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Microbial amylases should essentially remain active at higher temperatures, and in the alkaline pH and a range of surfactants to be suitable as detergent additives. In the present study, a thermophilic amylase producing bacterium, Bacillus licheniformis UDS-5 was isolated from Unai hot water spring in Gujarat, India. It was identified as a potent amylase producer during starch plate-based screening process. Therefore, the physicochemical parameters influencing amylase production were optimized using Plackett-Burman design and Central Composite Design. The amylase was purified through ammonium sulfate precipitation, size exclusion and ion exchange chromatography, achieving the purification fold and yield to be 9.2 and 40.6%, respectively. The enzyme displayed robust stability and activity across a wide range of temperatures and pHs, with an increased half-life and reduced deactivation rate constant. The amylase exhibited optimal catalysis at 70 °C and pH 8. The kinetic studies revealed Km and Vmax values of 0.58 mg/mL and 2528 μmol/mL/min, respectively. Besides, the purified amylase displayed stability in the presence of various metal ions, surfactants, and chelators suggesting its potential for industrial applications, particularly in the detergent industry. Moreover, detergent application studies demonstrated its efficacy in enhancing washing performance. A comparative profile on washing efficiency of the studied amylase and the commercial amylase with various detergents pointed towards its possible future use as a detergent additive., Competing Interests: Declarations Conflict of interest The authors declare no conflict of interest. Research involving human participants and/or animals This article does not contain any studies with human participants and/or animals performed by any of the authors., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacterial infections are evolving and one of the chief problems is emergence and prevalence of antibacterial resistance. Moreover, certain strains of Bacillus subtilis have become resistant to several antibiotics. To counteract this menace, the present work aimed to comprehend the antibacterial activity of synthesized two quinoline derivatives against Bacillus subtilis. Toxicity predictions via Protox II, SwissADME and T.E.S.T (Toxicity Estimation Software Tool) revealed that these derivatives were non-toxic and had little to no adverse effects. Molecular docking studies carried out in Schrodinger with two quinoline derivatives (referred Q1 and Q2) docked against selected target proteins (PDB IDs: 2VAM and1FSE) of B. subtilis demonstrated ideal binding energies (2VAM-Q1: - 4.63 kcal/mol and 2VAM-Q2: - 4.46 kcal/mol, and 1FSE-Q1: - 3.51 kcal/mol, 1FSE-Q2: - 6.34 kcal/mol). These complexes were simulated at 100 ns and the outcomes revealed their stability with slight conformational changes. Anti-microbial assay via disc diffusion method revealed zones of inhibition showing that B. subtilis was inhibited by both Q1 and Q2, with Q2 performing slightly better than Q1, pointing towards its effectiveness against this organism and necessitating further study on other bacteria in prospective studies. Thus, this study demonstrates that our novel quinoline derivatives exhibit antibacterial properties against Bacillus subtilis and can act as potent anti-bacterials., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Soft rot is one of the top ten most dangerous plant pathogens in agricultural production, storage, and transport, and the use of microorganisms and their metabolites to control soft rot is a current research hotspot. In this study, we identified the antimicrobial substance in the metabolite of Paenibacillus polymyxa KH-19, and determined that the antimicrobial substance of this strain was an active protein. The protein was completely precipitated at 40-60% ammonium sulphate saturation and showed good inhibitory effects against seven pathogenic bacteria including Pectobacterium carotovorum BC2 and seven pathogenic fungi including Pyricularia oryzae. The MIC of the protein was 51.563 µg/mL, temperature acid-base UV and light stability insensitive to protease, with high-temperature resistance. The antimicrobial protein was isolated and purified by DEAE-anion exchange column and Sephadex G-75 gel filtration chromatography, and the LC-MS/MS assay identified the protein as lysophosphatidyl esterase with a molecular weight of 25.255 kDa. The purified antimicrobial protein increased the inhibitory effect against P. carotovorum BC2, with the diameter of the circle of inhibition being 26.50 ± 0.915 mm. Bioinformatics analysis showed that the protein has the molecular formula of C 1117 H 1732 N 316 O 338 S 5 , encodes 224 amino acids, has an aliphatic index of 88.39, and belongs to the category of hydrophilic unstable proteins. The present study is the first report of an active protein with extreme thermoplastic and resistance to P. carotovorum BC2, which provides a reference for the preparation and application of the antimicrobial substances of P. polymyxa KH-19, as well as a theoretical basis for the study of the function of lysophosphodiesterase protein and its use as a microbial preparation., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Alkyl hydroperoxide reductase subunit C (AhpC) contributes to the cellular defense against reactive oxygen species. However, it remains understudied in psychrophiles. Amino acid comparison demonstrated that AhpC from Psychrobacter sp. ANT206 (ANT206) (PsAhpC) revealed fewer numbers of Lys and more numbers of Gly, which might have favored higher flexibility at low temperature. The recombinant PsAhpC (rPsAhpC) was most active at 25 °C and retained 35% of its residual activity at 0 °C, indicating that it was a cold-adapted enzyme. Additionally, rPsAhpC demonstrated significant salt tolerance, sustaining its activity in the presence of 4.0 M NaCl. Molecular dynamics simulations indicated that PsAhpC had comparatively loose conformation, which facilitated reactions at low temperatures. Subsequently, an ahpc knockout mutant was constructed, and the growth rate of the knockout mutant significantly decreased, suggesting that ahpc might be crucial for the growth of ANT206 at low temperatures. The findings provide a robust foundation for further investigation into the structural features and catalytic characterization of cold-adapted AhpC. The structural characteristics of PsAhpC and its cold tolerance and salt tolerance may be applied to stress resistance breeding of various organisms., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Klebsiella pneumoniae and Legionella pneumophila are common Gram-negative bacteria that can cause lung infections. The multidrug resistance of K. pneumoniae presents a significant challenge for treatment. This study focuses on isocitrate dehydrogenase (IDH), a key enzyme in the oxidative metabolic pathway of these two bacteria. KpIDH and LpIDH were successfully overexpressed and purified, and their biochemical characteristics were thoroughly investigated. The study revealed that KpIDH and LpIDH are homodimeric enzymes with molecular weights of approximately 70 kDa. They are completely dependent on the coenzyme NADP + and are inactive towards NAD + . KpIDH exhibits the highest catalytic activity at pH 8.0 in the presence of Mn 2+ and at pH 7.8 in the presence of Mg 2+ . Its optimal catalytic performance is achieved with both ions at 55 °C. LpIDH exhibited its highest activity at pH 7.8 in the presence of Mn 2+ and Mg 2+ , respectively, and exhibits optimal catalytic performance at 45 °C. Heat inactivation studies showed that KpIDH and LpIDH retained over 80% of their activity after being exposed to 45 °C for 20 min. Furthermore, we successfully altered the coenzyme specificity of KpIDH and LpIDH from NADP + to NAD + by replacing four key amino acid residues. This study provides a comprehensive biochemical characterization of two multidrug-resistant bacterial IDHs commonly found in hospital environments. It enhances our understanding of the characteristics of pathogenic bacteria and serves as a reference for developing new therapeutic strategies., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Cationic amino acid binding protein (CLasArgBP), one of the two amino acid binding receptor in Candidatus Liberibacter asiaticus (CLas), is predominately expressed in citrus psyllids as a part of ATP-binding cassette transport system. The present study describes characterization of CLasArgBP by various biophysical techniques and in silico study, to identify potential inhibitor molecules against CLasArgBP through virtual screening and MD simulations. Further, in planta study was carried out to assess the effect of selected inhibitors on Huanglongbing infected Mosambi plants. The results showed that CLasArgBP exhibits pronounced specificity for arginine, histidine and lysine. Surface plasmon resonance (SPR) study reports highest binding affinity for arginine (Kd, 0.14 µM), compared to histidine and lysine (Kd, 15 µΜ and 26 µΜ, respectively). Likewise, Differential Scanning Calorimetry (DSC) study showed higher stability of CLasArgBP for arginine, compared to histidine and lysine. N(omega)-nitro-L-arginine, Gamma-hydroxy-L-arginine and Gigartinine emerged as lead compounds through in silico study displaying higher binding energy and stability compared to arginine. SPR reports elevated binding affinities for N(omega)-nitro-L-arginine and Gamma-hydroxy-L-arginine (Kd, 0.038 µΜ and 0.061 µΜ, respectively) relative to arginine. DSC studies showed enhanced thermal stability for CLasArgBP in complex with selected inhibitors. Circular dichroism and fluorescence studies showed pronounced conformational changes in CLasArgBP with selected inhibitors than with arginine. In planta study demonstrated a substantial decrease in CLas titer in treated plants as compared to control plants. Overall, the study provides the first comprehensive characterization of cationic amino acid binding protein from CLas, as a potential drug target to manage HLB disease., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We have approached the construction of an artificial enzyme by employing a robust protein scaffold, lactococcal multidrug resistance regulator, LmrR, providing a structured secondary and outer coordination spheres around a molecular rhodium complex, [Rh I (P Et2 N gly P Et2 ) 2 ] - . Previously, we demonstrated a 2-3 fold increase in activity for one Rh-LmrR construct by introducing positive charge in the secondary coordination sphere. In this study, a series of variants was made through site-directed mutagenesis where the negative charge is located in the secondary sphere or outer coordination sphere, with additional variants made with increasingly negative charge in the outer coordination sphere while keeping a positive charge in the secondary sphere. Placing a negative charge in the secondary or outer coordination sphere demonstrates decreased activity by a factor of two compared to the wild-type Rh-LmrR. Interestingly, addition of positive charge in the secondary sphere, with the negatively charged outer coordination sphere restores activity. Vibrational and NMR spectroscopy suggest minimal changes to the electronic density at the rhodium center, regardless of inclusion of a negative or positive charge in the secondary sphere, suggesting another mechanism is impacting catalytic activity, explored in the discussion., (© 2024. The Author(s), under exclusive licence to Society for Biological Inorganic Chemistry (SBIC).)

