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Leishmaniasis is an infectious disease caused by obligate intracellular protozoa of the genus Leishmania with high infection and death rates in developing countries. New drugs with better pharmacological performance with regards to safety, efficacy, toxicity, and drug resistance than those/the ones currently used are urgently needed. Trypanothione synthetase (TryS) is an attractive target for the development of drugs against leishmaniasis because it is specific and essential to kinetoplastid parasites. In this study, Leishmaniamajor TryS was expressed and purified, and the kinetic parameters of purified TryS were determined. To identify novel inhibitors of LmTryS, a high-throughput screening (HTS) assay was developed and used to screen a library of 35,040 compounds. In the confirmatory assay, 42 compounds displayed half maximal inhibitory concentration (IC

Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS’s synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 μM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 μM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 μM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

Rimklb is a mammalian homologue of the E. coli enzyme RimK, which catalyzes addition of glutamic acid to the ribosomal protein S6. To date, no previous studies have shown any physiological role for Rimklb in mammals. In this study, using Western blotting, we found that Rimklb is distributed and expressed in mouse testis and heart. Rimklb was subsequently localized to the testicular Leydig cells using immunohistochemistry with an anti-Rimklb antibody. We generated a Rimklb mutant mouse in which a three-base deletion results in deletion of Ala 29 and substitution of Leu 30 with Val, which we named the RimklbA29del, L30V mutant mouse. RimklbA29del, L30V mutant mice show a decrease in testicular size and weight, and in vitro fertilization demonstrates complete male infertility. Furthermore, we found that a key factor in the mammalian target of the rapamycin/ribosomal protein S6 transcriptional pathway is hyperphosphorylated in the seminiferous tubules of the mutant testis. We conclude that Rimklb has important roles that include spermatogenesis in seminiferous tubules. In summary, male RimklbA29del, L30V mice are infertile.

A rational-based process was adopted for repurposing pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs bearing variable acyl chains, different stereochemical configuration and/or positional relationships. Structural features were highly influential on activity. Amongst, enantiomer 1e having 1,2-vicinal relationship for the -CH2O- and the N-acyl moieties, a saturated palmitoyl chain and an opposite stereochemical configuration to natural sphingolipids was the most potent hit compound against promastigotes showing IC50 value of 28.32 µM. The corresponding enantiomer 1a was 2-fold less potent showing a eudismic ratio of 0.54 in promastigotes. Compounds 1a and 1e inhibited the growth of amastigotes more potently relative to promastigotes. Amongst, enantiomer 1a as the more selective and safer. In silico docking study using a homology model of Leishmania donovani inositol phosphoceramide synthase (IPCS) provided plausible reasoning for the molecular factors underlying the found activity. Collectively, this study suggests compounds 1a and 1e as potential hit compounds for further development of new antileishmanial agents.

NAD synthetase (NadE) catalyzes the last step in NAD biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction with co-substrates ATP and amide donor ammonia. In this study, we report the crystal structure of Staphylococcus aureus NAD synthetase enzyme (saNadE) at 2.3 A resolution. We used this structure to perform molecular dynamics simulations of apo-enzyme, enzyme-substrate (NadE with ATP and NaAD) and enzyme-intermediate complexes (NadE with NaAD-AMP) to investigate key binding interactions and explore the conformational transitions and flexibility of the binding pocket. Our results show large shift of N-terminal region in substrate bound form which is important for ATP binding. Substrates drive the correlated movement of loop regions surrounding it as well as some regions distal to the active site and stabilize them at complex state. Principal component analysis of atomic projections distinguish feasible trajectories to delineate distinct motions in enzyme-substrate to enzyme-intermediate states. Our results suggest mixed binding involving dominant induced fit and conformational selection. MD simulation extracted ensembles of NadE could potentially be utilized for in silico screening and structure based design of more effective Methicillin Resistant Staphylococcus aureus (MRSA) inhibitors.

Leishmania and Trypanosoma parasites are responsible for the challenging neglected tropical diseases leishmaniases, Chagas disease, and human African trypanosomiasis, which account for up to 40,000 deaths annually mainly in developing countries. Current chemotherapy relies on drugs with significant limitations in efficacy and safety, prompting the urgent need to explore innovative approaches to improve the drug discovery pipeline. The unique trypanothione-based redox pathway, which is absent in human hosts, is vital for all trypanosomatids and offers valuable opportunities to guide the rational development of specific, broad-spectrum and innovative anti-trypanosomatid agents. Major efforts focused on the key metabolic enzymes trypanothione synthetase-amidase and trypanothione reductase, whose inhibition should affect the entire pathway and, finally, parasite survival. Herein, we will report and comment on the most recent studies in the search for enzyme inhibitors, underlining the promising opportunities that have emerged so far to drive the exploration of future successful therapeutic approaches.

