Is bismuth(III) able to inhibit the activity of urease? Puzzling results in the quest for soluble urease complexes for agrochemical and medicinal applications.

Bismuth(III) complexes have been reported to act as inhibitors of the enzyme urease, ubiquitously present in soils and implicated in the pathogenesis of several microorganisms. The general insolubility of Bi(III) complexes in water at neutral pH, however, is an obstacle to their utilization. In our quest to improve the solubility of Bi(III) complexes, we selected a compound reported to inhibit urease, namely [Bi(HEDTA)]·2H2O, and co-crystallized it with (i) racemic DL-histidine to obtain the conglomerate [Bi2(HEDTA)2(µ-D-His)2]·6H2O + [Bi2(HEDTA)2(µ-L-His)2]·6H2O, (ii) enantiopure L-histidine to yield [Bi2(HEDTA)2(µ-L-His)2]·6H2O, and (iii) cytosine to obtain [Bi(HEDTA)]·Cyt·2H2O. All compounds, synthesised by mechanochemical methods and by slurry, were characterized in the solid state by calorimetric (DSC and TGA) and spectroscopic (IR) methods, and their structures were determined using powder X-ray diffraction (PXRD) data. All compounds show an appreciable solubility in water, with values ranging from 6.8 mg mL-1 for the starting compound [Bi(HEDTA)]·2H2O to 36 mg mL-1 for [Bi2(HEDTA)2(µ-L-His)2]·6H2O. The three synthesized compounds as well as [Bi(HEDTA)]·2H2O were then tested for inhibition activity against urease. Surprisingly, no enzymatic inhibition was observed during in vitro assays using Canavalia ensiformis urease and in vivo assays using cultures of Helicobacter pylori, raising questions on the efficacy of Bi(III) compounds to counteract the negative effects of urease activity in the agro-environment and in human health.

An isothermal calorimetry assay for determining steady state kinetic and enzyme inhibition parameters for SARS-CoV-2 3CL-protease.

This manuscript describes the application of Isothermal Titration Calorimetry (ITC) to characterize the kinetics of 3CL pro from the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and its inhibition by Ensitrelvir, a known non-covalent inhibitor. 3CL pro is the main protease that plays a crucial role of producing the whole array of proteins necessary for the viral infection that caused the spread of COVID-19, responsible for millions of deaths worldwide as well as global economic and healthcare crises in recent years. The proposed calorimetric method proved to have several advantages over the two types of enzymatic assays so far applied to this system, namely Förster Resonance Energy Transfer (FRET) and Liquid Chromatography-Mass Spectrometry (LC-MS). The developed ITC-based assay provided a rapid response to 3CL pro activity, which was used to directly derive the kinetic enzymatic constants K M and k cat reliably and reproducibly, as well as their temperature dependence, from which the activation energy of the reaction was obtained for the first time. The assay further revealed the existence of two modes of inhibition of 3CL pro by Ensitrelvir, namely a competitive mode as previously inferred by crystallography as well as an unprecedented uncompetitive mode, further yielding the respective inhibition constants with high precision. The calorimetric method described in this paper is thus proposed to be generally and widely used in the discovery and development of drugs targeting 3CL pro .

Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori.

Essential trace metals play key roles in the survival, replication, and virulence of bacterial pathogens. Helicobacter pylori (H. pylori), the main bacterial cause of gastric ulcers, requires Ni(ii) to colonize and persist in the acidic environment inside the stomach, exploiting the nickel-containing enzyme urease to catalyze the hydrolysis of urea to ammonia and bicarbonate and create a pH-buffered microenvironment. Urease utilizes Ni(ii) as a catalytic cofactor for its activity. In ureolytic bacteria, unique transmembrane (TM) transporters evolved to guarantee the selective uptake and efflux of Ni(ii) across cellular membranes to meet the cellular requirements. NixA is an essential Ni(ii) transporter expressed by H. pylori when the extracellular environment experiences a drop in pH. This Class I nickel-cobalt transporter of the NiCoT family catalyzes the uptake of Ni(ii) across the inner membrane from the periplasm. In this study, we characterized NixA using a platform whereby, for the first time on a NiCoT transporter, recombinantly expressed and purified NixA and key mutants in the translocation pathway have been reconstituted in artificial lipid bilayer vesicles (proteoliposomes). Fluorescent sensors responsive to Ni(ii) transport (Fluozin-3-Zn(ii)), luminal pH changes (pyranine), and membrane potential (oxonol VI) were encapsulated in the proteoliposomes lumen to monitor, in real-time, NixA transport properties and translocation mechanism. Kinetic transport analysis revealed that NixA is highly selective for Ni(ii) with no substrate promiscuity towards Co(ii), the other putative metal substrate of the NiCoT family, nor Zn(ii). NixA-mediated Ni(ii) transport exhibited a Michaelis-Menten-type saturable substrate concentration dependence, with an experimental KM, Ni(ii) = 31.0 ± 1.2 µM. Ni(ii) transport by NixA was demonstrated to be electrogenic, and metal translocation did not require a proton motive force, resulting in the generation of a positive-inside transmembrane potential in the proteoliposome lumen. Mutation analysis characterized key transmembrane residues for substrate recognition, binding, and/or transport, suggesting the presence of a three-step transmembrane translocation conduit. Taken together, these investigations reveal that NixA is a Ni(ii)-selective Class I NiCoT electrogenic uniporter. The work also provides an in vitro approach to characterize the transport properties of metal transporters responsible for Ni(ii) acquisition and extrusion in prokaryotes.

