Biochemical and structural characterization of beta-carbonic anhydrase from the parasite Trichomonas vaginalis

Abstract

Trichomonas vaginalis is a unicellular parasite and responsible for one of the most common sexually transmittable infections worldwide, trichomoniasis. Carbonic anhydrases (CAs) are enzymes found in all lifeforms and are known to play a vital role in many biochemical processes in organisms including the maintenance of acid-base homeostasis. To date, eight evolutionarily divergent but functionally convergent forms of CAs (α, β, γ, δ, ζ, η, θ, and ι) have been discovered. The human genome contains only α-CAs, whereas many clinically significant pathogens express only β-CAs and/or γ-CAs. The characterization of pathogenic β- and γ-CAs provides important knowledge for targeting these biomolecules to develop novel anti-invectives against trichomoniasis. Here, we report the recombinant production and characterization of the second β-CA of T. vaginalis (TvaCA2). Light scattering analysis revealed that TvaCA2 is a dimeric protein, which was further supported with in silico modeling, suggesting similar structures between TvaCA2 and the first β-CA of T. vaginalis (TvaCA1). TvaCA2 exhibited moderate catalytic activity with the following kinetic parameters: k_cat of 3.8 × 10⁵ s^-1 and k_cat/K_M of 4.4 × 10⁷ M^-1 s^-1. Enzyme activity inhibition was studied with a set of clinically used sulfonamides and sulfonamide derivates. Twenty-seven out of the 39 compounds resulted in inhibition with a nanomolar range. These initial results encourage for future work entailing the design of more potent inhibitors against TvaCA2, which may provide new assets to fight trichomoniasis. KEY MESSAGES: • Protozoan parasite Trichomonas vaginalis has two β-carbonic anhydrases (TvaCA1/2). • TvaCA1/TvaCA2 represents promising targets for antitrichomonal drug development. • TvaCA2 is a dimer of 20.3 kDa and possesses moderate catalytic activity. • The most efficient inhibitor was clinical drug acetazolamide with K_I of 222.9 nM. • The 39 tested sulfonamides form the basis for the design of more potent inhibitors.

Keywords: Beta-carbonic anhydrase; Homology modeling; Inhibition; Kinetics; Protozoan; Trichomonas vaginalis.