Targeting Trypanothione Metabolism in Trypanosomatids.

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Discovery of novel Leishmania major trypanothione synthetase inhibitors by high-throughput screening.

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 (IC50) values < 50 µM and six of them corresponded to novel structures with IC50 ranging from 9 to 19 µM against LmTryS enzyme activity. Of the six inhibitors, TS001 showed the highest activity against growth of L. major promastigotes, L. donovani promastigotes, and Trypanosoma brucei brucei Lister 427 with IC50 values of 17, 26, and 31 µM, respectively. An in silico docking study using a homology model of LmTryS predicted the molecular interactions between LmTryS and the inhibitors.

Identification of L. infantum trypanothione synthetase inhibitors with leishmanicidal activity from a (non-biased) in-house chemical library.

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 trypanothione 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.

Evaluation of extracellular electron transfer in Pseudomonas aeruginosa by co-expression of intermediate genes in NAD synthetase production pathway.

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.

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening.

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.

Structural and molecular dynamics of ammonia transport in Staphylococcus aureus NH3-dependent NAD synthetase.

Ammonia dependent NAD+ synthetase from multi drug resistance Staphylococcus aureus catalyzes ATP dependent formation of NAD+ from deamido-NAD+ and ammonia at the synthetase active site. Binding of ATP accompanies a large movement of flexible loop region (205-225) acting as a lid to the catalytic core. A 17 Å long ammonia tunnel with an entry and exit radius of 3.5 Å and 3.2 Å respectively allows transfer of ammonia from surface to the active site of the enzyme in each monomer to attack the C7N=O7N linkage of transient intermediate NAD-adenylate thus releasing NAD+. In this study, we report structural details of ammonia transport tunnel in Staphylococcus aureus NH3-dependent NAD synthetase and compared their architecture and dynamics with other bacterial and eukaryotic enzymes. Tunnel shows conformational variations in apo and substrate complexes and is less intricate compared to glutamine dependent counterparts. We have also performed steered molecular dynamic simulations of ammonia transport across the tunnel in enzyme-intermediate complex which reveals critical bottleneck residues and structural determinants during ammonium migration. Ordered water molecules and conserved charged residues form a network of hydrogen bonds and electrostatic interaction which facilitate the ammonium movement towards the active center. Analysis of the sMD simulated structural snapshots delineates the conformational reshaping of ammonia tunnel at the different step of the enzymatic reaction. Tunnel architecture and environment could offer the new target site to design novel small molecule inhibitors for the development of more efficient therapeutics against multi drug resistant S. aureus strains.

Copper(II)-N-hydroxy-N,N'-diarylformamidine complexes: Synthesis, crystal structures, antibacterial and molecular docking studies.

A series of Cu(II) complexes were synthesized by using N-hydroxy-N,N'-diarylformamidine ligands: N-hydroxy-N,N'-(phenyl)formamidine (L1), N-hydroxy-N'-(4-methylphenyl)formamidine (L2), N-hydroxy-N,N'-(2,6-dimethylphenyl)formamidine (L3), N-hydroxy-N,N'-(2,6-diisopropylphenyl)formamidine (L4). Reaction of ligands L1-L4 with hydrated copper acetate furnished mononuclear Cu(II) complexes 1-4 with general formula [Cu-(L)2]. The molecular structures of complexes 3 and 4, as determined by single crystal X-ray diffraction, showed both to have square planar geometry with a near C2 symmetry. The antimicrobial potency of all four complexes was evaluated against three gram-(-) bacteria (S. typhimurium, P. aeruginosa, and E. coli) and two gram-(+) bacteria (Methicillin-resistant S. aureus (MRSA) and S. aureus), with ciprofloxacin as the reference drug. All tested complexes were inactive against gram-(+) bacteria strains except for complex 1, which displayed excellent activity when compared to the reference. Molecular docking studies showed that hydrogen bonding, pi-sigma and van der Waals interactions are prominent complex-protein connections, with complex 2 displaying good binding affinities with the studied biological targets.

Pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs as possible candidates against neglected tropical diseases (NTDs): identification of hit compounds towards development of potential treatment of Leishmania donovani.

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.

Ensemble learning application to discover new trypanothione synthetase 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.

Effect of Spermidine on Biofilm Formation in Escherichia coli K-12.

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.

Insights into the Relationship between Cobamide Synthase and the Cell Membrane.

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.IMPORTANCESalmonella 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. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.

Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine.

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 mutation causes male infertility in mice.

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.

Crystallographic and molecular dynamics simulation analysis of NAD synthetase from methicillin resistant Staphylococcus aureus (MRSA).

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 Å 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.

Recent Advancement in the Search of Innovative Antiprotozoal Agents Targeting Trypanothione Metabolism.

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 R2R3-MYB transcription factor CsMYB73 negatively regulates l-Theanine biosynthesis in tea plants (Camellia sinensis L.).

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.

A novel piperidine degradation mechanism in a newly isolated piperidine degrader Pseudomonas sp. strain KU43P.

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 Mg2+. These results clearly show that pipA encodes γ-glutamylpiperidide synthetase and that piperidine is first glutamylated and then hydroxylated in the piperidine degradation pathway of Pseudomonas sp. strain KU43P. This study has filled a void in the general knowledge of the microbial degradation of amine compounds.

Rv2037c, a stress induced conserved hypothetical protein of Mycobacterium tuberculosis, is a phospholipase: Role in cell wall modulation and intracellular survival.

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.

Development of a highly efficient and specific L-theanine synthase.

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.

Comprehensive analysis of iron utilization by Mycobacterium tuberculosis.

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.