A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Loop 6 and the ß-hairpin flap are structural hotspots that determine cofactor specificity in the FMN-dependent family of ene-reductases.

Flavin mononucleotide (FMN)-dependent ene-reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon-carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene-reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH-preferring 12-oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene-reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the ß-hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex with either NADPH4 or NADH4 corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the ß-hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the ß-hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the ß-hairpin flap favors NADH binding.

Identification of novel coenzyme Q10 biosynthetic proteins Coq11 and Coq12 in Schizosaccharomyces pombe.

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.

Gut Microbiome-Wide Search for Bacterial Azoreductases Reveals Potentially Uncharacterized Azoreductases Encoded in the Human Gut Microbiome.

The human gut is home to trillions of microorganisms that are responsible for the modification of many orally administered drugs, leading to a wide range of therapeutic outcomes. Prodrugs bearing an azo bond are designed to treat inflammatory bowel disease and colorectal cancer via microbial azo reduction, allowing for topical application of therapeutic moieties to the diseased tissue in the intestines. Despite the inextricable link between microbial azo reduction and the efficacy of azo prodrugs, the prevalence, abundance, and distribution of azoreductases have not been systematically examined across the gut microbiome. Here, we curated and clustered amino acid sequences of experimentally confirmed bacterial azoreductases and conducted a hidden Markov model-driven homolog search for these enzymes across 4644 genome sequences present in the representative Unified Human Gastrointestinal Genomes collection. We identified 1958 putative azo-reducing species, corroborating previous findings that azo reduction appears to be a ubiquitous function of the gut microbiome. However, through a systematic comparison of predicted and confirmed azo-reducing strains, we hypothesize the presence of uncharacterized azoreductases in 25 prominent strains of the human gut microbiome. Finally, we confirmed the azo reduction of Acid Orange 7 by multiple strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme Together, these results suggest the presence and activity of many uncharacterized azoreductases in the human gut microbiome and motivate future studies aimed at characterizing azoreductase genes in prominent members of the human gut microbiome. SIGNIFICANCE STATEMENT: This work systematically examined the prevalence, abundance, and distribution of azoreductases across the healthy and inflammatory bowel disease human gut microbiome, revealing potentially uncharacterized azoreductase genes. It also confirmed the reduction of Acid Orange 7 by strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme.

Probing the Interactions Responsible for the Structural Stability of Trypanothione Reductase Through Computer Simulation and Biophysical Characterization.

With the necessity to develop antileishmanial drugs with substrate specificity, trypanothione reductase (TryR) has gained popularity in parasitology. TryR is unique to be present only in trypanosomatids and is functionally similar to glutathione in mammals. It protects against oxidative stress exerted by the host defense mechanism. The TryR enzyme is essential for the survival of Leishmania parasites in the host as it reduces trypanothione and aids in neutralizing hydrogen peroxide produced by the host macrophages during infection. Henceforth, it becomes vital to decipher their functional stability and behaviour in the presence of denaturants. Our study is focused on structural, functional and behavioural stability aspects of TryR with different concentrations of Urea, Guanidinium chloride, alcohol based compounds followed by extensive molecular dynamics simulations in a lipid bilayer system. The results obtained from the study reveal an interesting insight into the possible mechanisms of modulation of the structure, function and stability of the TryR protein.

Anti-oxidative and anti-hyperglycemic properties of Agastache foeniculum essential oil and oily fraction in hyperglycemia-stimulated and lipopolysaccharide-stimulated macrophage cells: In vitro and in silico studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperglycemia (HG) and lipopolysaccharide (LPS) often promote superoxide accumulation, which may increase oxidative stress. Reducing superoxide production in hyperglycemia and the inflammatory condition is an emerging way to reduce protein and lipid oxidation and diabetes complication. AIM OF STUDY: To examine the effect of Agastache foeniculum essential oil (AFEO) and oil fraction (AFoil) on HG- and LPS-stimulated oxidative stress, the pathogenicity of AFEO and AFoil on oxidative stress was assessed. METHODS: The stimulatory effects of AFEO and AFoil on the activity and expression of NADH oxide (NOX), catalase (CAT), superoxide dismutase (SOD), and the expression of nuclear respiratory factor 2 (NRF2) and nuclear factor-kappa B (NF-kB) in the stimulated macrophage cell line, J774.A1, was studied. The interaction patterns of AFEO and AFoil components with NOX, SOD, CAT, NRF2, and NF-kB proteins were also deduced using molecular docking. RESULTS: Estragole was the main ingredient in AFEO (97%). Linolenic acid (32.10%), estragole (16.22%), palmitic acid (12.62%), linoleic acid (12.04%), and oleic acid (8.73%) were the major chemical components of the AFoil. NOX activation was stimulated in macrophage cells by HG and LPS. At 20 µg/mL, AFEO and AFoil decreased NOX activity while increased SOD and CAT activities in stimulated macrophages. AFoil with estragole and omega-3 fatty acids was better than AFEO with estragole in anti-hyperglycemic and anti-oxidative activity. According to molecular docking research, estragole, linoleic acid, and linolenic acid bind to different hydrophobic pockets of NOX, SOD, CAT, NFR2, and NF-kB using hydrogen bonds, van der Waals bonds, pi-alkyl, and pi-anion interactions, with different binding energies. CONCLUSION: AFEO and AFoil showed antioxidant and anti-diabetic activity. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes and up-regulating superoxide-removing enzymes at gene and protein levels. The AFoil emulsion can be used to reduce the detrimental impacts of hyperglycemia and oxidative stress.

