Glutathione S-transferase: A versatile and dynamic enzyme.

The dynamic and versatile group of enzymes referred to as glutathione S-transferases (GSTs) play diverse roles in cellular detoxification, safeguarding hosts from oxidative damage, and performing various other functions. This review explores different classes of GST, existence of polymorphisms in GST, functions of GST and utilizations of GST inhibitors in treatment of human diseases. The study indicates that the cytosolic GSTs, mitochondrial GSTs, microsomal GSTs, and bacterial proteins that provide resistance to Fosfomycin are the major classes. Given a GST, variation in its expression and function among individuals is due to the presence of polymorphic alleles that encode it. Genetic polymorphism might result in the modification of GST activity, thereby increasing individuals' vulnerability to harmful chemical compounds. GSTs have been demonstrated to play a regulatory function in cellular signalling pathways through kinases, S-Glutathionylation, and in detoxification processes. Various applications of bacterial GSTs and their potential roles in plants were examined. Targeting GSTs, especially GSTP1-1, is considered a potential therapeutic strategy for treating cancer and diseases linked to abnormal cell proliferation. Their role in cancer cell growth, differentiation, and resistance to anticancer agents makes them promising targets for drug development, offering prospects for the future.

Nuclear localisation sequences of chloride intracellular channels 1 and 4 facilitate nuclear import via interactions with import mediator importin-α: An empirical and theoretical perspective.

Chloride intracellular channel proteins (CLICs) display ubiquitous expression, with each member exhibiting specific subcellular localisation. While all CLICs, except CLIC3, exhibit a highly conserved putative nuclear localisation sequence (NLS), only CLIC1, CLIC3 and CLIC4 exist within the nucleus. The CLIC4 NLS, 199-KVVAKKYR-206, appears crucial for nuclear entry and interacts with mouse nuclear import mediator Impα isoform 1, omitting the IBB domain (mImpα1ΔIBB). The essential nature of the basic residues in the CLIC4 NLS has been established by the fact that mutating out these residues inhibits nuclear import, which in turn is linked to cutaneous squamous cell cancer. Given the conservation of the CLIC NLS, CLIC1 likely follows a similar import pathway to CLIC4. Peptides of the CLIC1 (Pep1; Pep1_S C/S mutant) and CLIC4 (Pep4) NLSs were designed to examine binding to human Impα isoform 1, omitting the IBB domain (hImpα1ΔIBB). Molecular docking indicated that the core CLIC NLS region (KKYR) forms a similar binding pattern to both mImpα1ΔIBB and hImpα1ΔIBB. Fluorescence quenching demonstrated that Pep1_S (Kd ≈ 237 µM) and Pep4 (Kd ≈ 317 µM) bind hImpα1ΔIBB weakly. Isothermal titration calorimetry confirmed the weak binding interaction between Pep4 and hImpα1ΔIBB (Kd ≈ 130 µM) and the presence of a proton-linked effect. This weak interaction may be due to regions distal from the CLIC NLS needed to stabilise and strengthen hImpα1ΔIBB binding. Additionally, this NLS may preferentially bind another hImpα isoform with different flexibility properties.

Coping with the ESKAPE pathogens: Evolving strategies, challenges and future prospects.

Globally, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are the major cause of nosocomial infections. These pathogens are multidrug resistant, and their negative impacts have brought serious health challenges and economic burden on many countries worldwide. Thus, this narrative review exploits different emerging alternative therapeutic strategies including combination antibiotics, antimicrobial peptides ((AMPs), bacteriophage and photodynamic therapies used in the treatment of the ESKAPE pathogens, their merits, limitations, and future prospects. Our findings indicate that ESKAPE pathogens exhibit resistance to drug using different mechanisms including drug inactivation by irreversible enzyme cleavage, drug-binding site alteration, diminution in permeability of drug or drug efflux increment to reduce accumulation of drug as well as biofilms production. However, the scientific community has shown significant interest in using these novel strategies with numerous benefits although they have some limitations including but not limited to instability and toxicity of the therapeutic agents, or the host developing immune response against the therapeutic agents. Thus, comprehension of resistance mechanisms of these pathogens is necessary to further develop or modify these approaches in order to overcome these health challenges including the barriers of bacterial resistance.