Chitinases are enzymes that can break down chitin, a major component of the exoskeleton of insects and fungi. This feature makes them potential biopesticides in agriculture since they are considered a safe and environmentally friendly alternative to synthetic pesticides. In this work, we performed a comparative study between two different bacterial expression strains to produce a recombinant chitinase with improved stability. Escherichia coli strains Origami B and BL21 (DE3) were selected for their distinct cytosolic environment to express BhChitA chitinase of Bacillus halodurans C-125 and to investigate the role of disulfide bond formation and proper folding on its stability and activity. Expression of the recombinant BhChitA in bacterial strain containing oxidative cytosol (Origami B) improved its activity and stability. Although both expression systems have comparable biochemical properties (temperature range 20-80 °C and pH spectrum 3-10), BhChitA expressed in Origami strain seems more stable than expressed in BL21. Furthermore, the optimal expression conditions of the recombinant BhChitA has been carried out at 30 °C during 6 h for the Origami strain, against 20 °C during 2 h for BL21. On the other hand, no significant differences were detected between the two enzymes when the effect of metal ions was tested. These findings correlate with the analysis of the overall structure of BhChitA. The model structure permitted to localize disulfide bond, which form a stable connection between the substrate-binding residues and the hydrophobic core. This link is required for efficient binding of the chitin insertion domain to the substrate. BhChitA exhibited in vitro antifungal effect against phytopathogenic fungi and suppressed necrosis of Botrytis cinerea on detached tomato leaves. In vitro assays showed the influence of BhChitA on growth suppression of Botrytis cinerea (53%) Aspergillus niger (65%), Fusarium graminearum (25%), and Fusarium oxysporum (34%). Our results highlight the importance of the bacterial expression system with oxidative cytosol in producing promising biopesticides that can be applied for post-harvest processing and crop protection., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The ring-like peripheral light-harvesting complex 2 (LH2) expressed by many phototrophic purple bacteria is a popular model system in biological light-harvesting research due to its robustness, small size, and known crystal structure. Furthermore, the availability of structural variants with distinct electronic structures and optical properties has made this group of light harvesters an attractive testing ground for studies of structure-function relationships in biological systems. LH2 is one of several pigment-protein complexes for which a link between functionality and effects such as excitonic coherence and vibronic coupling has been proposed. While a direct connection has not yet been demonstrated, many such interactions are highly sensitive to resonance conditions, and a dependence of intra-complex dynamics on detailed electronic structure might be expected. To gauge the sensitivity of energy-level structure and relaxation dynamics to naturally occurring structural changes, we compare the photo-induced dynamics in two structurally distinct LH2 variants. Using polarization-controlled 2D electronic spectroscopy at cryogenic temperatures, we directly access information on dynamic and static disorder in the complexes. The simultaneous optimal spectral and temporal resolution of these experiments further allows us to characterize the ultrafast energy relaxation, including exciton transport within the complexes. Despite the variations in PPC molecular structure manifesting as clear differences in electronic structure and disorder, the energy-transport and-relaxation dynamics remain remarkably similar. This indicates that the light-harvesting functionality of purple bacteria within a single LH2 complex is highly robust to structural perturbations and likely does not rely on finely tuned electronic- or electron-vibrational resonance conditions., (© 2024. The Author(s).)