The genome of Mycobacterium tuberculosis encode for several hypothetical proteins that needed to be characterized. Rv2037c, a hypothetical protein, was 25 and 4 folds upregulated under acidic and nutritive stress, respectively in M. tuberculosis H37Ra. The protein demonstrated lipolytic activity with pNP-decanoate with optimum pH 8.0 and temperature 40 °C. In addition, the protein demonstrated phospholipase activity. To understand the effect of rv2037c on mycobacterium physiology, the gene was cloned and expressed in M. smegmatis. The protein was found in membrane and extracellular fraction. The expression of rv2037c in M. smegmatis (MS_Rv2037c) altered colony morphology and cell surface features like enhanced biofilm and pellicle formation. MS_Rv2037c decreased cell-wall permeability, enhanced TDM content, resistance against various stresses and antibiotics. MS_Rv2037c demonstrated better infection and intracellular survival capability in infected THP-1 macrophage. Macrophages treated with Rv2037c demonstrated irregular cell membrane. Mice infected with MS_Rv2037c had higher bacterial load in lung, liver and spleen compared to control. Rv2037c induced the production of pro-inflammatory cytokines TNFα and IL12, suggesting its role in immune-modulation. Recombinant protein also generated humoral response in EPTB and MDR-TB patients. The results pointed towards the crucial role of this enzyme in cell-wall modulation, infection and intracellular survival of mycobacterium.

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.

Redox homeostasis in trypanosomatids is based on the low-molecular-weight trypanothione, an essential dithiol molecule that is synthetized by trypanothione synthetase (TryS) and maintained in its reduced state by trypa- nothione disulfide reductase (TryR). The fact that both enzymes are indispensable for parasite survival and absent in the mammalian hosts makes them ideal drug targets against leishmaniasis. Although many efforts have been directed to developing TryR inhibitors, much less attention has been focused on TryS. The screening of an in-house library of 144 diverse molecules using two parallel biochemical assays allowed us to detect 13 inhibitors of L. infantum TryS. Compounds 1 and 3 were characterized as competitive inhibitors with Ki values in the low micromolar range and plausible binding modes for them were identified by automated ligand docking against refined protein structures obtained through computational simulation of an entire catalytic cycle. The proposed binding site for both inhibitors overlaps the polyamine site in the enzyme and, additionally, 1 also occupies part of the ATP site. Compound 4 behaves as a mixed hyperbolic inhibitor with a Ki of 0.8 μM. The activity of 5 is clearly dependent on the concentration of the polyamine substrate, but its kinetic behavior is clearly not compatible with a competitive mode of inhibition. Analysis of the activity of the six best inhibitors against intracellular amastigotes identified 5 as the most potent leishmanicidal candidate, with an EC50 value of 0.6 μM and a selectivity index of 35., This work has been supported by the Spanish MICINN (Projects PID2019-104070RB-C21 and PID2019-104070RBC22), the Spanish National Research Council (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE-201980E028), and the Comunidad de Madrid (PLATESA2-CM ref S-2018/BAA-4370). The MCIN is also acknowledged for the pre- doctoral fellowship to M.A.C.

Ammonia dependent NAD

Pseudomonas aeruginosa (PA) is an electrogenic bacterium, in which extracellular electron transfer (EET) is mediated by microbially-produced phenazines, especially pyocyanin. Increasing EET rate in electrogenic bacteria is key for the development of biosensors and bioelectrofermentation processes. In this work, the production of pyocyanin, Nicotinamide Adenine Dinucleotide (NAD) and NAD synthetase by the electrogenic strain PA-A4 is determined using a Microbial Fuel Cell (MFC). Effects of metabolic inhibition and enhancement of pyocyanin and NAD synthetase on NAD/NADH levels and electrogenicity was demonstrated by short chronoamperometry measurements (0-48 h). Combined overexpression of two intermediate NAD synthetase production genes-nicotinic acid mononucleotide adenyltransferase (nadD) and quinolic acid phosphoribosyltransferase (nadC) genes, which are distant on the PA genomic map, enabled co-transcription and increased NAD synthetase activity. The resulting PA-A4 nadD + nadC shows increases in pyocyanin concentration, NAD synthetase activity, NAD/NADH levels, and MFC potential, all significantly higher than its wild type. Extracellular respiratory mechanisms in PA are linked with NAD metabolism, and targeted increased yield of NAD could directly lead to enhanced EET. A previous attempt at enhancing NAD synthetase for electrogenicity by targeting the terminal NAD synthetase gene (nadE) in standard P. aeruginosa PA01 had earlier been reported. Our work however, poses another route to electrogenicity enhancement in PA using; a combination of nadD and nadC. Further experiments are needed to understand specific intracellular mechanisms governing how over-expression of nadD and nadC induced activity of NadE protein. These findings significantly advance the knowledge of the versatility of NAD biosynthetic genes in PA electrogenicity.