The future of cassava in the era of biotechnology in Southern Africa.

Cassava (Manihot esculenta) is a major staple food and the world's fourth source of calories. Biotechnological contributions to enhancing this crop, its advances, and present issues must be assessed regularly. Functional genomics, genomic-assisted breeding, molecular tools, and genome editing technologies, among other biotechnological approaches, have helped improve the potential of economically important crops like cassava by addressing some of its significant constraints, such as nutrient deficiency, toxicity, poor starch quality, disease susceptibility, low yield capacity, and postharvest deterioration. However, the development, improvement, and subsequent acceptance of the improved cultivars have been challenging and have required holistic approaches to solving them. This article provides an update of trends and gaps in cassava biotechnology, reviewing the relevant strategies used to improve cassava crops and highlighting the potential risk and acceptability of improved cultivars in Southern Africa.

Comparative structural analysis of the human and Schistosoma glutathione transferase dimer interface using selective binding of bromosulfophthalein.

The binding channel of Schistosoma glutathione transferase (SGST) has been identified to possess a non-substrate site implicated in enzyme inhibition. This binding channel is formed by the interface of the GST dimer. We produced a comparative characterization of the SGST dimer interface with respect to that of human GST (hGST) analogues using the selective binding of bromosulfophthalein (BSP). First, two SGST and three hGST structures were used as search queries to assemble a data set of 48 empirical GST structures. Sequence alignment to generate a universal residue indexing scheme was then performed, followed by local superposition of the dimer interface. Principal component analysis revealed appreciable variation of the dimer interface, suggesting the potential for selective inhibition of SGST. BSP was found to dock invariably in the dimer interface core pocket, placing it in proximity to the GST catalytic domains, through which it may exert its inhibitory behavior. Binding poses across the GST forms were distinguished with ligand interaction profiling, where SGST complexes showed stabilization of ligand aromatic- and sulfonate moieties, which altogether anchor the ligand and produce a tight association. In comparison, missing aromatic stabilization in the hGST complexes impart large bonding distances, causing mobile poses likely to dissociate. Altogether, this study illustrates the potential for selective inhibition of SGST, rationalizes the selective behavior of the BSP inhibitor, and produces a reliable metric for construction and validation of pharmacophore models of the SGST binding channel.

Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein.

South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD600 of ~0.45 at 37 °C for four hours. Denatured protein fractions (dialysed in 4 M urea), passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS-PAGE. The secondary structure of SACMV MP presented as predominantly ß-strands. An ANS (1-anilino-8-naphthalene sulphonate)-binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS (8-anilino-1-naphthalenesulphonic acid). A 2' (3')-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity.

Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies.

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

Caseinolytic Proteins (Clp) in the Genus Klebsiella: Special Focus on ClpK.

Caseinolytic proteins (Clp), which are present in both prokaryotes and eukaryotes, play a major role in cell protein quality control and survival of bacteria in harsh environmental conditions. Recently, a member of this protein family, ClpK was identified in a pathogenic strain of Klebsiella pneumoniae which was responsible for nosocomial infections. ClpK is linked to the thermal stress survival of this pathogen. The genome wide analysis of Clp proteins in Klebsiella spp. indicates that ClpK is present in only 34% of the investigated strains. This suggests that the uptake of the clpk gene is selective and may only be taken up by a pathogen that needs to survive harsh environmental conditions. In silico analyses and molecular dynamic simulations show that ClpK is mainly α-helical and is highly dynamic. ClpK was successfully expressed and purified to homogeneity using affinity and anion exchange chromatography. Biophysical characterization of ClpK showed that it is predominantly alpha-helical, and this is in agreement with in silico analysis of the protein structure. Furthermore, the purified protein is biologically active and hydrolyses ATP in a concentration- dependent manner.