Chromosomal replication is a ubiquitous and essential cellular process. In bacteria, the master replication initiator DnaA plays a key role in promoting an open complex at the origin (oriC) and recruiting helicase in a tightly regulated process. The C-terminal domain IV specifically recognises consensus sequences of double-stranded DNA in oriC, termed DnaA-boxes, thereby facilitating the initial engagement of DnaA to oriC. Here, we report the 13 Cβ and backbone 1 H, 15 N, and 13 C chemical shift assignments of soluble DnaA domain IV from Bacillus subtilis at pH 7.6 and 298 K., (© 2024. The Author(s).)

Diverse extracellular sensor domains enable cells to regulate their behavior, physiological processes, and interspecies interactions in response to environmental stimuli. These sensing mechanisms facilitate the ultimate adaptation of organisms to their surrounding conditions. Pseudomonas aeruginosa (PAO1) is a clinically significant opportunistic pathogen in hospital infection. The CHASE4 domain, a putative extracellular sensing module, is found in the N-terminus of GGDEF-EAL-containing PA2072, a transmembrane receptor from P. aeruginosa. However, the signal identification and sensing mechanism of monomeric PA2072 CHASE4 remains largely unknown. Here, we report backbone and side chain resonance assignments of PA2072 CHASE4 as a basis for studying the structural mechanism of CHASE4-mediated signal recognition., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27 kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2 H, 13 C, 15 N enriched samples of CcdB_G100T. Analysis of 1 H, 13 C α , 13 C β secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

