Crystal structure and functional studies of an unusual L-cysteine desulfurase from Archaeoglobus fulgidus.

L-Cysteine desulfurase IscS and scaffold IscU proteins are universally involved in Fe/S cluster synthesis. The Archaeoglobus fulgidus (Af) genome encodes proteins having a high degree of primary structure similarity to IscS and IscU from other organisms. However, AfIscS is unusual because it lacks the active site lysine residue that normally forms an internal Schiff base with pyridoxal-phosphate (PLP) and serves as a base during catalysis. Our as-isolated recombinant AfIscS contains pyridoxamine phosphate (PMP) instead of the expected PLP and lacks desulfurase activity. We have solved its structure to 1.43 Å resolution and found that PMP binds non-covalently at the PLP site of the enzyme and displays significant disorder. However, the previously reported structure of recombinant Af(IscU-D35A-IscS)(2) contains an in vivo generated [Fe(2)S(2)] species within AfIscU and the question arises as to how its sulfides were generated. Here, we report that adding PLP to AfIscS produces an enzyme that displays in vitro L-cysteine desulfurase activity mediating the synthesis of a stable holo Af(IscU-D35A-IscS) complex.

Enterococcus faecalis SufU scaffold protein enhances SufS desulfurase activity by acquiring sulfur from its cysteine-153.

Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry.

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis ( Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials.

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of betaalphabetaalphabetabeta-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future.

Shikimate kinase: a potential target for development of novel antitubercular agents.

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

Slow-onset inhibition of 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis by an inorganic complex.

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. The reemergence of tuberculosis as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, and the proliferation of multi-drug-resistant strains have created a need for the development of new antimycobacterial agents. Mycolic acids, the hallmark of mycobacteria, are high-molecular-weight alpha-alkyl, beta-hydroxy fatty acids, which appear mostly as bound esters in the mycobacterial cell wall. The product of the M. tuberculosis inhA structural gene (InhA) has been shown to be the primary target for isoniazid (INH), the most prescribed drug for active TB and prophylaxis. InhA was identified as an NADH-dependent enoyl-ACP reductase specific for long-chain enoyl thioesters. InhA is a member of the mycobacterial Type II fatty acid biosynthesis system, which elongates acyl fatty acid precursors of mycolic acids. Although the history of chemotherapeutic agent development demonstrates the remarkably successful tinkering of a few structural scaffolds, it also emphasizes the ongoing, cyclical need for innovation. The main focus of our contribution is on new data describing the rationale for the design of a pentacyano(isoniazid)ferrateII compound that requires no KatG-activation, its chemical characterization, in vitro activity studies against WT and INH-resistant I21V M. tuberculosis enoyl reductases, the slow-onset inhibition mechanism of WT InhA by the inorganic complex, and molecular modeling of its interaction with WT InhA. This inorganic complex represents a new class of lead compounds to the development of anti-tubercular agents aiming at inhibition of a validated target.

Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis.

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M.tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3A, 2.2A, 2.0 A, and 1.9A. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T, and S94A INH-resistant mutants of InhA as compared to INH-sensitive wild-type InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes.

Functional shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv: purification and characterization.

Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents.

DAHP synthase from Mycobacterium tuberculosis H37Rv: cloning, expression, and purification of functional enzyme.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now "super strains," resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47U mg(-1) under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development.

Electron transfer kinetics and mechanistic study of the thionicotinamide coordinated to the pentacyanoferrate(III)/(II) complexes: a model system for the in vitro activation of thioamides anti-tuberculosis drugs.