Double trouble? Gag in conjunction with double insert in HIV protease contributes to reduced DRV susceptibility.

HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

Structural and biochemical characterization of Plasmodium falciparum Hsp70-x reveals functional versatility of its C-terminal EEVN motif.

Plasmodium falciparum, the main agent of malaria expresses six members of the heat shock protein 70 (Hsp70) family. Hsp70s serve as protein folding facilitators in the cell. Amongst the six Hsp70 species that P. falciparum expresses, Hsp70-x (PfHsp70-x), is partially exported to the host red blood cell where it is implicated in host cell remodeling. Nearly 500 proteins of parasitic origin are exported to the parasite-infected red blood cell (RBC) along with PfHsp70-x. The role of PfHsp70-x in the infected human RBC remains largely unclear. One of the defining features of PfHsp70-x is the presence of EEVN residues at its C-terminus. In this regard, PfHsp70-x resembles canonical eukaryotic cytosol-localized Hsp70s which possess EEVD residues at their C-termini in place of the EEVN residues associated with PfHsp70-x. The EEVD residues of eukaryotic Hsp70s facilitate their interaction with co-chaperones. Characterization of the role of the EEVN residues of PfHsp70-x could provide insights into the function of this protein. In the current study, we expressed and purified recombinant PfHsp70-x (full length) and its EEVN minus form (PfHsp70-xT ). We then conducted structure- function assays towards establishing the role of the EEVN motif of PfHsp70-x. Our findings suggest that the EEVN residues of PfHsp70-x are important for its ATPase activity and chaperone function. Furthermore, the EEVN residues are crucial for the direct interaction between PfHsp70-x and human Hsp70-Hsp90 organizing protein (hHop) in vitro. Hop facilitates functional cooperation between Hsp70 and Hsp90. However, it remains to be established if PfHsp70-x and hHsp90 cooperate in vivo.

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma.

The ß-subunit of the human eukaryotic elongation factor 1 complex (heEF1ß) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α- and γ-subunits (eEF1γ). To biophysically characterize heEF1ß, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL-heEF1ß), N-terminus (NT-heEF1ß), and the C-terminus (CT-heEF1ß) regions of the protein. Our results suggest that heEF1ß is predominantly alpha-helical and possesses an accessible hydrophobic cavity in the CT-heEF1ß. Both FL-heEF1ß and NT-heEF1ß form dimers of size 62 and 30 kDa, respectively, but the CT-heEF1ß is monomeric. FL-heEF1ß interacts with the N-terminus glutathione transferase-like domain of heEF1γ (NT-heEF1γ) to form a 195-kDa complex or a 230-kDa complex in the presence of oxidized glutathione. On the other hand, NT-heEF1ß forms a 170-kDa complex with NT-heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL-heEF1ß associated with monomeric or dimeric NT-heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (KD  = 9.6 nM for monomeric or 11.3 nM for dimeric NT-heEF1γ). We postulate that the N-terminus region of heEF1ß may be responsible for its dimerization and the C-terminus region of heEF1ß modulates the formation of an ordered heEF1ß-γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.

The isomerization of Δ5-androstene-3,17-dione by the human glutathione transferase A3-3 proceeds via a conjugated heteroannular diene intermediate.

The seemingly simple proton abstraction reactions underpin many chemical transformations, including isomerization reactions, and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid Δ(5)-androstene-3,17-dione by the glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process, and the hydrogen bonds postulated to be the stabilizing forces of these transient species. The UV-visible detection of the intermediate places this species in the catalytic pathway, whereas fluorescence spectroscopy is used to obtain the binding constant of the analog intermediate, equilenin. Solvent isotope exchange reveals that proton abstraction from the substrate to form the intermediate is rate-limiting. Analysis of the data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mol. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.

Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma.

The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, ß and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the ß subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) µM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03µmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.

Describing the ligandin properties of Plasmodium falciparum and vivax glutathione transferase towards bromosulfophthalein from empirical and computational modelling viewpoints.