ProXp-ala is a key component of the translational machinery in all three Domains of life. This enzyme helps to maintain the fidelity of proline codon translation through aminoacyl-tRNA Pro proofreading. In the first step of tRNA aminoacylation, the cognate aminoacyl-tRNA synthetase (aaRS) binds and activates an amino acid in the enzyme's synthetic active site. If a non-cognate amino acid passes this first selection step and is charged onto the tRNA, a distinct aaRS editing active site may recognize the mischarged tRNA and deacylate it. Alternatively, this editing reaction may be carried out by a separate enzyme that deacylates the mischarged tRNA in trans. ProXp-ala is responsible for editing Ala mischarged onto tRNA Pro . Since trans-editing domains such as ProXp-ala bind their substrates after release from the synthetase, they must recognize not only the mischarged amino acid, but also the specific tRNA. Previous studies showed that Caulobacter crescentus (Cc) ProXp-ala distinguishes tRNA Pro from tRNA Ala , in part, based on the unique tRNA Pro acceptor stem base pair C1:G72. Previous crystallographic and NMR data also revealed a role for conformational selection by the ProXp-ala α2 helix in Ala- versus Pro-tRNA Pro substrate discrimination. The α2 helix makes lattice contacts in the crystal, which left some uncertainty as to its position in solution. We report resonance assignments for the substrate-free Cc ProXp-ala and the NMR-derived three-dimensional structure of the protein. These data reveal the position of the α2 helix in solution, with implications for substrate binding and recognition., (© 2024. The Author(s).)

The contribution of Fe(II)-oxidizing bacteria to iron cycling in freshwater, groundwater, and marine environments has been widely recognized in recent years. These organisms perform extracellular electron transfer (EET), which constitutes the foundations of bioelectrochemical systems for the production of biofuels and bioenergy. It was proposed that the Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes soluble ferrous Fe(II) at the surface of the cell and performs EET through the Mto redox pathway. This pathway is composed by the periplasmic monoheme cytochrome MtoD that is proposed to bridge electron transfer between the cell exterior and the cytoplasm. This makes its functional and structural characterization, as well as evaluating the interaction process with its physiological partners, essential for understanding the mechanisms underlying EET. Here, we report the complete assignment of the heme proton and carbon signals together with a near-complete assignment of 1 H, 13 C and 15 N backbone and side chain resonances for the reduced, diamagnetic form of the protein. These data pave the way to identify and structurally map the molecular interaction regions between the cytochrome MtoD and its physiological redox partners, to explore the EET processes of S. lithotrophicus ES-1., (© 2024. The Author(s).)

Signalosomes are high-order protein machineries involved in complex mechanisms controlling regulated immune defense and cell death execution. The immune response is initiated by the recognition of exogeneous or endogenous signals, triggering the signalosome assembly process. The final step of signalosome fate often involves membrane-targeting and activation of pore-forming execution domains, leading to membrane disruption and ultimately cell death. Such cell death-inducing domains have been thoroughly characterized in plants, mammals and fungi, notably for the fungal cell death execution protein domain HeLo. However, little is known on the mechanisms of signalosome-based immune response in bacteria, and the conformation of cell death executors in bacterial signalosomes is still poorly characterized. We recently uncovered the existence of NLR signalosomes in various multicellular bacteria and used genome mining approaches to identify putative cell death executors in Streptomyces olivochromogenes. These proteins contain a C-terminal amyloid domain involved in signal transmission and a N-terminal domain, termed BELL for Bacteria analogous to fungal HeLL (HeLo-like), presumably responsible for membrane-targeting, pore-forming and cell death execution. In the present study, we report the high yield expression of S. olivochromogenes BELL2 and its characterization by solution NMR spectroscopy. BELL is folded in solution and we report backbone and sidechain assignments. We identified five α-helical secondary structure elements and a folded core much smaller than its fungal homolog HeLo. This study constitutes the first step toward the NMR investigation of the full-length protein assembly and its membrane targeting., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917 bp, encoding 638 amino acids with a predicted molecular mass of 62.3 kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50 °C, respectively, and it was stable within pH 4.5‒7.0 and up to 45 °C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4 °C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Although the genome of Mycobacterium tuberculosis (Mtb) H37Rv, the causative agent of tuberculosis, has been repeatedly annotated and updated, a range of proteins from this human pathogen have unknown functions. Mtb Rv0439c, a member of the short-chain dehydrogenase/reductases superfamily, has yet to be cloned and characterized, and its function remains unclear. In this work, we present for the first time the optimized expression and purification of this enzyme, as well as bioinformatic analysis to unveil its potential coenzyme and substrate. Optimized expression in Escherichia coli yielded soluble Rv0439c, while certain tag fusions resulted in insolubility. Sequence and docking analyses strongly suggested that Rv0439c has a clear preference for NADP + , with Arg53 being a key residue that confers coenzyme specificity. Furthermore, functional prediction using CLEAN and DEEPre servers suggested that this protein is a potential NADP + -retinol dehydrogenase (EC No. 1.1.1.300) in retinol metabolism, and this was supported by a BLASTp search and docking studies. Collectively, our findings provide a solid basis for future functional characterization and structural studies of Rv0439c, which will contribute to enhanced understanding of Mtb biology., Competing Interests: Declarations Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein's N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the 13 C, 15 N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176-181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies., (© 2024. The Author(s).)

