Interaction and inhibition mechanism of urease in vitro and soil system by a natural benzylisothiocyanate isolated from Moringa oleifera

Abstract

Urease catalyzes urea hydrolysis in NH3 and CO2 in the soil, leading to N-urea losses by volatilization (30–70%); thus, urease inhibitor addition is an alternative that reduces these losses. In this sense, biophysical studies are important to evaluate potential urease inhibitors and biotechnological applications since currently, the N-(n-butyl)thiophosphoric triamide (NBPT) is the only commercial urease inhibitor for agriculture purposes. Thus, this work focused on evaluating the mechanism of urease inhibition by 4-(4′-O-acetyl-α-L-rhamnosyloxy)benzylisothiocyanate (MFC) isolated from Moringa oleifera L. (Moringaceae). The experiment consisted of spectroscopic studies of enzyme kinetics and activity (urease 12 mU and MFC 0–800 µM); assays to evaluate conformational changes in the enzyme using ultraviolet–visible molecular absorption and molecular fluorescence (5 µM urease and MFC 0–200 µM); the evaluation of thiols and amino/guanidine groups using 5,5′-dithiobis(2-nitrobenzoic acid) (50 µM) and fluorescamine (100 µM). Inhibition of soil ureases using Ferralsol soil samples was performed with MFC compared with thiourea, hydroxyurea, and NBPT (0.5 mM for all). The results of enzymatic studies indicated that MFC is a mixed inhibitor with concentration necessary to produce 50% inhibition (IC50) of 487 μM. From the spectrofluorimetric titration were obtained Ksv (1.30 ×103 M−1) and Kb (1.80 ×102 M−1), which reflect the magnitude of fluorescence suppression and the interaction between urease and MFC, while the n value 0.77 indicates that the stoichiometry of the complex is 1:1. Besides, the interaction process MFC-urease changes protein structure, especially tryptophan (Trp) residues' microenvironment, highlighting MFC action as a mixed inhibitor since the Trp495 and Trp648 residues are far away from the urease catalytic site. The monitoring of the residues containing thiol and amino/guanidine groups indicated covalent interaction of isothiocyanate with these groups forming dithiocarbamates and thiourea (urease inhibitor group) was confirmed as majority products by mass spectrometry. Finally, MFC was applied to the soil samples, and the residual activity of soil ureases was determined, with inhibition of 49.4% (soil A) and 32.0% (soil B), noting that soil inhibition is like NBPT (54.4%, soil A) and can be considered a potential compound for biotechnology application.