Design,synthesis and biological activity of novel substituted 3-benzoic acid derivatives as MtDHFR inhibitors

The enzyme dihydrofolate reductase from M. tuberculosis (MtDHFR) has a high unexploited potential to be a target for new drugs against tuberculosis (TB), due to its importance for pathogen survival. Preliminary studies have obtained fragment-like molecules with low affinity to MtDHFR which can potentially become lead compounds. Taking this into account, the fragment MB872 was used as a prototype for analogue development by bioisosterism/retro-bioisosterism, which resulted in 20 new substituted 3-benzoic acid derivatives. Compounds were active against MtDHFR, with IC₅₀ values ranging from 7 to 40 μM, where compound 4e not only had the best inhibitory activity (IC₅₀ = 7 μM), but also was 71-fold more active than the original fragment MB872. The 4e inhibition kinetics indicated an uncompetitive mechanism, which was supported by molecular modeling which suggested that the compounds can access an independent backpocket from the substrate and competitive inhibitors. Thus, based on these results, substituted 3-benzoic acid derivatives have strong potential to be developed as novel MtDHFR inhibitors and also anti-TB agents.     

Ligand specificity of group I biotin protein ligase of Mycobacterium tuberculosis

Background

Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC.

Methodology/Principal Findings

BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the ∼29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5′AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5′AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5′AMP. Docking simulations also suggest that bio-5′AMP hydrogen bonds to the conserved ‘GRGRRG’ sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K_m for BCCP was ∼5.2 µM and ∼420 nM for biotin. MtBPL has low affinity (K_b = 1.06×10⁻⁶ M) for biotin relative to EcBirA but their K_m are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5′AMP by EcBirA is channeled for its repressor activity.

Conclusions/Significance

These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.     

A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase

Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, ¹HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation.     

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H₂O₂-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.     

Incrassamarin A–D: Four new 4-substituted coumarins from Calophyllum incrassatum and their biological activities

Four new 4-substituted coumarins, incrassamarin A (1), B (2), C (3) and D (4) with (7S,8S)-7,8-dihydro-5-hydroxy-7,8-dimethyl-4-propyl-2H,6H-benzo[1,2-b;5,4-b’]dipyran-2,6-dione (5), friedelin, carpachromene, amentoflavone, epiafzelechin and L-quercitrin were isolated from the barks and leaves of Calophyllum incrassatum (Guttiferae). The compounds were isolated and purified by size-exclusion recycling HPLC and column chromatographic techniques. The structures of the compounds were determined by spectroscopic means. The compounds were tested for their cytotoxic activity towards MCF-7 and A-549 cell lines and α-glucosidase enzymatic inhibitory activity. Compound (1) displayed cytotoxic activity against A-549 cell lines with IC₅₀ 87.71 μg/mL and showed inhibition towards α-glucosidase enzymatic activity with IC₅₀ 93.25 μM. This is the first report on the isolation of phytochemicals from the barks and leaves of C. incrassatum and their bioactivities.     

Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit

Background

The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas'' disease chemotherapy is sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.

Methodology/Principal Finding

In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51_Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51_Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51_Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site.

Conclusions/Significance

CYP51_Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51_Tc.     

Solution structure of subunit γ (γ1-204) of the Mycobacterium tuberculosis F-ATP synthase and the unique loop of γ165-178, representing a novel TB drug target

Tuberculosis, caused by the strain Mycobacterium tuberculosis, is in focus of interest due to the emergence of multi- and extensive drug-resistant TB strains. The F₁F_O ATP synthase is one of the essential enzymes in energy requirement of both proliferating aerobic and hypoxic dormant stage of mycobacterium life cycle, and therefore a potential TB drug target. Subunit γ of F-ATP synthases plays an important role in coupling and catalysis via conformational transitions of its N- and C-termini as well as the bottom segment of the globular domain of γ, which is in close proximity to the rotating and ion-pumping c-ring. Here we describe the first production, purification and low resolution solution structure of subunit γ (γ_1–204, Mtγ_1–204) of the M. tuberculosis F-ATP synthase. Mtγ_1–204 is a pear-like shaped protein with a molecular weight of 23?±?2 kDa. Protein sequence analysis of Mtγ revealed differences in the amino acid composition to γ subunits from other sources, in particular the presence of a unique stretch of 13 amino acid residues (Mtγ_165–178). NMR studies showed that Mtγ_165–178 forms a loop of polar residues. Mtγ_165–178 has been aligned at the bottom of the globular domain of the Escherichia coli subunit γ, being in close vicinity to the polar residues R41, Q42, E44 and Q46 (M. tuberculosis nomenclature) of the c-ring. The putative role(s) of Mtγ_165–178 in coupling and as a potential drug target are discussed.     