Proteus penneri (P. penneri) is a bacillus-shaped, gram-negative, facultative anaerobe bacterium that is primarily an invasive pathogen and the etiological agent of several hospital-associated infections. P. penneri strains are naturally resistant to macrolides, amoxicillin, oxacillin, penicillin G, and cephalosporins; in addition, no vaccines are available against these strains. This warrants efforts to propose a theoretical based multi-epitope vaccine construct to prevent pathogen infections. In this research, reverse vaccinology bioinformatics and immunoinformatics approaches were adopted for vaccine target identification and construction of a multi-epitope vaccine. In the first phase, a core proteome dataset of the targeted pathogen was obtained using the NCBI database and subjected to bacterial pan-genome analysis using bacterial pan-genome analysis (BPGA) to predict core protein sequences which were then used to find good vaccine target candidates. This identified two proteins, Hcp family type VI secretion system effector and superoxide dismutase family protein, as promising vaccine targets. Afterward using the IEDB database, different B-cell and T-cell epitopes were predicted. A set of four epitopes "KGSVNVQDRE, NTGKLTGTR, IIHSDSWNER, and KDGKPVPALK" were chosen for the development of a multi-epitope vaccine construct. A 183 amino acid long vaccine design was built along with "EAAAK" and "GPGPG" linkers and a cholera toxin B-subunit adjuvant. The designed vaccine model comprised immunodominant, non-toxic, non-allergenic, and physicochemical stable epitopes. The model vaccine was docked with MHC-I, MHC-II, and TLR-4 immune cell receptors using the Cluspro2.0 web server. The binding energy score of the vaccine was - 654.7 kcal/mol for MHC-I, - 738.4 kcal/mol for MHC-II, and - 695.0 kcal/mol for TLR-4. A molecular dynamic simulation was done using AMBER v20 package for dynamic behavior in nanoseconds. Additionally, MM-PBSA binding free energy analysis was done to test intermolecular binding interactions between docked molecules. The MM-GBSA net binding energy score was - 148.00 kcal/mol, - 118.00 kcal/mol, and - 127.00 kcal/mol for vaccine with TLR-4, MHC-I, and MHC-II, respectively. Overall, these in silico-based predictions indicated that the vaccine is highly promising in terms of developing protective immunity against P. penneri. However, additional experimental validation is required to unveil the real immune response to the designed vaccine., Competing Interests: Declarations Conflict of Interest The authors declare no conflict of interest., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual D-lysine in addition to the typical D-alanine and D-glutamate. Previously, we identified the D-lysine and D-glutamate biosynthetic pathways of T. maritima. Additionally, we reported some multifunctional enzymes involved in amino acid metabolism. In the present study, we characterized the enzymatic properties of TM1744 (threonine aldolase) to probe both its potential multifunctionality and D-amino acid metabolizing activities. TM1744 displayed aldolase activity toward both L-allo-threonine and L-threonine, and exhibited higher activity toward L-threo-phenylserine. It did not function as an aldolase toward D-allo-threonine or D-threonine. Furthermore, TM1744 had racemase activity toward two amino acids, although its racemase activity was lower than its aldolase activity. TM1744 did not have other amino acid metabolizing activities. Therefore, TM1744 is a low-specificity L-threonine aldolase with limited racemase activity., (© 2024. The Author(s), under exclusive licence to Springer Nature Japan KK.)

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 Mg 2+ 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 Mg 2+ . Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg 2+ , while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg 2+ 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 Mg 2+ . Conclusively, the presence of Mg 2+ induces a conformation in KpPrAT that favours nucleotide binding., (© 2024. The Author(s).)

C-phycocyanin (C-PC) is the main component of water-soluble light-harvesting complexes (phycobilisomes, PBS) of cyanobacteria. PBS are involved in the absorption of quantum energy and the transfer of electronic excitation energy to the photosystems. A specific environment of C-PC chromophoric groups is provided by the protein matrix structure including protein-protein contacts between different subunits. Registration of C-PC spectral characteristics and the fluorescence anisotropy decay have revealed a significant pH influence on the chromophore microenvironment: at pH 5.0, a chromophore is more significantly interacts with the solvent, whereas at pH 9.0 the chromophore microenvironment becomes more viscous. Conformations of chromophores and the C-PC protein matrix have been studied by Raman and infrared spectroscopy. A decrease in the medium pH results in changes in the secondary structure either the C-PC apoproteins and chromophores, the last one adopts a more folded conformation., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

To investigate the effect of α3 and α5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (Δbtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the Δα3α5 lipase was purified by Ni-NTA column chromatography. Next, the biochemical properties of the Δα3α5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and Δα3α5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and Δα3α5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of Δα3α5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and Δα3α5 lipases by 2.1- and 2.5-fold, respectively., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/β hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome., (© 2024. His Majesty the King in Right of Canada as represented by the Minister of Agriculture and Agri-Food, and Liam Worrel.)