Research has spotlighted glutathione transferase (GST) as a promising target for antimalarial drug development due to its pivotal role in cellular processes, including metabolizing toxins and managing oxidative stress. This interest arises from GST's potential to combat multidrug resistance in existing antimalarial drugs. Plasmodium falciparum GST (PfGST) and Plasmodium vivax GST (PvGST) are key targets; inhibiting them not only disrupt detoxification but also reduce their antioxidant capacity, a critical feature for potent antimalarials. Bromosulfophthalein (BSP), a clinical liver function dye, emerged as a potent cytosolic GST inhibitor. This study explored BSP's inhibitory properties on PfGST and PvGST, showcasing its binding capabilities through empirical and computational analyses. The study revealed BSP's ability to significantly inhibit GST activity, altering the proteins' structures and stability. Specifically, BSP binding induced spectral changes and impacted the proteins' thermal stability, reducing their melting temperatures. Computational simulations highlighted BSP's strong binding to PfGST and PvGST at their dimer interface, stabilized by various interactions, including hydrogen bonds and van der Waals forces. Notably, BSP's binding altered the proteins' compactness and conformational dynamics, suggesting a potential non-competitive, allosteric inhibition mechanism. This study provided novel insights into BSP's candidacy as an antimalarial drug by targeting PfGST and PvGST. Its ability to disrupt crucial functions of these enzymes' positions BSP as a promising candidate for further drug development in combating malariaCommunicated by Ramaswamy H. Sarma.

Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches.

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

Exploring the evolutionary trajectory and functional landscape of cannabinoid receptors: A comprehensive bioinformatic analysis.

The bioinformatic analysis of cannabinoid receptors (CBRs) CB1 and CB2 reveals a detailed picture of their structure, evolution, and physiological significance within the endocannabinoid system (ECS). The study highlights the evolutionary conservation of these receptors evidenced by sequence alignments across diverse species including humans, amphibians, and fish. Both CBRs share a structural hallmark of seven transmembrane (TM) helices, characteristic of class A G-protein-coupled receptors (GPCRs), which are critical for their signalling functions. The study reports a similarity of 44.58 % between both CBR sequences, which suggests that while their evolutionary paths and physiological roles may differ, there is considerable conservation in their structures. Pathway databases like KEGG, Reactome, and WikiPathways were employed to determine the involvement of the receptors in various signalling pathways. The pathway analyses integrated within this study offer a detailed view of the CBRs interactions within a complex network of cannabinoid-related signalling pathways. High-resolution crystal structures (PDB ID: 5U09 for CB1 and 5ZTY for CB2) provided accurate structural information, showing the binding pocket volume and surface area of the receptors, essential for ligand interaction. The comparison between these receptors' natural sequences and their engineered pseudo-CBRs (p-CBRs) showed a high degree of sequence identity, confirming the validity of using p-CBRs in receptor-ligand interaction studies. This comprehensive analysis enhances the understanding of the structural and functional dynamics of cannabinoid receptors, highlighting their physiological roles and their potential as therapeutic targets within the ECS.

Unveiling the potential of Muscadine grape Skin extract as an innovative therapeutic intervention in cancer treatment.

The use of muscadine grape extracts (MGSE). in cancer treatment has gained attention due to its distinctive composition of polyphenols and antioxidants. This review analyses the reported anti-cancer properties of MGSE. The study commences by reviewing the phytochemical composition of MGSE, highlighting the presence of resveratrol and ellagic acid. Furthermore, the review underscores the mechanism of action of these active compounds in MGSE in combating cancer cells. The anti-cancer potential of MGSE compared to other plant extracts is also discussed. In addition, it highlights MGSE's superiority and distinct phytochemical composition in preventing cancer growth by comparing its anti-cancer compounds with those of other anti-cancer medicinal plants. Lastly, the combinatory approaches of MGSE with traditional cancer therapies, its safety, and its possible side effects were highlighted. This work provides an understanding of the anti-cancer properties of MGSE, positioning it as a valuable and unique challenge within the field of cancer therapy.

Combating Aminoglycoside Resistance: From Structural and Functional Characterisation to Therapeutic Challenges with RKAAT.