Background: Leishmaniasis is caused by a protozoan parasite, Leishmania. It is common in more than 98 countries throughout the world. Due to insufficient availability of antileishmanial chemotherapeutics, it is an urgent need to search for new molecules which have better efficacy, low toxicity and are available at low cost. Objectives: There is a high rate of diabetic cases throughout the world that is why we planned to test the antileishmanial activity of glyburide, an effective sugar lowering drug used for the treatment of diabetes. In this study, glyburide showed a significant decrease in the parasite growth and survival in vitro in a dose-dependent manner. Methods: Anti-leishmanial activity of glyburide was checked by culturing Leishmania donovani promastigotes in the presence of glyburide in a dose and time dependent manner. Docking study against Leishmania donovani-Trypanothione synthetase (LdTrySyn) protein was performed using Autodock Vina tool. Results: Growth reversibility assay shows that growth of treated parasite was not reversed when transferred to fresh culture media after 7 days. Moreover, docking studies show efficient interactions of glyburide with key residues in the catalytic site of Leishmania donovani- Trypanothione synthetase (LdTrySyn), a very important leishmanial enzyme involved in parasite’s survival by detoxification of Nitric Oxide (NO) species, generated by the mammalian host as a defense molecule. Thus this study proves that the drug-repurposing is a beneficial strategy for identification of new and potent antileishmanial molecules. Conclusion: The results suggest that glyburide binds to LdTrySyn and inhibits its activity which further leads to the altered parasite morphology and inhibition of parasite growth. Glyburide may also be used in combination with other anti-leishmanial drugs to potentiate the response of the chemotherapy. Overall this study provides information about combination therapy as well as a single drug treatment for the infected patients suffering from diabetes. This study also provides raw information for further in vivo disease model studies to confirm the hypothesis.

Polyamines are essential for biofilm formation in Escherichia coli, but it is still unclear which polyamines are primarily responsible for this phenomenon. To address this issue, we constructed a series of E. coli K-12 strains with mutations in genes required for the synthesis and metabolism of polyamines. Disruption of the spermidine synthase gene (speE) caused a severe defect in biofilm formation. This defect was rescued by the addition of spermidine to the medium but not by putrescine or cadaverine. A multidrug/spermidine efflux pump membrane subunit (MdtJ)-deficient strain was anticipated to accumulate more spermidine and result in enhanced biofilm formation compared to the MdtJ(+) strain. However, the mdtJ mutation did not affect intracellular spermidine or biofilm concentrations. E. coli has the spermidine acetyltransferase (SpeG) and glutathionylspermidine synthetase/amidase (Gss) to metabolize intracellular spermidine. Under biofilm-forming conditions, not Gss but SpeG plays a major role in decreasing the too-high intracellular spermidine concentrations. Additionally, PotFGHI can function as a compensatory importer of spermidine when PotABCD is absent under biofilm-forming conditions. Last, we report here that, in addition to intracellular spermidine, the periplasmic binding protein (PotD) of the spermidine preferential ABC transporter is essential for stimulating biofilm formation. IMPORTANCE Previous reports have speculated on the effect of polyamines on bacterial biofilm formation. However, the regulation of biofilm formation by polyamines in Escherichia coli has not yet been assessed. The identification of polyamines that stimulate biofilm formation is important for developing novel therapies for biofilm-forming pathogens. This study sheds light on biofilm regulation in E. coli. Our findings provide conclusive evidence that only spermidine can stimulate biofilm formation in E. coli cells, not putrescine or cadaverine. Last, ΔpotD inhibits biofilm formation even though the spermidine is synthesized inside the cells from putrescine. Since PotD is significant for biofilm formation and there is no ortholog of the PotABCD transporter in humans, PotD could be a target for the development of biofilm inhibitors.