Structural exploration with AlphaFold2-generated STAT3α structure reveals selective elements in STAT3α-GRIM-19 interactions involved in negative regulation.

STAT3, an important transcription factor constitutively activated in cancers, is bound specifically by GRIM-19 and this interaction inhibits STAT3-dependent gene expression. GRIM-19 is therefore, considered as an inhibitor of STAT3 and may be an effective anti-cancer therapeutic target. While STAT3 exists in a dimeric form in the cytoplasm and nucleus, it is mostly present in a monomeric form in the mitochondria. Although GRIM-19-binding domains of STAT3 have been identified in independent experiments, yet the identified domains are not the same, and hence, discrepancies exist. Human STAT3-GRIM-19 complex has not been crystallised yet. Dictated by fundamental biophysical principles, the binding region, interactions and effects of hotspot mutations can provide us a clue to the negative regulatory mechanisms of GRIM-19. Prompted by the very nature of STAT3 being a challenging molecule, and to understand the structural basis of binding and interactions in STAT3α-GRIM-19 complex, we performed homology modelling and ab-initio modelling with evolutionary information using I-TASSER and avant-garde AlphaFold2, respectively, to generate monomeric, and subsequently, dimeric STAT3α structures. The dimeric form of STAT3α structure was observed to potentially exist in an anti-parallel orientation of monomers. We demonstrate that during the interactions with both unphosphorylated and phosphorylated STAT3α, the NTD of GRIM-19 binds most strongly to the NTD of STAT3α, in direct contrast to the earlier works. Key arginine residues at positions 57, 58 and 68 of GRIM-19 are mainly involved in the hydrogen-bonded interactions. An intriguing feature of these arginine residues is that these display a consistent interaction pattern across unphosphorylated and phosphorylated monomers as well as unphosphorylated dimers in STAT3α-GRIM-19 complexes. MD studies verified the stability of these complexes. Analysing the binding affinity and stability through free energy changes upon mutation, we found GRIM-19 mutations Y33P and Q61L and among GRIM-19 arginines, R68P and R57M, to be one of the top-most major and minor disruptors of binding, respectively. The proportionate increase in average change in binding affinity upon mutation was inclined more towards GRIM-19 mutants, leading to the surmise that GRIM-19 may play a greater role in the complex formation. These studies propound a novel structural perspective of STAT3α-GRIM-19 binding and inhibitory mechanisms in both the monomeric and dimeric forms of STAT3α as compared to that observed from the earlier experiments, these experimental observations being inconsistent among each other.

MSMEG_3955 from Mycobacterium smegmatis is a FMN bounded homotrimeric NAD(P)H:Flavin mononucleotide (FMN) oxidoreductase.

BACKGROUND: Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb. RESULTS: In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% ß-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways. CONCLUSION: MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Nanomicelle-enhanced, asymmetric ERED-catalyzed reductions of activated olefins. Applications to 1-pot chemo- and bio-catalysis sequences in water.

Bio-catalytic reactions involving ene-reductases (EREDs) in tandem with chemo-catalysis in water can be greatly enhanced by the presence of nanomicelles derived from the surfactant TPGS-750-M. Transformations are provided that illustrate the variety of sequences now possible in 1-pot as representative examples of this environmentally attractive approach to organic synthesis.

Modular Assembly of Phosphite Dehydrogenase and Phenylacetone Monooxygenase for Tuning Cofactor Regeneration.