A comprehensive knowledge of aminoglycoside-modifying enzymes (AMEs) and their role in bacterial resistance mechanisms is urgently required due to the rising incidence of antibiotic resistance, particularly in Klebsiella pneumoniae infections. This study explores the essential features of AMEs, including their structural and functional properties, the processes by which they contribute to antibiotic resistance, and the therapeutic importance of aminoglycosides. The study primarily examines the Recombinant Klebsiella pneumoniae Aminoglycoside Adenylyl Transferase (RKAAT), particularly emphasizing its biophysical characteristics and the sorts of resistance it imparts. Furthermore, this study examines the challenges presented by RKAAT-mediated resistance, an evaluation of treatment methods and constraints, and options for controlling infection. The analysis provides a prospective outlook on strategies to address and reduce antibiotic resistance. This extensive investigation seeks to provide vital insights into the continuing fight against bacterial resistance, directing future research efforts and medicinal approaches.

Exploring NAD+ metabolism and NNAT: Insights from structure, function, and computational modeling.

Nicotinamide Adenine Dinucleotide (NAD+), a coenzyme, is ubiquitously distributed and serves crucial functions in diverse biological processes, encompassing redox reactions, energy metabolism, and cellular signalling. This review article explores the intricate realm of NAD + metabolism, with a particular emphasis on the complex relationship between its structure, function, and the pivotal enzyme, Nicotinate Nucleotide Adenylyltransferase (NNAT), also known as nicotinate mononucleotide adenylyltransferase (NaMNAT), in the process of its biosynthesis. Our findings indicate that NAD + biosynthesis in humans and bacteria occurs via the same de novo synthesis route and the pyridine ring salvage pathway. Maintaining NAD homeostasis in bacteria is imperative, as most bacterial species cannot get NAD+ from their surroundings. However, due to lower sequence identity and structurally distant relationship of bacteria, including E. faecium and K. pneumonia, to its human counterpart, inhibiting NNAT, an indispensable enzyme implicated in NAD + biosynthesis, is a viable alternative in curtailing infections orchestrated by E. faecium and K. pneumonia. By merging empirical and computational discoveries and connecting the intricate NAD + metabolism network with NNAT's crucial role, it becomes clear that the synergistic effect of these insights may lead to a more profound understanding of the coenzyme's function and its potential applications in the fields of therapeutics and biotechnology.

In silico screening of selective ATP mimicking inhibitors targeting the Plasmodium falciparum Grp94.

Plasmodium falciparum parasites export more than 400 proteins to remodel the host cell environment and increase its chances of surviving and reproducing. The endoplasmic reticulum (ER) plays a central role in protein export by facilitating protein sorting and folding. The ER resident member of the Hsp90 family, glucose-regulated protein 94 (Grp94), is a molecular chaperone that facilitates the proper folding of client proteins in the ER lumen. In P. falciparum, Grp94 (PfGrp94) is essential for parasite survival, rendering it a promising anti-malarial drug target. Despite this, its druggability has not been fully explored. Consequently, this study sought to identify small molecule inhibitors targeting the PfGrp94. Potential small molecule inhibitors of PfGrp94 were designed and screened using in silico studies. Molecular docking studies indicate that two novel compounds, Compound S and Compound Z selectively bind to PfGrp94 over its human homologues. Comparatively, Compound Z had a higher affinity for PfGrp94 than Compound S. Further interrogation of the inhibitor binding using molecular dynamics (MD) analysis confirmed that Compound Z formed stable binding poses within the ATP-binding pocket of the PfGrp94 N-terminal domain (NTD) during the 250 ns simulation run. PfGrp94 interacted with Compound Z through hydrogen bonding and hydrophobic interactions with residues Asp 148, Asn 106, Gly 152, Ile 151 and Lys 113. Based on the findings of this study, Compound Z could serve as a competitive and selective inhibitor of PfGrp94 and may be useful as a starting point for the development of a potential drug for malaria.Communicated by Ramaswamy H. Sarma.