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.     

Mycobacterial Nicotinate Mononucleotide Adenylyltransferase: STRUCTURE,MECHANISM, AND IMPLICATIONS FOR DRUG DISCOVERY*

Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD (MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 3₁₀ helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity (K_i ∼ 25 μm) and antimycobacterial activity (MIC₈₀) ∼ 40–80 μm). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. These findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.     

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PP_i). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP_i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.     

Synthesis,biological evaluations and computational studies of N-(3-(-2-(7-Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors

In the present investigation, new chloroquinoline derivatives bearing vinyl benzylidene aniline substituents at 2nd position were synthesized and screed for biofilm inhibitory, antifungal and antibacterial activity. The result of biofilm inhibition of C. albicans suggested that compounds 5j (IC₅₀ value?=?51.2?μM) and 5a (IC₅₀ value?=?66.2?μM) possess promising antibiofilm inhibition when compared with the standard antifungal drug fluconazole (IC₅₀?=?40.0?μM). Two compounds 5a (MIC?=?94.2?μg/mL) and 5f (MIC?=?98.8?μg/mL) also exhibited good antifungal activity comparable to standard drug fluconazole (MIC?=?50.0?μg/mL). The antibacterial screening against four strains of bacteria viz. E. coli, P. aeruginosa, B. subtilis, and S. aureus suggested their potential antibacterial activity and especially all the compounds except 5g were found more active than the standard drug ciprofloxacin against B. subtilis. To further gain insights into the possible mechanism of these compounds in biofilm inhibition through the agglutinin like protein (Als), molecular docking and molecular dynamics simulation studies were carried out. Molecular modeling studies suggested the clear role in inhibition of this protein and the resulting biofilm inhibitory activity.     

Nuclear metabolism. II. Further studies on epoxide hydrolase activity

Apparent K_m- and V_max-values of nuclear styrene 7,8-oxide hydrolase were determined at different protein concentrations. In the protein concentrations range used no significant differences in the apparent K_m-values were observed. The influence of the incubation with different modifiers (i.e. SKF-525A, metyrapone, 1,2-epoxy-3,3,3 trichloropropane, cyclohexene oxide) at two different concentrations on this enzyme activity was also determined. Cyclohexene oxide and 1,2-epoxy-3,3,3-trichloropropane, two well known inhibitors of the microsomal epoxide hydrolase(s) caused a marked inhibition, metyrapone had a strong activating effect whereas SKF-525A had no effect. In vivo pretreatment with phenobarbital significantly induced the nuclear epoxide hydrolase whereas β-naphthoflavone caused a lower degree of induction. This pattern is quantitatively different but qualitatively very similar to the microsomal one. Moreover a toxifying to detoxifying enzymatic activity balance is attempted for the metabolization of the alkenic double bond of styrene, taking into account the ratio between the styrene monooxygenase (toxifying enzyme) and the styrene 7,8-oxide hydrolase (detoxifying enzyme) after the above mentioned pretreatments, both in the microsomal and nuclear fractions.     

The Pentapeptide Repeat Proteins MfpAMt and QnrB4 Exhibit Opposite Effects on DNA Gyrase Catalytic Reactions and on the Ternary Gyrase-DNA-Quinolone Complex