Trypanosomatid-caused diseases are among the neglected infectious diseases with the highest disease burden, affecting about 27 million people worldwide and, in particular, socio-economically vulnerable populations. Trypanothione synthetase (TryS) is considered one of the most attractive drug targets within the thiol-polyamine metabolism of typanosomatids, being unique, essential and druggable. Here, we have compiled a dataset of 401 T. brucei TryS inhibitors that includes compounds with inhibitory data reported in the literature, but also in-house acquired data. QSAR classifiers were derived and validated from such dataset, using publicly available and open-source software, thus assuring the portability of the obtained models. The performance and robustness of the resulting models were substantially improved through ensemble learning. The performance of the individual models and the model ensembles was further assessed through retrospective virtual screening campaigns. At last, as an application example, the chosen model-ensemble has been applied in a prospective virtual screening campaign on DrugBank 5.1.6 compound library. All the in-house scripts used in this study are available on request, whereas the dataset has been included as supplementary material.

Salmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis., Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The “late steps” of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5′ phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis.

The degradation pathways in microorganisms for piperidine, a secondary amine with various applications, are not yet fully understood, especially in non-Mycobacterium species. In this study, we have identified a piperidine-degrading isolate (KU43P) from a soil sample collected in a cultivation field in Osaka, Japan, and characterized its mechanisms of piperidine degradation, thereby furthering current understanding of the process. The genome of isolate KU43P consists of a 5,869,691-bp circular chromosome with 62.67% GC content and with 5,294 predicted protein-coding genes, 77 tRNA genes, and 22 rRNA genes. 16S rRNA gene sequence analysis and average nucleotide identity analysis suggest that the isolate is a novel species of the Pseudomonas putida group in the genus Pseudomonas. The genomic region encoding the piperidine degradation pathway, designated as the pip gene cluster, was identified using transposon mutagenesis and reverse transcription polymerase chain reaction. Deletion analyses of pipA, which encodes a glutamine synthetase (GS)-like protein, and pipBa, which encodes a cytochrome P450 monooxygenase, indicate that pipA and pipBa are involved in piperidine metabolism and suggest that pipA is involved in the first step of the piperidine metabolic pathway. Escherichia coli whole cells overexpressing PipA converted piperidine and glutamate to γ-glutamylpiperidide, and crude cell extract enzyme assays of PipA showed that this reaction requires ATP and Mg

Buthionine sulfoximine (BSO) induces decreased GSH and trypanothione [T(SH)2] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2-metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or glutathione, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not glutathione synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the glutathione site. Furthermore, parasites overexpressing γECS and TryS showed ~50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS. This article is protected by copyright. All rights reserved.

Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure-activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50 = 1000 μM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90 μM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19-100 μg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.

Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by certain metabolites, especially l-theanine, in C. sinensis. To determine why more l-theanine accumulates in C. sinensis than in other plants, we compare l-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic route. We quantify relevant intermediates and metabolites by mass spectrometry. l-Glutamic acid, a precursor of l-theanine, is present in most plants, while ethylamine, another precursor of l-theanine, specifically accumulates in Camellia species, especially C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and l-glutamic acid to l-theanine. After supplementation with [2H5]ethylamine, all the plants produce [2H5]l-theanine, which suggests that ethylamine availability is the...

XimA is a unique amide synthetase that belongs to the ANL superfamily of adenylating enzymes, but with a special structural fold. In order to improve the enzyme promiscuity, we engineered XimA by site-directed mutagenesis at a specific position based on our theoretical model of XimA. Thus, we were able to produce diverse benzopyran derivatives with up to 15 different l-form and d-form amino acid substitutions, catalyzed by several XimA variants. Molecular docking and molecular dynamics simulations conducted for various XimA systems provide further structural insights into the substitution effects of the phenylalanine-201 as an active site residue on protein dynamics and enzyme catalysis.

The amide functional group is ubiquitous in nature and one of the most important motifs in pharmaceuticals, agrochemicals, and other valuable products. While coupling amides and carboxylic acids is a trivial synthetic transformation, it often requires protective group manipulation, along with stoichiometric quantities of expensive and deleterious coupling reagents. Nature has evolved a range of enzymes to construct amide bonds, the vast majority of which utilize adenosine triphosphate to activate the carboxylic acid substrate for amine coupling. Despite the fact that these enzymes operate under mild conditions, as well as possessing chemoselectivity and regioselectivity that obviates the need for protecting groups, their synthetic potential has been largely unexplored. In this review, we discuss recent research into the discovery, characterization, and development of amide bond forming enzymes, with an emphasis on stand-alone ligase enzymes that can generate amides directly from simple carboxylic acid and amine substrates.