Kinetic and structural details of urease inactivation by thiuram disulphides.

This paper reports on the molecular details of the reactivity of urease, a nickel-dependent enzyme that catalyses the last step of organic nitrogen mineralization, with thiuram disulphides, a class of molecules known to inactivate the enzyme with high efficacy but for which the mechanism of action had not been yet established. IC50 values of tetramethylthiuram disulphide (TMTD or Thiram) and tetraethylthiuram disulphide (TETD or Disulfiram) in the low micromolar range were determined for plant and bacterial ureases. The X-ray crystal structure of Sporosarcina pasteurii urease inactivated by Thiram, determined at 1.68 Å resolution, revealed the presence of a covalent modification of the catalytically essential cysteine residue. This is located on the flexible flap that modulates the size of the active site channel and cavity. Formation of a Cys-S-S-C(S)-N(CH3)2 functionality responsible for enzyme inactivation was observed. Quantum-mechanical calculations carried out to rationalise the large reactivity of the active site cysteine support the view that a conserved histidine residue, adjacent to the cysteine in the active site flap, modulates the charge and electron density along the thiol SH bond by shifting electrons towards the sulphur atom and rendering the thiol proton more reactive. We speculate that this proton could be transferred to the nickel-coordinated urea amide group to yield a molecule of ammonia from the generated Curea-NH3+ functionality during catalysis.

Protocol for production and purification of SARS-CoV-2 3CLpro.

3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L-1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay. For complete details on the use and execution of this protocol, please refer to Bafna et al.1.

The structure of the high-affinity nickel-binding site in the Ni,Zn-HypA•UreE2 complex.

The maturation pathway for the nickel-dependent enzyme urease utilizes the protein UreE as a metallochaperone to supply Ni(II) ions. In Helicobacter pylori urease maturation also requires HypA and HypB, accessory proteins that are commonly associated with hydrogenase maturation. Herein we report on the characterization of a protein complex formed between HypA and the UreE2 dimer. Nuclear magnetic resonance (NMR) coupled with molecular modelling show that the protein complex apo, Zn-HypA•UreE2, forms between the rigorously conserved Met-His-Glu (MHE motif) Ni-binding N-terminal sequence of HypA and the two conserved His102A and His102B located at the dimer interface of UreE2. This complex forms in the absence of Ni(II) and is supported by extensive protein contacts that include the use of the C-terminal sequences of UreE2 to form additional strands of ß-sheet with the Ni-binding domain of HypA. The Ni-binding properties of apo, Zn-HypA•UreE2 and the component proteins were investigated by isothermal titration calorimetry using a global fitting strategy that included all of the relevant equilibria, and show that the Ni,Zn-HypA•UreE2 complex contains a single Ni(II)-binding site with a sub-nanomolar KD. The structural features of this novel Ni(II) site were elucidated using proteins produced with specifically deuterated amino acids, protein point mutations, and the analyses of X-ray absorption spectroscopy, hyperfine shifted NMR features, as well as molecular modeling coupled with quantum-mechanical calculations. The results show that the complex contains a six-coordinate, high-spin Ni(II) site with ligands provided by both component proteins.

Optimized Ebselen-Based Inhibitors of Bacterial Ureases with Nontypical Mode of Action.