The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD-RIAD) system. These peptides allow self-assembly based on specific protein-protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer-Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in E. coli and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMORIAD-PTDHRIDD and PAMORIAD/RIAD-PTDHRIDD). These enzyme complexes displayed similar catalytic activity when compared with the PTDH-PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO-PTDH complex less PTDH was required for the same performance when compared with the PTDH-PAMO fusion enzyme.

Druggable hot spots in trypanothione reductase: novel insights and opportunities for drug discovery revealed by DRUGpy.

Assessment of target druggability guided by search and characterization of hot spots is a pivotal step in early stages of drug-discovery. The raw output of FTMap provides the data to perform this task, but it relies on manual intervention to properly combine different sets of consensus sites, therefore allowing identification of hot spots and evaluation of strength, shape and distance among them. Thus, the user's previous experience on the target and the software has a direct impact on how data generated by FTMap server can be explored. DRUGpy plugin was developed to overcome this limitation. By automatically assembling and scoring all possible combinations of consensus sites, DRUGpy plugin provides FTMap users a straight-forward method to identify and characterize hot spots in protein targets. DRUGpy is available in all operating systems that support PyMOL software. DRUGpy promptly identifies and characterizes pockets that are predicted by FTMap to bind druglike molecules with high-affinity (druggable sites) or low-affinity (borderline sites) and reveals how protein conformational flexibility impacts on the target's druggability. The use of DRUGpy on the analysis of trypanothione reductases (TR), a validated drug target against trypanosomatids, showcases the usefulness of the plugin, and led to the identification of a druggable pocket in the conserved dimer interface present in this class of proteins, opening new perspectives to the design of selective inhibitors.

Efficient Dimerization Disruption of Leishmania infantum Trypanothione Reductase by Triazole-phenyl-thiazoles.

Inhibition of Leishmania infantum trypanothione disulfide reductase (LiTryR) by disruption of its homodimeric interface has proved to be an alternative and unexploited strategy in the search for novel antileishmanial agents. Proof of concept was first obtained by peptides and peptidomimetics. Building on previously reported dimerization disruptors containing an imidazole-phenyl-thiazole scaffold, we now report a new 1,2,3-triazole-based chemotype that yields noncompetitive, slow-binding inhibitors of LiTryR. Several compounds bearing (poly)aromatic substituents dramatically improve the ability to disrupt LiTryR dimerization relative to reference imidazoles. Molecular modeling studies identified an almost unexplored hydrophobic region at the interfacial domain as the putative binding site for these compounds. A subsequent structure-based design led to a symmetrical triazole analogue that displayed even more potent inhibitory activity over LiTryR and enhanced leishmanicidal activity. Remarkably, several of these novel triazole-bearing compounds were able to kill both extracellular and intracellular parasites in cell cultures.

Optimal pH shift of the NADH oxidase from Lactobacillus rhamnosus with a single mutation.

OBJECTIVE: To improve the activity of a water-forming NADH oxidase from Lactobacillus rhamnosus under neutral or alkaline pH for coupling NAD+-dependent dehydrogenases with an alkaline optimal pH. RESULTS: The water-forming NADH oxidase from Lactobacillus rhamnosus was engineered by replacing the aspartic acid or glutamic acid with arginine on the surface. The mutant D251R improved the activity with a 112%, 111%, and 244% relative activity to the wild-type at pH 6.5, pH 7.0, and pH 7.5, respectively. Docking substrate into the D251R mutant reveals that the NADH is access to the substrate-binding site with a larger substrate loop due to the enhanced electrostatic repulsion between ARG-251 and ARG-243. In the D251R-NADH complex, the carboxyl of NADH additionally forms two hydrogen bonds (2.6 and 2.9 Å) with G154 due to the changed interaction of substrate and the residues in the catalytic sites, and the hydrogen bond with the oxygen of carbonyl in P295 is shortened from 2.9 to 2.0 Å, which could account for the enhanced specific activity. CONCLUSIONS: The D251R mutant displayed higher catalytic activity than the wild-type in the pH range 6.5-7.5, and further insight into those shorter and newly formed hydrogen bonds in substrate docking analysis could account for the higher bind affinity and catalytic efficiency of D251R mutant.

Elucidating the role of GRIM-19 as a substrate and allosteric activator of pro-apoptotic serine protease HtrA2.