NADH (NAD

NAD+ synthetase is an essential enzyme of de novo and recycling pathways of NAD+ biosynthesis in Mycobacterium tuberculosis but not in humans. This bifunctional enzyme couples the NAD+ synthetase and glutaminase activities through an ammonia tunnel but free ammonia is also a substrate. Here we show that the Homo sapiens NAD+ synthetase (hsNadE) lacks substrate specificity for glutamine over ammonia and displays a modest activation of the glutaminase domain compared to tbNadE. We report the crystal structures of hsNadE and NAD+ synthetase from M. tuberculosis (tbNadE) with synthetase intermediate analogues. Based on the observed exclusive arrangements of the domains and of the intra- or inter-subunit tunnels we propose a model for the inter-domain communication mechanism for the regulation of glutamine-dependent activity and NH3 transport. The structural and mechanistic comparison herein reported between hsNadE and tbNadE provides also a starting point for future efforts in the development of anti-TB drugs., M. tuberculosis NAD+ synthetase (tbNadE) is of interest as a drug target. Here the authors present the actively trapped Homo sapiens NAD+ synthetase (hsNadE) and tbNadE structures and show key differences in the synthetase active site and in structural elements possibly involved in the allosteric regulation of catalysis to be leveraged for the development of M. tuberculosis selective inhibitors.

Plants are frequently exposed to variable environmental stresses that adversely affect plant growth, development and agricultural production. In this study, a trypanothione synthetase gene from Trypanosoma cruzi, TcTryS, was chemically synthesized and its roles in tolerance to multiple abiotic stresses were functionally characterized by generating transgenic rice overexpressing TcTryS. Overexpression of TcTryS in rice endows transgenic plants with hypersensitivity to ABA, hyposensitivity to NaCl- and mannitol-induced osmotic stress at the seed germination stage. TcTryS overexpression results in enhanced tolerance to drought, salt, cadmium, and 2,4,6-trichlorophenol stresses in transgenic rice, simultaneously supported by improved physiological traits. The TcTryS-overexpression plants also accumulated greater amounts of proline, less malondialdehyde and more transcripts of stress-related genes than wild-type plants under drought and salt stress conditions. In addition, TcTryS might play a positive role in maintaining chlorophyll content under 2,4,6-trichlorophenol stress. Histochemical staining assay showed that TcTryS renders transgenic plants better ROS-scavenging capability. All of these results suggest that TcTryS could function as a key regulator in modulation of abiotic stress tolerance in plant, and may have applications in the engineering of economically important crops.

Background: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids. Methods and Results: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000 M) and heavy metal (1-500 M) toxicity. Treatment with TryS inhibitors (5-30 M) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. Conclusions: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used antitrypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease. Fil: Mesias, Andrea Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Sasoni, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Orban, Oliver C. F.. Technische Universitat Carolo Wilhelmina Zu Braunschweig.; Alemania Fil: Kunick, Conrad. Technische Universitat Carolo Wilhelmina Zu Braunschweig.; Alemania Fil: Robello, Carlos. Instituto Pasteur de Montevideo; Uruguay Fil: Comini, Marcelo. Instituto Pasteur de Montevideo; Uruguay Fil: Garg, Nisha J.. University of Texas Medical Branch; Estados Unidos Fil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina

NAD (nicotinamide adenine dinucleotide) synthase catalyses the biochemical synthesis of NAD, from nicotinic acid adenine dinucleotide (NAAD). NAD may be synthesized through the

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase.

l-Theanine, a non-proteinaceous amino acid abundantly present in tea (Camellia sinensis), contributes to the umami flavor of tea and has beneficial effects on human health. While key l-theanine biosynthetic genes have been well documented, their transcriptional regulation remains poorly understood. In this study, we determined the l-theanine contents in tea leaves of two cultivars at three developmental stages and investigated the expression patterns of the l-theanine biosynthetic genes CsGS1 and CsGS2. Additionally, we identified an R2R3-MYB transcription factor, CsMYB73, belonging to subgroup 22 of the R2R3-MYB family. CsMYB73 expression negatively correlated with l-theanine accumulation during leaf maturation. We found that CsMYB73, as a nuclear protein, binds to the promoter regions of CsGS1 and CsGS2 via MYB recognition sequences and represses the transcription of CsGS1 and CsGS2 in tobacco leaves. Collectively, our results demonstrate that CsMYB73 is a transcriptional repressor involved in l-theanine biosynthesis in tea plants. Our findings might contribute to future tea plant breeding strategies.