MfpA_Mt and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA_Mt gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA_Mt on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 μM, MfpA_Mt inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 μM. We showed that the D87 residue in GyrA has a major role in the MfpA_Mt-gyrase interaction, as D87H and D87G substitutions abolished MfpA_Mt inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpA_Mt and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 μM as described for other Qnr proteins, but MfpA_Mt did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA_Mt also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpA_Mt and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.The pentapeptide repeat protein (PRP) family includes more than 500 proteins in the prokaryotic and eukaryotic kingdoms (45). PRPs are characterized by the repetition of the pentapeptide repeat motif [S,T,A,V][D,N][L,F][S,T,R][G] (6), which results in a right-handed β-helical structure (8, 17). The functions of the majority of the members of this large and heterogeneous family remain unknown, but three PRPs, McbG (from Escherichia coli), MfpA_Mt (from Mycobacterium tuberculosis), and Qnr (from Klebsiella pneumoniae and other enterobacteria) were reported to interact with DNA gyrase, at least with the E. coli enzyme (17, 33, 35, 44). McbG was shown to protect E. coli DNA gyrase from the toxic action of microcin B17 (33). Qnr and MfpA_Mt were involved in resistance to fluoroquinolones, which are synthetic antibacterial agents prescribed worldwide for the treatment of various infectious diseases, including tuberculosis (7).DNA gyrase is an essential ATP-dependent enzyme that transiently cleaves a segment of double-stranded DNA, passes another piece of DNA through the break, and reseals it (12). DNA gyrase is unique in catalyzing the negative supercoiling of DNA in order to facilitate the progression of RNA polymerase. Most eubacteria, such as E. coli, have two type II DNA topoisomerases, i.e., DNA gyrase and topoisomerase IV, but a few, such as M. tuberculosis, harbor only DNA gyrase (11).Quinolones target type II topoisomerases, and their activity is measured by the inhibition of supercoiling by gyrase or decatenation by topoisomerase IV and stabilization of complexes composed of topoisomerase covalently linked to cleaved DNA (16). The DNA gyrase active enzyme is a GyrA₂GyrB₂ heterotetramer. The quinolone-gyrase interaction site in gyrase is thought to be located at the so-called quinolone resistance-determining regions (QRDR) in the A subunit (amino acids 57 to 196 in GyrA) and the B subunit (amino acids 426 to 466 in GyrB), which contain the majority of mutations conferring quinolone resistance (19). The GyrB QRDR is thought to interact with the GyrA QRDR to form a drug-binding pocket (18). Resistance to quinolones is usually due to chromosomal mutations either in the structural genes encoding type II topoisomerases (QRDR) (19, 22) or in regulatory genes producing decreased cell wall permeability or enhancement of efflux pumps (36). The recent emergence of plasmid-borne resistance genes, such as qnr (9, 13, 31, 38, 46), aac(6′)-Ib-cr (32, 39) and qepA (34, 47), renewed interest in quinolone resistance, and especially interest in the new Qnr-based mechanism. Three qnr determinants have been identified so far: qnrA (variants A1 to A6), qnrB (variants B1 to B19), and qnrS (variants S1 and S2) (15, 21, 23, 27). Qnr confers a new mechanism of quinolone resistance by mediating DNA gyrase protection (42): in vitro, QnrA1 and QnrB1 protect E. coli DNA gyrase and topoisomerase IV from the inhibitory effect of fluoroquinolones in a concentration-dependent manner (23, 42-44). Although Qnr was shown to bind GyrA and GyrB and compete with DNA binding, the consequences of Qnr binding for enzyme performance are not yet clear.mfpA, a chromosomal gene that encodes a 192-amino-acid PRP, is an intrinsic quinolone resistance determinant of Mycobacterium smegmatis (29). A similar gene, mfpA_Mt, was found in the M. tuberculosis genome, and MfpA_Mt shows 67% identity with MfpA. Recent crystallography analysis of MfpA_Mt showed that its atomic structure displays size, shape, and electrostatic similarity to B-form DNA, and MfpA_Mt has been suggested to interact with DNA gyrase via DNA mimicry (17). The effect of MfpA_Mt was studied by testing E. coli DNA gyrase, and MfpA_Mt showed catalytic inhibition (17, 37), but whether it protects gyrase from quinolones was not assessed. Because the structure and functions of the M. tuberculosis gyrase, as well as its interaction with quinolones, differ from those of the E. coli gyrase (2, 3, 20, 26, 28), we suspected that the PRP-topoisomerase interaction exhibits species specificity, i.e., depends on the proteins issued from the same host.Our objective was to compare the effects of MfpA_Mt and Qnr on their respective targets, i.e., the effect of MfpA_Mt on the M. tuberculosis gyrase and the effect of Qnr on the E. coli gyrase, by assessing (i) the catalytic reactions of the enzyme and (ii) the interaction with the DNA gyrase-DNA-fluoroquinolone ternary complex. Among the Qnr proteins, we selected the QnrB4 protein, which is a frequent variant of QnrB and has not yet been purified and studied. We cloned, expressed, and purified the two PRPs, MfpA_Mt and QnrB4, as recombinant His tag fusion proteins and assessed their functions under the same experimental conditions.     