The mechanism of activation thioamide-pyridine anti-tuberculosis prodrugs is poorly described in the literature. It has recently been shown that ethionamide, an important component of second-line therapy for the treatment of multi-drug-resistant tuberculosis, is activated through an enzymatic electron transfer (ET) reaction. In an attempt to shed light on the activation of thioamide drugs, we have mimicked a redox process involving the thionicotinamide (thio) ligand, investigating its reactivity through coordination to the redox reversible [Fe(III/II)(CN)(5)(H(2)O)](2-/3-) metal center. The reaction of the Fe(III) complex with thionicotinamide leads to the ligand conversion to the 3-cyanopyridine species coordinated to a Fe(II) metal center. The rate constant, k(et)=10 s(-1), was determined for this intra-molecular ET reaction. A kinetic study for the cross-reaction of thionicotinamide and [Fe(CN)(6)](3-) was also carried out. The oxidation of thionicotinamide by [Fe(CN)(6)](3-) leads to formation of mainly 3-cyanopyridine and [Fe(CN)(6)](4-) with a k(et)=(5.38+/-0.03) M(-1)s(-1) at 25 degrees C, pH 12.0. The rate of this reaction is strongly dependent on pH due to an acid-base equilibrium related to the deprotonation of the R-SH functional group of the imidothiol form of thionicotinamide. The kinetic results reinforced the assignment of an intra-molecular mechanism for the ET reaction of [Fe(III)(CN)(5)(H(2)O)](2-) and the thioamide ligand. These results can be valuable for the design of new thiocarbonyl-containing drugs against resistant strains of Mycobacterium tuberculosis by a self-activating mechanism.

Crystallographic structure of PNP from Mycobacterium tuberculosis at 1.9A resolution.

Even being a bacterial purine nucleoside phosphorylase (PNP), which normally shows hexameric folding, the Mycobacterium tuberculosis PNP (MtPNP) resembles the mammalian trimeric structure. The crystal structure of the MtPNP apoenzyme was solved at 1.9 A resolution. The present work describes the first structure of MtPNP in complex with phosphate. In order to develop new insights into the rational drug design, conformational changes were profoundly analyzed and discussed. Comparisons over the binding sites were specially studied to improve the discussion about the selectivity of potential new drugs.

An inorganic iron complex that inhibits wild-type and an isoniazid-resistant mutant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis.

The in vitro kinetics of inactivation of both wild-type and I21V InhA enzymes by [FeII(CN)5(INH)]3- indicate that this process requires no activation by KatG, and no need for the presence of NADH. This inorganic complex may represent a new class of lead compounds to the development of anti-tubercular agents aiming at inhibition of a validated target.

Protective immune response against methicillin resistant Staphylococcus aureus in a murine model using a DNA vaccine approach.

Methicillin resistant Staphylococcus aureus (MRSA) are a major pathogen responsible for serious hospital infections worldwide. These bacteria are resistant to all beta-lactam antibiotics due to the production of an additional penicillin binding protein, the PBP2a, encoded by the mecA gene, which shows low affinity for this class of antibiotics. In this study, we cloned an internal region from the transpeptidase domain from the PBP2a into a mammalian expression vector, to be used as DNA vaccine in a Murine model. After three sets of DNA vaccination, the immune response represented by antibodies against a fragment of PBP2a was evaluated by enzyme linked immunosorbent assay (ELISA), showing a significant antibody response. The antibacterial effect of the DNA vaccine was evaluated by intraperitoneal immunization and challenge with a sublethal dose of MRSA for 7 days in mice. After the challenge, the number of bacteria from kidneys from immunized and non-immunized mice were determined. Kidneys from immunized mice had 1000 times less on bacteria than the positive controls (non-immunized mice). The response specificity indicates no effects against the normal PBPs from staphylococci and no effects against Gram positive rods from normal intestinal flora. Our results indicate that the immunization against the PBP2a from MRSA using a DNA vaccine approach could be used as a new strategy to efficiently fight these multiresistant bacteria.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis.

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aroA-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1L of LB cell culture, with a specific activity value of approximately 18 Umg(-1). The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (P(i)) and continuous assay of reactions that generate P(i) such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P(i) detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P(i) detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1L cell culture, with a specific activity value of 80 Umg(-1).