Iron is essential for nearly all bacterial pathogens, including Mycobacterium tuberculosis (Mtb), but is severely limited in the human host. To meet its iron needs, Mtb secretes siderophores, small molecules with high affinity for iron, and takes up iron-loaded mycobactins (MBT) and carboxymycobactins (cMBT), from the environment. Mtb is also capable of utilizing heme and hemoglobin which contain more than 70% of the iron in the human body. However, many components of these iron acquisition pathways are still unknown. In this study, a high-density transposon mutagenesis coupled with deep sequencing (TnSeq) showed that Mtb exhibits nearly opposite requirements for 165 genes in the presence of heme and hemoglobin versus MBT and cMBT as iron sources. The ESX-3 secretion system was assessed as essential for siderophore-mediated iron uptake and, surprisingly, also for heme utilization by Mtb. Predictions derived from the TnSeq analysis were validated by growth experiments with isogenic Mtb mutants. These results showed that (i) the efflux pump MmpL5 plays a dominant role in siderophore secretion, (ii) the Rv2047c protein is essential for growth of Mtb in the presence of mycobactin, and (iii) the transcriptional repressor Zur is required for heme utilization by Mtb. The novel genetic determinants of iron utilization revealed in this study will stimulate further experiments in this important area of Mtb physiology., Author summary Tuberculosis is caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious disease, resulting in approximately 1.5 million deaths per year worldwide. M. tuberculosis resides in granulomas which are formed in the lung in an attempt to wall off the infection. Recently, it was shown that iron-sequestering proteins accumulate in these granulomas to high concentrations, establishing an environment devoid of free iron. In this study, we examined the contribution of each gene towards the utilization of different iron sources by M. tuberculosis. We identified 165 genes involved in iron utilization by Mtb, 66 of which were classified as essential or as required for optimal growth of M. tuberculosis in the presence of different iron sources. Our study provides an unprecedented insight into the genetic determinants of iron utilization by M. tuberculosis.

Objectives Green tea contains a predominant set of polyphenolic compounds with biological activities. The aim of this study was to investigate the antileishmanial activities of the main components of green tea, including catechin, (−)-epicatechin, epicatechin gallate (ECG) and (−)-epigallocatechin 3-O-gallate (EGCG), against Leishmania infantum promastigotes. Methods Green tea ligands and the control drug pentamidine were docked using AutoDock 4.3 software into the active sites of trypanothione synthetase and arginase, which were modelled using homology modelling programs. The colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to measure L. infantum promastigotes at different concentrations of green tea compounds in a concentration- and time-dependent manner. Results were expressed as 50% and 90% inhibitory concentrations (IC50 and IC90, respectively). Results In silico and in vitro assays showed that all of the green tea compounds have antileishmanial activity. EGCG and ECG were the most active compounds against L. infantum promastigotes, with IC50 values of 27.7 μM and 75 μM and IC90 values of 88.4 μM and 188.7 μM, respectively. Pentamidine displayed greater growth inhibition than all of the other tested compounds in a concentration- and time-dependent manner. Conclusion In this study, in silico and docking results were in accordance with the in vitro activity of the compounds. Moreover, EGCG and ECG showed reasonable levels of selectivity for Leishmania.

In actinobacteria, post-translational modification of proteins with prokaryotic ubiquitin-like protein Pup targets them for degradation by a bacterial proteasome assembly consisting of the 20S core particle (CP) and the mycobacterial proteasomal ATPase (Mpa). Modification of hundreds of cellular proteins with Pup at specific surface lysines is carried out by a single Pup-ligase (PafA, proteasome accessory factor A). Pupylated substrates are recruited to the degradative pathway by binding of Pup to the N-terminal coiled-coil domains of Mpa. Alternatively, pupylation can be reversed by the enzyme Dop (deamidase of Pup). Although pupylated substrates outcompete free Pup in proteasomal degradation, potential discrimination of the degradation complex between the various pupylated substrates has not been investigated. Here we show that Mpa binds stably to an open-gate variant of the proteasome (oCP) and associates with bona fide substrates with highly similar affinities. The proteasomal degradation of substrates differing in size, structure and assembly state was recorded in real-time, showing that the pupylated substrates are processed by the Mpa-oCP complex with comparable kinetic parameters. Furthermore, the members of a complex, pupylated proteome (pupylome) purified from Mycobacterium smegmatis are degraded evenly as followed by western blotting. In contrast, analysis of the depupylation behavior of several pupylome members suggests substrate-specific differences in enzymatic turnover, leading to the conclusion that largely indiscriminate degradation competes with differentiated depupylation to control the ultimate fate of pupylated substrates., PLoS ONE, 14 (4), ISSN:1932-6203

