinium and adenosine 5'-[α,β-methylene]diphosphate (AMPCP) inhibited the hydrolysis of rTvNTPDase4. The inhibition of ATPase/ADPase activity was more effective with gadolinium, while the inhibition of AMPase activity was more effective with AMPCP. The enzyme rTvNTPDase4 was not cytotoxic to HMVII cells. In molecular dynamics, we observed that the ability of TvNTPDase4 to hydrolyze ATP, ADP, and AMP substrates occurs through direct interactions with the apyrase-conserved regions (ACR), especially ACR1 and ACR4. In this work, we did not find any candidate sequence for ecto-5'-nucleotidase (E-5'-N) in T. vaginalis, which leads us to believe that the parasite does not have this enzyme in its proteomic repertoire. Finally, we report that rTvNTPDase4 expressed and purified from a bacterial is active and has potential for biotechnological applications.