Clostridium tyrobutyricum strain NRRL B-67062 was previously isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, the cell-free supernatant of the fermentation broth from NRRL B-67062 contained antibacterial activity against certain Gram-positive bacteria. To determine the source of this antibacterial activity, we report the genome and genome mining of this strain. The complete genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3114 predicted coding sequences, 79 RNA genes, and a G+C content of 31.0%. Analyses of the genome data for genes potentially associated with antimicrobial features were sought after by using BAGEL-4 and anti-SMASH databases. Among the leads, a polypeptide of 66 amino acids (PEG 446) contains the DUF4177 domain, which is an uncharacterized highly conserved domain (pfam13783). The cloning and expression of the peg446 gene in Escherichia coli and Bacillus subtilis confirmed the antibacterial property against Lactococcus lactis LM 0230, Limosilactobacillus fermentum 0315-25, and Listeria innocua NRRL B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested that PEG 446 contains one alpha-helix and three anti-parallel short beta-sheets. These results will aid further functional studies and facilitate simultaneously fermentative production of both butyric acid and a putative bacteriocin from agricultural waste and lignocellulosic biomass materials., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Antimicrobial peptides have gradually gained advantages over small molecule inhibitors for their multifunctional effects, synthesising accessibility and target specificity. The current study aims to determine an antimicrobial peptide to inhibit PknB, a serine/threonine protein kinase (STPK), by binding efficiently at the helically oriented hinge region. A library of 5626 antimicrobial peptides from publicly available repositories has been prepared and categorised based on the length. Molecular docking using ADCP helped to find the multiple conformations of the subjected peptides. For each peptide served as input the tool outputs 100 poses of the subjected peptide. To maintain an efficient binding for relatively a longer duration, only those peptides were chosen which were seen to bind constantly to the active site of the receptor protein over all the poses observed. Each peptide had different number of constituent amino acid residues; the peptides were classified based on the length into five groups. In each group the peptide length incremented upto four residues from the initial length form. Five peptides were selected for Molecular Dynamic simulation in Gromacs based on higher binding affinity. Post-dynamic analysis and the frame comparison inferred that neither the shorter nor the longer peptide but an intermediate length of 15 mer peptide bound well to the receptor. Residual substitution to the selected peptides was performed to enhance the targeted interaction. The new complexes considered were further analysed using the Elastic Network Model (ENM) for the functional site's intrinsic dynamic movement to estimate the new peptide's role. The study sheds light on prospects that besides the length of peptides, the combination of constituent residues equally plays a pivotal role in peptide-based inhibitor generation. The study envisages the challenges of fine-tuned peptide recovery and the scope of Machine Learning (ML) and Deep Learning (DL) algorithm development. As the study was primarily meant for generation of therapeutics for Tuberculosis (TB), the peptide proposed by this study demands meticulous invitro analysis prior to clinical applications., (© 2024. The Author(s).)

Anaplasma phagocytophilum is an intracellular tick-transmitted bacterial pathogen that infects neutrophils in mammals and causes granulocytic anaplasmosis. In this study, we investigated the molecular chaperones ClpB and DnaK from A. phagocytophilum. In Escherichia coli, ClpB cooperates with DnaK and its co-chaperones DnaJ and GrpE in ATP-dependent reactivation of aggregated proteins. Since ClpB is not produced in metazoans, it is a promising target for developing antimicrobial therapies, which generates interest in studies on that chaperone's role in pathogenic bacteria. We found that ClpB and DnaK are transcriptionally upregulated in A. phagocytophilum 3-5 days after infection of human HL-60 and tick ISE6 cells, which suggests an essential role of the chaperones in supporting the pathogen's intracellular life cycle. Multiple sequence alignments show that A. phagocytophilum ClpB and DnaK contain all structural domains that were identified in their previously studied orthologs from other bacteria. Both A. phagocytophilum ClpB and DnaK display ATPase activity, which is consistent with their participation in the ATP-dependent protein disaggregation system. However, despite a significant sequence similarity between the chaperones from A. phagocytophilum and those from E. coli, the former were not as effective as their E. coli orthologs during reactivation of aggregated proteins in vitro and in supporting the survival of E. coli cells under heat stress. We conclude that the A. phagocytophilum chaperones might have evolved with distinct biochemical properties to maintain the integrity of pathogenic proteins under unique stress conditions of an intracellular environment of host cells., Competing Interests: Declarations of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Xylanases derived from Bacillus species hold significant importance in various large-scale production sectors, with increasing demand driven by biofuel production. However, despite their potential, the extreme environmental conditions often encountered in production settings have led to their underutilisation. To address this issue and enhance their efficacy under adverse conditions, we conducted a theoretical investigation on a group of five Bacillus species xylanases belonging to the glycoside hydrolase GH11 family. Bacillus sp. NCL 87-6-10 (sp_NCL 87-6-10) emerged as a potent candidate among the selected biocatalysts; this Bacillus strain exhibited high thermal stability and achieved a transition state with minimal energy requirements, thereby accelerating the biocatalytic reaction process. Our approach aims to provide support for experimentalists in the industrial sector, encouraging them to employ structural-based reaction modelling scrutinisation to predict the ability of targeted xylanases., Methods: Utilising crystal structure data available in the Carbohydrate-Active enzymes database, we aimed to analyse their structural capabilities in terms of thermal-stability and activity. Our investigation into identifying the most prominent Bacillus species xylanases unfolds with the help of the semi-empirical quantum mechanics MOPAC method integrated with the DRIVER program is used in calculations of reaction pathways to understand the activation energy. Additionally, we scrutinised the selected xylanases using various analyses, including constrained network analyses, intermolecular interactions of the enzyme-substrate complex and molecular orbital assessments calculated using the AM1 method with the MO-G model (MO-G AM1) to validate their reactivity., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The primary photochemical reaction of photosynthesis in green sulfur bacteria occurs in the homodimer PscA core proteins by a special chlorophyll pair. The light induced excited state of the special pair producing P 840 + is rapidly reduced by electron transfer from one of the two PscC subunits. Molecular dynamics (MD) simulations are combined with bioinformatic tools herein to provide structural and dynamic insight into the complex between the two PscA core proteins and the two PscC subunits. The microscopic dynamic model involves extensive sampling at atomic resolution and at a cumulative time-scale of 22µs and reveals well defined protein-protein interactions. The membrane complex is composed of the two PscA and the two PscC subunits and macroscopic connections are revealed within a putative electron transfer pathway from the PscC subunit to the special pair P 840 located within the PscA subunits. Our results provide a structural basis for understanding the electron transport to the homodimer RC of the green sulfur bacteria. The MD based approach can provide the basis to further probe the PscA-PscC complex dynamics and observe electron transfer therein at the quantum level. Furthermore, the transmembrane helices of the different PscC subunits exert distinct dynamics in the complex., (© 2024. The Author(s).)