Summary Background Vitamin D deficiency has been associated with non‐alcoholic fatty liver disease (NAFLD). However, the role of polymorphisms determining vitamin D status remains unknown. Objectives The objectives of this study were to determine in UK children with biopsy‐proven NAFLD (i) their vitamin D status throughout a 12‐month period and (ii) interactions between key vitamin D‐related genetic variants (nicotinamide adenine dinucleotide synthase‐1/dehydrocholesterol reductase‐7, vitamin D receptor, group‐specific component, CYP2R1) and disease severity. Methods In 103 paediatric patients with NAFLD, serum 25‐hydroxyvitamin D (25OHD) levels and genotypes were determined contemporaneously to liver biopsy and examined in relation to NAFLD activity score and fibrosis stage. Results Only 19.2% of children had adequate vitamin D status; most had mean 25OHD levels considered deficient (

Staphylococcus aureusis an important human and animal pathogen that causes a wide range of infections. The prevalence of multidrug-resistantS. aureusstrains in both hospital and community settings makes it imperative to characterize new drug targets to combatS. aureusinfections. In this context, enzymes involved in NAD metabolism and synthesis are significant drug targets as NAD is a central player in several cellular processes. NAD synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide, making it a crucial intermediate enzyme linked to the biosynthesis of several amino acids, purine and pyrimidine nucleotides, coenzymes and antibiotics.

This study was aimed to determine the abundance of four antibiotic resistance genes (blaTEM, ermB, qnrS and sulI), as well as bacterial community composition associated with the intestinal mucus of wild freshwater fish species collected from the Foix and La Llosa del Cavall reservoirs, which represent ecosystems with high and low anthropogenic disturbance, respectively. Water and sediments from these reservoirs were also collected and analyzed to determine the pollution level by antibiotics. The blaTEM gene was only detected in brown trout and Ebro barbel, which were collected from La Llosa del Cavall reservoir. In contrast, the sulI and qnrS genes were only detected in common carp, which were collected from the Foix reservoir. Although the ermB gene was also detected in common carp, the values were below the limit of quantification. Likewise, water and sediment samples from the Foix reservoir had higher concentrations and more classes of antibiotics than those from La Llosa del Cavall. Pyrosequencing analysis of 16S rRNA genes revealed significant differences in bacterial communities associated with the intestinal mucus of fish species. Therefore, these findings suggest that anthropogenic activities are not only increasing the pollution of aquatic environments, but also contributing to the emergence and spread of antibiotic resistance in organisms that inhabit such environments.

Nicotinamide mononucleotide adenylyl transferases (NMNATs) are a family of highly conserved proteins indispensable for cellular homeostasis. NMNATs are classically known for their enzymatic function of catalyzing NAD+ synthesis, but also have gained a reputation as essential neuronal maintenance factors. NMNAT deficiency has been associated with various human diseases with pronounced consequences on neural tissues, underscoring the importance of the neuronal maintenance and protective roles of these proteins. New mechanistic studies have challenged the role of NMNAT-catalyzed NAD+ production in delaying Wallerian degeneration and have specified new mechanisms of NMNAT's chaperone function critical for neuronal health. Progress in understanding the regulation of NMNAT has uncovered a neuronal stress response with great therapeutic promise for treating various neurodegenerative conditions.

Streptococcus pyogenes, also known as Group A Strep (GAS), is an obligate human pathogen that is responsible for millions of infections and numerous deaths per year. Infection manifestations can range from simple, acute pharyngitis to more complex, necrotizing fasciitis. To date, most treatments for GAS infections involve the use of common antibiotics including tetracycline and clindamycin. Unfortunately, new strains have been identified that are resistant to these drugs therefore, new targets must be identified to treat drug resistant strains. This work is focused on the structural and functional characterization of three proteins: spNadC, spNadD, and spNadE. These enzymes are involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). The structures of spNadC and spNadE were determined. SpNadC is suggested to play a role in GAS virulence, while spNadE, functions as an NAD synthetase and is considered to be a new drug target. Determination of the spNadE structure uncovered a putative, NH3 channel, which may provide insight into the mechanistic details of NH3-dependent NAD+ synthetases in prokaryotes.