HtrA2 (high-temperature requirement A2) and GRIM-19 (gene associated with retinoic and interferon-induced mortality 19 protein) are involved in various biological functions with their deregulation leading to multiple diseases. Although it is known that the interaction between GRIM-19 with HtrA2 promotes the pro-apoptotic activity of the latter, the mechanistic details remained elusive till date. Moreover, designing allosteric modulators of HtrA2 remains obscure due to lack of adequate information on the mode of interaction with its natural substrates cum binding partners. Therefore, in this study, we have unfolded the interaction between HtrA2 and GRIM-19 so as to understand its subsequent functional repercussions. Using in silico analyses and biochemical assays, we identified the region in GRIM-19 that is involved in protein-protein interaction with HtrA2. Furthermore, we have presented a comprehensive illustration of HtrA2's cleavage site specificity. Quantitative analysis using enzyme kinetics underscored the role of GRIM-19 in significant allosteric activation of HtrA2. Overall, this is an extensive study that not only defines HtrA2-GRIM-19 interaction, but also creates a framework for developing strategies toward allosteric regulation of HtrA2 for future therapeutic interventions.

Spectral deconvolution of redox species in the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii. A flavin-dependent bifurcating enzyme.

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.

Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin.

The substrates O2 and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH2 to reduce the ferric iron, which forms the stable ferric-superoxide-TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 1010 M-1 s-1 forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.

Design of the Enzyme-Carrier Interface to Overcome the O2 and NADH Mass Transfer Limitations of an Immobilized Flavin Oxidase.

Understanding how the immobilization of enzymes on solid carriers affects their performance is paramount for the design of highly efficient heterogeneous biocatalysts. An efficient supply of substrates onto the solid phase is one of the main challenges to maximize the activity of the immobilized enzymes. Herein, we apply advanced single-particle analysis to decipher the optimal design of an immobilized NADH oxidase (NOX) whose activity depends both on O2 and NADH concentrations. Carrier physicochemical properties and its functionality along with the enzyme distribution across the carrier were implemented as design variables to study the effects of the intraparticle concentration of substrates (O2 and NADH) on the activity. Intraparticle O2-sensing analysis revealed the superior performance of the enzyme immobilized at the outer surface in terms of effective supply of O2. Furthermore, the co-immobilization of NADH and NOX within the tuned surface of porous microbeads increases the effective concentration of NADH in the surroundings of the enzyme. As a result, the optimal spatial organization of NOX and its confinement with NADH allow a 100% recovery of the activity of the soluble enzyme upon the immobilization process. By engineering these variables, we increase the NADH oxidation activity of the heterogeneous biocatalyst by up to 650% compared to NOX immobilized under suboptimal conditions. In conclusion, this work highlights the rational design and engineering of the enzyme-carrier interface to maximize the efficiency of heterogeneous biocatalysts.

Bioassay-based Corchorus capsularis L. leaf-derived ß-sitosterol exerts antileishmanial effects against Leishmania donovani by targeting trypanothione reductase.

Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of Corchorus capsularis L. leaves exhibits a significant effect against Leishmania donovani promastigotes. In the current study, bioassay-guided fractionation results for Corchorus capsularis L. leaf-derived ß-sitosterol (ß-sitosterolCCL) were observed by spectroscopic analysis (FTIR, 1H NMR, 13C NMR and GC-MS). The inhibitory efficacy of this ß-sitosterolCCL against L. donovani promastigotes was measured (IC50 = 17.7 ± 0.43 µg/ml). ß-SitosterolCCL significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of ß-sitosterolCCL was validated by enzyme inhibition and an in silico study in which ß-sitosterolCCL was found to inhibit Leishmania donovani trypanothione reductase (LdTryR). Overall, ß-sitosterolCCL appears to be a novel inhibitor of LdTryR and might represent a successful approach for treatment of VL in the future.

Structure/activity virtual screening and in vitro testing of small molecule inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from gut methanogenic bacteria.

Virtual screening techniques and in vitro binding/inhibitory assays were used to search within a set of more than 8,000 naturally occurring small ligands for candidate inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase (FNO) from Methanobrevibacter smithii, the enzyme that catalyses the bidirectional electron transfer between NADP+ and F420H2 during the intestinal production of CH4 from CO2. In silico screening using molecular docking classified the ligand-enzyme complexes in the range between - 4.9 and - 10.5 kcal/mol. Molecular flexibility, the number of H-bond acceptors and donors, the extent of hydrophobic interactions, and the exposure to the solvent were the major discriminants in determining the affinity of the ligands for FNO. In vitro studies on a group of these ligands selected from the most populated/representative clusters provided quantitative kinetic, equilibrium, and structural information on ligands' behaviour, in optimal agreement with the predictive computational results.

Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme.

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.