S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called "last-resort" drugs. Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant S. aureus is high. M23B family peptidoglycan hydrolases, enzymes that can kill S. aureus by cleaving glycine-glycine peptide bonds in S. aureus cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. We report here the near complete 1 H/ 13 C/ 15 N resonance assignment of the catalytic domain of LytM, residues 185-316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall., (© 2023. The Author(s).)

The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24-48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0., (© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Universal stress proteins (USPs) are widely distributed and play crucial roles in cellular responses to biotic and abiotic stresses. These roles include regulating cell growth and development, cell motility, hypoxia responses, and ion sequestration. With the increasing frequency and intensity of extreme weather events due to climate change, pathogens have developed different strategies to withstand environmental stresses, in which USPs play a significant role in their survival and virulence. In this study, we analyzed the importance of USPs in various organisms, such as archaea, plants, and fungi, as a parameter that influences their survival. We discussed the different types Of USPs and their role, aiming to carry out fundamental research in this field to identify significant constraints for better understanding of USP functions at molecular level. Additionally, we discussed concepts and research techniques that could help overcome these hurdles and facilitate new molecular approaches to better understand and target USPs as important stress adaptation and survival regulators. Although the precise characteristics of USPs are still unclear, numerous innovative uses have already been developed, tested, and implemented. Complementary approaches to basic research and applications, as well as new technology and analytical techniques, may offer insights into the cryptic but crucial activities of USPs in various living systems., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA replication is a tightly coordinated event carried out by a multiprotein replication complex. An essential factor in the bacterial replication complex is the ring-shaped DNA sliding clamp, β-clamp, ensuring processive DNA replication and DNA repair through tethering of polymerases and DNA repair proteins to DNA. β -clamp is a hub protein with multiple interaction partners all binding through a conserved clamp binding sequence motif. Due to its central role as a DNA scaffold protein, β-clamp is an interesting target for antimicrobial drugs, yet little effort has been put into understanding the functional interactions of β-clamp. In this review, we scrutinize the β-clamp structure and dynamics, examine how its interactions with a plethora of binding partners are regulated through short linear binding motifs and discuss how contexts play into selection. We describe the dynamic process of clamp loading onto DNA and cover the recent advances in drug development targeting β-clamp. Despite decades of research in β-clamps and recent landmark structural insight, much remains undisclosed fostering an increased focus on this very central protein., (© 2024. The Author(s).)

Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (T m ) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Exchange of B800 bacteriochlorophyll (BChl) a in light-harvesting complex 2 (LH2) is promising for a better understanding of the mechanism on intracomplex excitation energy transfer of this protein. Structural and spectroscopic properties of LH2 lacking B800 BChl a (B800-depleted LH2), which is an important intermediate protein in the B800 exchange, will be useful to tackle the energy transfer mechanism in LH2 by the B800 exchange strategy. In this study, we report a unique spectral change of B800-depleted LH2, in which the Q y absorption band of B800 BChl a is automatically recovered under neutral pH conditions. This spectral change was facilitated by factors for destabilization of LH2, namely, a detergent, lauryl dimethylamine N-oxide, and an increase in temperature. Spectral analyses in the preparation of an LH2 variant denoted as B800-recovered LH2 indicated that most BChl a that was released by decomposition of part of B800-depleted LH2 was a source of the production of B800-recovered LH2. Characterization of purified B800-recovered LH2 demonstrated that its spectroscopic and structural features was quite similar to those of native LH2. The current results indicate that the recovery of the B800 Q y band of B800-depleted LH2 originates from the combination of decomposition of part of B800-depleted LH2 and in situ reconstitution of BChl a into the B800 binding pockets of residual B800-depleted LH2, resulting in the formation of stable B800-recovered LH2., (© 2024. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

