Leonardo Kras Borges Martinelli

Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme–ligand binary complex formation

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.

Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme.

Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van’t Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

Synthesis, Inhibition of Mycobacterium tuberculosis Enoyl-acyl Carrier Protein Reductase and Antimycobacterial Activity of Novel Pentacyanoferrate(II)-isonicotinoylhydrazones

Tuberculosis remains among the top causes of death triggered by a single pathogen. Herein, a greener synthetic approach for isonicotinoylhydrazones is described using ultrasound energy. These compounds were used as starting materials for synthesizing pentacyanoferrate(II)isonicotinoylhydrazones, which inhibited the reaction catalyzed by Mycobacterium tuberculosis 2-trans-enoyl-ACP(CoA) reductase (MtInhA) in a time-dependent manner. The most active coordination complex showed an increase of more than ten-fold in the MtInhA inhibition rate constant compared with lead pentacyano(isoniazid)ferrate(II) (IQG607). Additionally, the new series of metal-based compounds demonstrated antitubercular activity against a drug-susceptible Mycobacterium tuberculosis (Mtb) strain and was devoid of toxicity to mammalian cells (IC50 > 20 μmol L, half maximal inhibitory concentration). Finally, one of the synthesized compounds showed intracellular activity similar to isoniazid in a macrophage model of Mtb infection, indicating that this chemical class may furnish novel structures to embark on the preclinical phase of anti-tuberculosis drug development.

Mode of action of recombinant hypoxanthine–guanine phosphoribosyltransferase from Mycobacterium tuberculosis

Tuberculosis (TB) is the second most important cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. A better understanding of the purine salvage pathway can unveil details of the biology of M. tuberculosis that might be used to develop new strategies to combat this pathogen. Hypoxanthine–guanine phosphoribosyltransferase (HGPRT) is an enzyme from the purine phosphoribosyltransferase (PRTase) family and catalyzes the conversion of hypoxanthine or guanine and 5-phospho-α-D-ribose 1-diphosphate (PRPP) to, respectively, inosine 5′-monophosphate (IMP) or guanosine 5′-monophosphate (GMP), and pyrophosphate (PPi). Gel filtration chromatography has shown that recombinant M. tuberculosis HGPRT (MtHGPRT) is homodimeric. A sequential compulsory ordered enzyme mechanism with PRPP as the substrate that binds to free MtHGPRT enzyme and PPi as the first product to dissociate is proposed based on kinetic data and thermodynamics of ligand binding from isothermal titration calorimetry (ITC) results. ITC data have also provided thermodynamic signatures of non-covalent interactions for PRPP, IMP and GMP binding to free MtHGPRT. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the MtHGPRT-catalyzed chemical reaction, pre-steady-state kinetics, solvent kinetic isotope effects, equilibrium constants and pH-rate profiles are also presented. Pre-steady-state analysis reveals that there is an initial rapid phase (burst) followed by a slower phase, suggesting that product release is rate limiting. The data here described provide a better understanding of the mode of action of MtHGPRT.

Thermodynamics, functional and structural characterization of inosine–uridine nucleoside hydrolase from Leishmania braziliensis

Leishmaniasis is considered one of the main endemic diseases in the world, and Brazil is among the countries with the highest incidence of cutaneous and mucocutaneous forms of leishmaniasis caused mainly by Leishmania braziliensis. The first-line drugs used in the treatment of leishmaniasis have several limitations: parenteral administration, long duration of treatment, and serious toxicity. One key metabolic characteristic of these parasites is the lack of a de novo purine biosynthesis pathway, making them auxotrophic to purines. Accordingly, they rely solely on the purine salvage pathway for nucleotide synthesis. A better understanding of the purine salvage pathway can reveal details of the biology of L. braziliensis that could, in turn, be used to develop new strategies to combat this parasite. The inosine–uridine nucleoside hydrolase from L. braziliensis (LbIU-NH) plays an important role in the salvage process and is an attractive drug target as there is no similar catalytic activity in mammals. Here is described cloning, heterologous protein expression, and a three-step purification protocol that yielded homogenous recombinant protein. The determination of LbIU-NH steady-state kinetic constants for inosine, adenosine, cytidine, uridine and p-nitrophenyl β-D-ribofuranoside is also reported. These data suggest that LbIU-NH displays characteristics of a nonspecific hydrolase. The thermodynamic profile suggests that D-ribose can bind to free enzyme with favorable enthalpic (ΔH) and entropic (ΔS) contributions. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the LbIU-NH-catalyzed chemical reaction, pre-steady-state kinetics, solvent kinetic isotope effects, and pH-rate profiles are also presented. In addition, the crystal structure of LbIU-NH in complex with β-D-ribose and Ca2+ at 1.5 A resolution is described.

Biochemical, thermodynamic and structural studies of recombinant homotetrameric adenylosuccinate lyase from Leishmania braziliensis

Adenylosuccinate lyase (ASL) is involved in both de novo and salvage pathways of purine biosynthesis. ASL belongs to the argininosuccinate lyase/fumarase C superfamily of enzymes which share a general acid–base catalytic mechanism with β-elimination of fumarate as the common product. Cloning, expression, and a method to obtain homogeneous recombinant ASL from Leishmania braziliensis (LbASL) are described. Mass spectrometry analysis of recombinant LbASL, oligomeric state determination and multiple sequence alignment are presented. Steady-state kinetics of LbASL showed a Michaelis–Menten pattern. Isothermal titration calorimetry binding assays suggested that LbASL follows a Uni-Bi ordered kinetic mechanism, in which release of fumarate is followed by AMP to yield free enzyme. Initial velocity data for the reverse reaction and the Haldane relationship allowed calculation of an unfavorable equilibrium constant for the LbASL-catalyzed chemical reaction. The activation energy and thermodynamic activation parameters were estimated. Solvent kinetic isotope effects V/K and V suggest a modest contribution of solvent proton transference during the rate-limiting step of the reaction. Proton inventory data show that the modest normal effect on V arises from a single protonic site, and the transition state fractionation factor value of 0.74 suggests participation of solvent proton transfer in transition-state vibrations perpendicular to the reaction coordinate. pH-rate profiles for kcat and kcat/KM suggested amino acid residues involved in, respectively, catalysis and substrate binding. A model of LbASL was built to provide a structural basis for the experimental data. A better understanding of the mode of action of LbASL is useful for the rational design of antileishmaniasis agents.

Mycobacterium tuberculosis histidinol dehydrogenase: biochemical characterization and inhibition studies

HisD-Encoded histidinol dehydrogenase (HisD) catalyzes the two last chemical reactions of the L-histidine biosynthetic pathway, namely the conversion of L-histidinol (L-Hol) to L-histidinaldehyde (L-Hal) and to L-histidine (L-His). The hisD gene product has been shown to be essential for Mycobacterium tuberculosis survival in vitro. Herein, we describe a series of biochemical studies on recombinant Mycobacterium tuberculosis HisD (MtHisD). The synthesis of hydrazones derived from L-histidine yielded inhibitors in the low micromolar range, one of which showed moderate anti-Mtb activity. The compounds described here are, to the best of our knowledge, the first inhibitors of MtHisD activity reported in the literature, and they could become promising candidates for future development.