Screening of 25 analogs of Ebselen, diversified at the N-aromatic residue, led to the identification of the most potent inhibitors of Sporosarcina pasteurii urease reported to date. The presence of a dihalogenated phenyl ring caused exceptional activity of these 1,2-benzisoselenazol-3(2H)-ones, with Ki value in a low picomolar range (<20 pM). The affinity was attributed to the increased π-π and π-cation interactions of the dihalogenated phenyl ring with αHis323 and αArg339 during the initial step of binding. Complementary biological studies with selected compounds on the inhibition of ureolysis in whole Proteus mirabilis cells showed a very good potency (IC50 < 25 nM in phosphate-buffered saline (PBS) buffer and IC90 < 50 nM in a urine model) for monosubstituted N-phenyl derivatives. The crystal structure of S. pasteurii urease inhibited by one of the most active analogs revealed the recurrent selenation of the Cys322 thiolate, yielding an unprecedented Cys322-S-Se-Se chemical moiety.

The Ni(II)-Binding Activity of the Intrinsically Disordered Region of Human NDRG1, a Protein Involved in Cancer Development.

Nickel exposure is associated with tumors of the respiratory tract such as lung and nasal cancers, acting through still-uncharacterized mechanisms. Understanding the molecular basis of nickel-induced carcinogenesis requires unraveling the mode and the effects of Ni(II) binding to its intracellular targets. A possible Ni(II)-binding protein and a potential focus for cancer treatment is hNDRG1, a protein induced by Ni(II) through the hypoxia response pathway, whose expression correlates with higher cancer aggressiveness and resistance to chemotherapy in lung tissue. The protein sequence contains a unique C-terminal sequence of 83 residues (hNDRG1*C), featuring a three-times-repeated decapeptide, involved in metal binding, lipid interaction and post-translational phosphorylation. In the present work, the biochemical and biophysical characterization of unmodified hNDRG1*C was performed. Bioinformatic analysis assigned it to the family of the intrinsically disordered regions and the absence of secondary and tertiary structure was experimentally proven by circular dichroism and NMR. Isothermal titration calorimetry revealed the occurrence of a Ni(II)-binding event with micromolar affinity. Detailed information on the Ni(II)-binding site and on the residues involved was obtained in an extensive NMR study, revealing an octahedral paramagnetic metal coordination that does not cause any major change of the protein backbone, which is coherent with CD analysis. hNDRG1*C was found in a monomeric form by light-scattering experiments, while the full-length hNDRG1 monomer was found in equilibrium between the dimer and tetramer, both in solution and in human cell lines. The results are the first essential step for understanding the cellular function of hNDRG1*C at the molecular level, with potential future applications to clarify its role and the role of Ni(II) in cancer development.

Inhibition of Urease by Hydroquinones: A Structural and Kinetic Study.

Hydroquinones are a class of organic compounds abundant in nature that result from the full reduction of the corresponding quinones. Quinones are known to efficiently inhibit urease, a NiII -containing enzyme that catalyzes the hydrolysis of urea to yield ammonia and carbonate and acts as a virulence factor of several human pathogens, in addition to decreasing the efficiency of soil organic nitrogen fertilization. Here, we report the molecular characterization of the inhibition of urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) by 1,4-hydroquinone (HQ) and its methyl and tert-butyl derivatives. The 1.63-Å resolution X-ray crystal structure of the SPU-HQ complex discloses that HQ covalently binds to the thiol group of αCys322, a key residue located on a mobile protein flap directly involved in the catalytic mechanism. Inhibition kinetic data obtained for the three compounds on JBU reveals the occurrence of an irreversible inactivation process that involves a radical-based autocatalytic mechanism.

Pro5 is not essential for the formation of 'Ni-hook' in nickel superoxide dismutase.

The N-terminus of nickel-dependent superoxide dismutase (NiSOD) forms a structural motif known as the "Ni-hook," where the peptide wraps around the metal to bring cysteine-2 and cysteine-6 into spatial proximity, allowing these residues to coordinate in a cis-geometry. A highly conserved proline-5 residue in the Ni-hook adopts a cis-conformation that is widely considered important for its formation. Herein, we investigate this role by point mutation of Pro5 to alanine. The results obtained show that the variant exhibits wild-type-like redox catalysis and features a Ni(III) center very similar to that found in enzyme. Structural analysis using X-ray absorption spectroscopy of the nickel sites in as-isolated P5A-NiSOD reveals changes in the variant and are consistent with a six-coordinate Ni site with (N/O)4S2 coordination. These changes are attributed to changes in the Ni(II) site structure. Nickel-binding studies using isothermal titration calorimetry reveal two binding events with Kd = 25(20) nM, and 250(60) nM. These events are attributed to i) Ni(II) binding to a preformed Ni-hook containing cis-Pro5 and ii) the combination of trans- to cis- isomerization upon Ni(II) binding, respectively. The higher-affinity binding event is absent in P5A-NiSOD, an observation attributed to the low abundance of the cis-Ala5 isomer in the apo-protein.

