The nickel-dependent enzyme urease is a virulence factor for a large number of pathogenic and antibiotic-resistant bacteria, as well as a negative factor for the efficiency of soil nitrogen fertilization for crop production. The use of urease inhibitors to offset these effects requires knowledge, at a molecular level, of their mode of action. The 1.28 Å resolution structure of the enzyme-inhibitor complex obtained upon incubation of Sporosarcina pasteurii urease with N-(n-butyl)thiophosphoric triamide (NBPT), a molecule largely utilized in agriculture, reveals the presence of the monoamidothiophosphoric acid (MATP) moiety, obtained upon enzymatic hydrolysis of the diamide derivative of NBPT (NBPD) to yield n-butyl amine. MATP is bound to the two Ni(II) ions in the active site of urease using a μ-bridging O atom and terminally bound O and NH groups, with the S atom of the thiophosphoric amide pointing away from the metal center. The mobile flap modulating the size of the active site cavity is found in the closed conformation. Docking calculations suggest that the interaction between urease in the open flap conformation and NBPD involves a role for the conserved αArg339 in capturing and orienting the inhibitor prior to flap closure. Calorimetric and spectrophotometric determinations of the kinetic parameters of this inhibition indicate the occurrence of a reversible slow inhibition mode of action, characterized, for both bacterial and plant ureases, by a very small value of the dissociation constant of the urease-MATP complex. No need to convert NBPT to its oxo derivative NBPTO, as previously proposed, is necessary for urease inhibition.

The nickel-dependent enzyme urease represents a negative element for the efficiency of soil nitrogen fertilization as well as a virulence factor for a large number of pathogenic and antibiotic-resistant bacteria. The development of ever more efficient urease inhibitors demands knowledge of their modes of action at the molecular level. N-(n-Butyl)-phosphoric triamide (NBPTO) is the oxo-derivative of N-(n-butyl)-thiophosphoric triamide (NBPT), which is extensively employed in agriculture to increase the efficiency of urea-based fertilizers. The 1.45 Å resolution structure of the enzyme–inhibitor complex obtained upon incubation of Sporosarcina pasteurii urease (SPU) with NBPTO shows the presence of diamido phosphoric acid (DAP), generated upon enzymatic hydrolysis of NBPTO with the release of n-butyl amine. DAP is bound in a tridentate binding mode to the two Ni­(II) ions in the active site of urease via two O atoms and an amide NH2 group, whereas the second amide group of DAP points away from the metal center into the active-site channel. The mobile flap modulating the size of the active-site cavity is found in a disordered closed–open conformation. A kinetic characterization of the NBPTO-based inhibition of both bacterial (SPU) and plant (Canavalia ensiformis or jack bean, JBU) ureases, carried out by calorimetric measurements, indicates the occurrence of a reversible slow-inhibition mode of action. The latter is characterized by a very small value of the equilibrium dissociation constant of the urease–DAP complex caused, in turn, by the large rate constant for the formation of the enzyme–inhibitor complex. The much greater capability of NBPTO to inhibit urease, as compared with that of NBPT, is thus not caused by the presence of a PO moiety versus a PS moiety, as previously suggested, but rather by the readiness of NBPTO to react with urease without the need to convert one of the P–NH2 amide moieties to its P–OH acid derivative, as in the case of NBPT. The latter process is indeed characterized by a very small equilibrium constant that reduces drastically the concentration of the active form of the inhibitor in the case of NBPT. This indicates that high-efficiency phosphoramide-based urease inhibitors must have at least one O atom bound to the central P atom in order for the molecule to efficiently and rapidly bind to the dinickel center of the enzyme.

Carbon monoxide dehydrogenase reversibly catalyzes the oxidation of CO into CO2. The monofunctional enzyme from Rhodospirillum rubrum (RrCODH) has been extensively characterized in the past, although its use and investigation by bioelectrochemistry have been limited. Here, we developed a heterologous system yielding a highly stable and active recombinant RrCODH in one-step purification, with a CO oxidation activity reaching a maximum of 26,500 U mg -1 , making RrCODH the most active CODH under ambient conditions described so far. Electron Paramagnetic Resonance was used to precisely characterize the recombinant RrCODH, demonstrating the integrity of the active site. Selective CO2/CO interconversion with maximum turnover frequencies of 150 s -1 for CO oxidation (1.5 mA cm -2 at 250 mV overpotential) and 420 s -1 for CO2 reduction (4.2 mA cm -2 at 180 mV overpotential) are catalyzed by the recombinant RrCODH immobilized on MWCNT electrodes modified with 1-pyrenebutyric acid adamantyl amide (MWCNT ADA ), either in classic three-electrode cell or in specifically-designed CO2/CO-diffusing electrodes. This functional device is stable for hours, and for at least 800,000 turnover numbers. The performances of recombinant RrCODH-modified MWCNT ADA are closed to the best metal-based and molecular-based catalysts. These results greatly increase the benchmark for bioelectrocatalysis of reversible CO2 conversion.

The inhibition of urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) by a class of six aromatic poly‐hydroxylated molecules, namely mono‐ and dimethyl‐substituted catechols, was investigated on the basis of the inhibitory efficiency of the catechol scaffold. The aim was to probe the key step of a mechanism proposed for the inhibition of SPU by catechol, namely the sulfanyl radical attack on the aromatic ring, as well as to obtain critical information on the effect of substituents of the catechol aromatic ring on the inhibition efficacy of its derivatives. The crystal structures of all six SPU‐inhibitors complexes, determined at high resolution, as well as kinetic data obtained on JBU and theoretical studies of the reaction mechanism using quantum mechanical calculations, revealed the occurrence of an irreversible inactivation of urease by means of a radical‐based autocatalytic multistep mechanism, and indicate that, among all tested catechols, the mono‐substituted 3‐methyl‐catechol is the most efficient inhibitor for urease.