Design and synthesis of novel thiadiazole-thiazolone hybrids as potential inhibitors of the human mitotic kinesin Eg5

A novel series of 1,3,4-thiadiazole-thiazolone hybrids 5a–v were designed, synthesized, characterized, and evaluated against the basal and the microtubule (MT)-stimulated ATPase activity of Eg5. From the evaluated derivatives, 5h displayed the highest inhibition with an IC₅₀ value of 13.2?µM against the MT-stimulated Eg5 ATPase activity. Similarly, compounds 5f and 5i also presented encouraging inhibition with IC₅₀ of 17.2?µM and 20.2?µM, respectively. A brief structure–activity relationship (SAR) analysis indicated that 2-chloro and 4-nitro substituents on the phenyl ring of the thiazolone motif contributed significantly to enzyme inhibition. An in silico molecular docking study using the crystal structure of Eg5 further supported the SAR and reasoned the importance of crucial molecular protein–ligand interactions in influencing the inhibition of the ATPase activity of Eg5. The magnitude of the electron-withdrawing functionalities over the hybrids and the critical molecular interactions contributed towards higher in vitro potency of the compounds. The drug-like properties of the synthesized compounds 5a–v were also calculated based on the Lipinski’s rule of five and in silico computation of key pharmacokinetic parameters (ADME). Thus, the present work unveils these hybrid molecules as novel Eg5 inhibitors with promising drug-like properties for future development.     

Regulation by oligomerization in a mycobacterial folate biosynthetic enzyme

Folate derivatives are essential cofactors in the biosynthesis of purines, pyrimidines and amino acids across all forms of life. Mammals uptake folate from their diets, whereas most bacteria must synthesize folate de novo. Therefore, the enzymes in the folate biosynthetic pathway are attractive drug targets against bacterial pathogens such as Mycobacterium tuberculosis, the cause of the world's most deadly infectious disease, tuberculosis (TB). M.tuberculosis 7,8-dihydroneopterin aldolase (Mtb FolB, DHNA) is the second enzyme in the folate biosynthetic pathway, which catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and glycoaldehyde. The 1.6A X-ray crystal structure of Mtb FolB complexed with its product, 6-hydroxymethyl-7,8-dihydropterin, reveals an octameric assembly similar to that seen in crystal structures of other FolB homologs. However, the 2.5A crystal structure of unliganded Mtb FolB reveals a novel tetrameric oligomerization state, with only partially formed active sites. A substrate induced conformational change appears to be necessary to convert the inactive tetramer to the active octamer. Ultracentrifugation confirmed that in solution unliganded Mtb FolB is mainly tetrameric and upon addition of substrate FolB is predominantly octameric. Kinetic analysis of substrate binding gives a Hill coefficient of 2.0, indicating positive cooperativity. We hypothesize that Mtb FolB displays cooperativity in substrate binding to regulate the cellular concentration of 7,8-dihydroneopterin, so that it may function not only as a precursor to folate but also as an antioxidant for the survival of M.tuberculosis against host defenses.     

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease’s causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli’s nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (E_a, ΔG^#, ΔS^#, ΔH^#) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.     

Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77

BackgroundFungal aryl-alcohol oxidases (AAOx) are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids, with concomitant production of hydrogen peroxide (H₂O₂) as by-product. The H₂O₂ can be provided to lignin degradation pathway, a biotechnological property explored in biofuel production. In the thermophilic fungus Thermothelomyces thermophilus (formerly Myceliophthora thermophila), just one AAOx was identified in the exo-proteome.MethodsThe glycosylated and non-refolded crystal structure of an AAOx from T. thermophilus at 2.6 Å resolution was elucidated by X-ray crystallography combined with small-angle X-ray scattering (SAXS) studies. Moreover, biochemical analyses were carried out to shed light on enzyme substrate specificity and thermostability.ResultsThis flavoenzyme harbors a flavin adenine dinucleotide as a cofactor and is able to oxidize aromatic substrates and 5-HMF. Our results also show that the enzyme has similar oxidation rates for bulky or simple aromatic substrates such as cinnamyl and veratryl alcohols. Moreover, the crystal structure of MtAAOx reveals an open active site, which might explain observed specificity of the enzyme.ConclusionsMtAAOx shows previously undescribed structural differences such as a fully accessible catalytic tunnel, heavy glycosylation and Ca²⁺ binding site providing evidences for thermostability and activity of the enzymes from AA3_2 subfamily.General significanceStructural and biochemical analyses of MtAAOx could be important for comprehension of aryl-alcohol oxidases structure-function relationships and provide additional molecular tools to be used in future biotechnological applications.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.