Structure, dynamics, and function of SrnR, a transcription factor for nickel-dependent gene expression.

Streptomyces griseus, a bacterium producing antibacterial drugs and featuring possible application in phytoremediation, expresses two metal-dependent superoxide dismutase (SOD) enzymes, containing either Fe(II) or Ni(II) in their active site. In particular, the alternative expression of the two proteins occurs in a metal-dependent mode, with the Fe(II)-enzyme gene (sodF) repressed at high intracellular Ni(II) concentrations by a two-component system (TCS). This complex involves two proteins, namely SgSrnR and SgSrnQ, which represent the transcriptional regulator and the Ni(II) sensor of the system, respectively. SgSrnR belongs to the ArsR/SmtB family of metal-dependent transcription factors; in the apo-form and in the absence of SgSrnQ, it can bind the DNA operator of sodF, upregulating gene transcription. According to a recently proposed hypothesis, Ni(II) binding to SgSrnQ would promote its interaction with SgSrnR, causing the release of the complex from DNA and the consequent downregulation of the sodF expression. SgSrnQ is predicted to be highly disordered, thus the understanding, at the molecular level, of how the SgSrnR/SgSrnQ TCS specifically responds to Ni(II) requires the knowledge of the structural, dynamic, and functional features of SgSrnR. These were investigated synergistically in this work using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, atomistic molecular dynamics calculations, isothermal titration calorimetry, and in silico molecular docking. The results reveal that the homodimeric apo-SgSrnR binds to its operator in a two-step process that involves the more rigid globular portion of the protein and leaves its largely disordered regions available to possibly interact with the disordered SgSrnQ in a Ni-dependent process.

Nickel import and export in the human pathogen Helicobacter pylori, perspectives from molecular modelling.

The uptake of essential metal ions and the ability to extrude them when their excess causes toxicity are crucial processes for all living beings. Nickel is a virulence factor for several human pathogens and in particular for the human gastric pathogen Helicobacter pylori because of its crucial role in the catalytic activity of two Ni-dependent enzymes, urease and hydrogenase. H. pylori requires efficient uptake mechanisms to import Ni(II) because of its scarcity in the human body, but the molecular details of Ni(II) homeostasis are not fully known. Here we offer a structural framework for the machinery of Ni(II) import/export in H. pylori, obtained through comparative modelling and macromolecular docking. The model structures reported in this perspective are initial steps towards the understanding of these processes at the molecular level and in the direction to exploit them to eradicate infections caused by this family of pathogens. The differences between the structural models obtained by using both the recently released neural network-based approach implemented in AlphaFold2 and a more classical user-driven modelling procedure are also discussed.

Medicinal Au(I) compounds targeting urease as prospective antimicrobial agents: unveiling the structural basis for enzyme inhibition.

A few gold compounds were recently found to show antimicrobial properties in vitro, holding great promise for the discovery of new drugs to overcome antibiotic resistance. Here, the inhibition of the bacterial virulence factor urease by four Au(I)-compounds, namely Au(PEt3)Cl, Au(PEt3)Br, Au(PEt3)I and [Au(PEt3)2]Cl, obtained from the antiarthritic Au(I)-drug Auranofin and earlier reported to act as antimicrobials, is investigated. The three monophosphino Au(I) complexes showed IC50 values in the 30-100 nM range, while the diphosphino Au(I) complex, though being less active, still showed a IC50 value of 7 µM. The structural basis for this inhibition was provided by solving the crystal structures of urease co-crystallized with Au(PEt3)I and [Au(PEt3)2]Cl: at least two Au(I) ions bind the enzyme in a flap domain involved in the catalysis, thus obliterating enzyme activity. Peculiar changes observed in the two structures reveal implications for the mechanism of soft metal binding and enzyme inactivation.

Revisiting the CooJ family, a potential chaperone for nickel delivery to [NiFe]­carbon monoxide dehydrogenase.