The antilarval mushroom polyenes 18-methyl-19-oxoicosaoctaenoic acid and 20-methyl-21-oxodocosanonaenoic acid appear in response to injury of the mycelium of the stereaceous mushroom BY1. We identified a polyketide synthase (PPS1) which belongs to a hitherto completely uncharacterized clade of polyketide synthases. Expression of the PPS1 gene is massively upregulated following mycelial damage. The synthesis of the above polyenes was reconstituted in the mold Aspergillus niger as a heterologous host. This demonstrates that PPS1 1) synchronously produces branched-chain polyketides of varied lengths, and 2) catalyzes the unprecedented shift of eight or nine double bonds. This study represents the first characterization of a reducing polyketide synthase from a mushroom. We also show that injury-induced de novo synthesis of polyketides is a fungal response strategy.

Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response., Scientific Reports, 7 (1), ISSN:2045-2322

Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps−/− knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys−/−/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.

A great number of sesquiterpenes are reported in the available literature as good antileishmanial leads. However, their mode of action at the molecular level has not been elucidated. The lack of molecular studies could be considered an impediment for studies seeking to improve sesquiterpene-based drug design. The present in silico study allows us to make important observations about the molecular details of the binding modes of a set of antileishmanial sesquiterpenes against four drug-enzyme targets [pteridine reductase-1 (PTR1), N-myristoyl transferase (NMT), cysteine synthase (CS), trypanothione synthetase (TryS)]. Through molecular docking it was found that two sesquiterpene coumarins are promising leads for the PTR1 and TryS inhibition purposes, and some xanthanolides also exhibited better affinity towards PTR1 and CS binding. In addition, the affinity values were clustered by Principal Component Analysis and drug-like properties were analyzed for the strongest-docking sesquiterpenes. The results are an excellent starting point for future studies of structural optimization of this kind of compounds.

Vitamin D has an important immunoregulatory function,1 ,2 and there is suggestive evidence for its role in the aetiology of autoimmune diseases, including rheumatoid arthritis (RA).3 Vitamin D levels are reduced in individuals with severe RA,4 ,5 but it is difficult to determine whether this reduction is cause or effect. The onset of arthritis during winter is associated with greater radiographic progression at 6 months, which would be compatible with a causative role.6 Single nucleotide polymorphisms (SNPs) in four vitamin D metabolism genes (GC, DHCR7/NADSYN1, CYP2R1 and CYP24A1) are associated at genome-wide significance with circulating vitamin D levels.7 ,8 Since vitamin D level is associated with RA severity, an association between those SNPs and RA severity would establish causality. Testing the association of genetic variants in genes that control circulating vitamin D levels provides, therefore, an instrument to obtain an unbiased test of the hypothesis that vitamin D levels are aetiologically linked to RA outcome by controlling unmeasured confounding (Mendelian randomisation9). Hence, we tested SNPs previously associated with vitamin D metabolism,7 ,8 and SNPs in these …

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60–74% and 15–31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64–73% by TXN1, 17–20% by TXNPx and 11–16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity. Keywords: Trypanothione, Gamma-glutamylcysteine synthetase, Trypanothione synthetase, Tryparedoxin, Trypanothione reductase, Flux control coefficient, Benznidazol

Antibacterial drug development suffers from a paucity of targets whose inhibition kills replicating and nonreplicating bacteria. The latter include phenotypically dormant cells, known as persisters, which are tolerant to many antibiotics and often contribute to failure in the treatment of chronic infections. This is nowhere more apparent than in tuberculosis caused by Mycobacterium tuberculosis, a pathogen that tolerates many antibiotics once it ceases to replicate. We developed a strategy to identify proteins that Mycobacterium tuberculosis requires to both grow and persist and whose inhibition has the potential to prevent drug tolerance and persister formation. This strategy is based on a tunable dual-control genetic switch that provides a regulatory range spanning three orders of magnitude, quickly depletes proteins in both replicating and nonreplicating mycobacteria, and exhibits increased robustness to phenotypic reversion. Using this switch, we demonstrated that depletion of the nicotinamide adenine dinucleotide synthetase (NadE) rapidly killed Mycobacterium tuberculosis under conditions of standard growth and nonreplicative persistence induced by oxygen and nutrient limitation as well as during the acute and chronic phases of infection in mice. These findings establish the dual-control switch as a robust tool with which to probe the essentiality of Mycobacterium tuberculosis proteins under different conditions, including those that induce antibiotic tolerance, and NadE as a target with the potential to shorten current tuberculosis chemotherapies.