Nickel insertion into nickel-dependent carbon monoxide dehydrogenase (CODH) represents a key step in the enzyme activation. This is the last step of the biosynthesis of the active site, which contains an atypical heteronuclear NiFe4S4 cluster known as the C-cluster. The enzyme maturation is performed by three accessory proteins, namely CooC, CooT and CooJ. Among them, CooJ from Rhodospirillum rubrum is a histidine-rich protein containing two distinct and spatially separated Ni(II)-binding sites: a N-terminal high affinity site (HAS) and a histidine tail at the C-terminus. In 46 CooJ homologues, the HAS motif was found to be strictly conserved with a H(W/F)XXHXXXH sequence. Here, a proteome database search identified at least 150 CooJ homologues and revealed distinct motifs for HAS, featuring 2, 3 or 4 histidines. The purification and biophysical characterization of three representative members of this protein family showed that they are all homodimers able to bind Ni(II) ions via one or two independent binding sites. Initially thought to be present only in R. rubrum, this study strongly suggests that CooJ could play a significant role in CODH maturation or in nickel homeostasis.

Probing the transport of Ni(II) ions through the internal tunnels of the Helicobacter pylori UreDFG multimeric protein complex.

The survival of several pathogenic bacteria, such as Helicobacter pylori (Hp), relies on the activity of the nickel-dependent enzyme urease. Nickel insertion into urease is mediated by a multimeric chaperone complex (HpUreDFG) that is responsible for the transport of Ni(II) from a conserved metal binding motif located in the UreG dimer (CPH motif) to the catalytic site of the enzyme. The X-ray structure of HpUreDFG revealed the presence of water-filled tunnels that were proposed as a route for Ni(II) translocation. Here, we probe the transport of Ni(II) through the internal tunnels of HpUreDFG, from the CPH motif to the external surface of the complex, using microsecond-long enhanced molecular dynamics simulations. The results suggest a "bucket-brigade" mechanism whereby Ni(II) can be transported through a series of stations found along these internal pathways.

Nickel as a virulence factor in the Class I bacterial carcinogen, Helicobacter pylori.

Helicobacter pylori is a human bacterial pathogen that causes peptic ulcers and has been designated a Class I carcinogen by the International Agency for Research on Cancer (IARC). Its ability to survive in the acid environment of the stomach, to colonize the stomach mucosa, and to cause cancer, are linked to two enzymes that require nickel-urease and hydrogenase. Thus, nickel is an important virulence factor and the proteins involved in nickel trafficking are potential antibiotic targets. This review summarizes the nickel biochemistry of H. pylori with a focus on the roles of nickel in virulence, nickel homeostasis, maturation of urease and hydrogenase, and the unique nickel trafficking that occurs between the hydrogenase maturation pathway and urease nickel incorporation that is mediated by the metallochaperone HypA and its partner, HypB.

Kinetic and structural analysis of the inactivation of urease by mixed-ligand phosphine halide Ag(I) complexes.

Soft metal ions can inactivate urease, a Ni(II)-dependent enzyme whose hydrolytic activity has significant implications in agro-environmental science and human health. Kinetic and structural studies of the reaction of Canavalia ensiformis urease (JBU) and Sporosarcina pasteurii urease (SPU) with Ag(I) compounds of general formula [Ag(PEt3)X]4 (X = Cl, Br, I), and with the ionic species [Ag(PEt3)2]NO3, revealed the role of the Ag(I) ion and its ligands in modulating the metal-enzyme interaction. The activity of JBU is obliterated by the [Ag(PEt3)X]4 complexes, with IC50 values in the nanomolar range; the efficiency of the inhibition increases in the Cl- < Br- < I- order. The activity of JBU upon [Ag(PEt3)2]NO3 addition decreases to a plateau corresponding to ca. 60% of the original activity and decreases with time at a reduced rate. Synchrotron X-ray crystallography on single crystals obtained after the incubation of SPU with the Ag(I) complexes yielded high-resolution (1.63-1.97 Å) structures. The metal-protein adducts entail a dinuclear Ag(I) cluster bound to the conserved residues αCys322, αHis323, and αMet367, with a bridging cysteine thiolate atom, a weak Ag…Ag bond, and a quasi-linear Ag(I) coordination geometry. These observations suggest a mechanism that involves the initial substitution of the phosphine ligand, followed by a structural rearrangement to yield the dinuclear Ag(I) cluster. These findings indicate that urease, in addition to the active site dinuclear Ni(II) cluster, possesses a secondary metal binding site, located on the mobile flap domain, capable of recognizing pairs of soft metal ions and controlling catalysis.

Inhibition of Urease, a Ni-Enzyme: The Reactivity of a Key Thiol With Mono- and Di-Substituted Catechols Elucidated by Kinetic, Structural, and Theoretical Studies.

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.