The hydrolysis of deacylated glycerophospholipids into sn-glycerol 3-phosphate and alcohol is facilitated by evolutionarily conserved proteins known as glycerophosphodiester phosphodiesterases (GDPDs). These proteins are crucial for the pathogenicity of bacteria and for bioremediation processes aimed at degrading organophosphorus esters that pose a hazard to both humans and the environment. Additionally, GDPDs are enzymes that respond to multiple nutrients and could potentially serve as candidate genes for addressing deficiencies in zinc, iron, potassium, and especially phosphate in important plants like rice. In mammals, glycerophosphodiesterases (GDEs) play a role in regulating osmolytes, facilitating the biosynthesis of anandamine, contributing to the development of skeletal muscle, promoting the differentiation of neurons and osteoblasts, and influencing pathological states. Due to their capacity to enhance a plant's ability to tolerate various nutrient deficiencies and their potential as pharmaceutical targets in humans, GDPDs have received increased attention in recent times. This review provides an overview of the functions of GDPD families as vital and resilient enzymes that regulate various pathways in bacteria, plants, and humans., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The distinctive morphology characteristics of microfold cells (M cells) allow the vaccine antigen not only to interact with immune cells directly, but also to effectively stimulate mucosal immune responses via receptors on its apical surface. Human prion protein, a transmembrane receptor for Brucella abortus Hsp60, is highly expressed on the M cell surface. Nonetheless, this protein tends to express in inclusion body in prokaryotic hosts. In this study, the shorter interacting regions of human prion protein were identified via computational methods such as docking and molecular dynamics simulations to minimize its aggregation tendency. The computational calculations revealed three novel human prion protein-interacting regions, namely PrP125, PrP174, and PrP180. In accordance with in silico prediction, the biologically synthesized peptides fusing with GST tag demonstrated their specific binding to Hsp60 protein via pull-down assay. Hence, this finding laid the groundwork for M-cell targeting candidate validation through these newly identified interacting regions., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cold shock proteins (CSPs) are small, acidic proteins which contain a conserved nucleic acid-binding domain. These perform mRNA translation acting as "RNA chaperones" when triggered by low temperatures initiating their cold shock response. CSP- RNA interactions have been predominantly studied. Our focus will be CSP-DNA interaction examination, to analyse the diverse interaction patterns such as electrostatic, hydrogen and hydrophobic bonding in both thermophilic and mesophilic bacteria. The differences in the molecular mechanism of these contrasting bacterial proteins are studied. Computational techniques such as modelling, energy refinement, simulation and docking were operated to obtain data for comparative analysis. The thermostability factors which stabilise a thermophilic bacterium and their effect on their molecular regulation is investigated. Conformational deviation, atomic residual fluctuations, binding affinity, Electrostatic energy and Solvent Accessibility energy were determined during stimulation along with their conformational study. The study revealed that mesophilic bacteria E. coli CSP have higher binding affinity to DNA than thermophilic G. stearothermophilus. This was further evident by low conformation deviation and atomic fluctuations during simulation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Implementation of computational tools in the identification of novel drug targets for Tuberculosis (TB) has been a promising area of research. TB has been a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) localized primarily on the lungs and it has been one of the most successful pathogen in the history of mankind. Extensively arising drug resistivity in TB has made it a global challenge and need for new drugs has become utmost important.The involvement of Nucleoid-Associated Proteins (NAPs) in maintaining the structure of the genomic material and regulating various cellular processes like transcription, DNA replication, repair and recombination makes significant, has opened a new arena to find the drugs targeting Mtb. The current study aims to identify potential inhibitors of NAPs through a computational approach. In the present work we worked on the eight NAPs of Mtb, namely, Lsr2, EspR, HupB, HNS, NapA, mIHF and NapM. The structural modelling and analysis of these NAPs were carried out. Moreover, molecular interaction were checked and binding energy was identified for 2500 FDA-approved drugs that were selected for antagonist analysis to choose novel inhibitors targeting NAPs of Mtb. Drugs including Amikacin, streptomycin, kanamycin, and isoniazid along with eight FDA-approved molecules that were found to be potential novel targets for these mycobacterial NAPs and have an impact on their functions. The potentiality of several anti-tubercular drugs as therapeutic agents identified through computational modelling and simulation unlocks a new gateway for accomplishing the goal to treat TB., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Eis (Enhanced intracellular survival) protein is an aminoglycoside acetyltransferase enzyme classified under the family - GNAT (GCN5-related family of N-acetyltransferases) secreted by Mycobacterium tuberculosis (Mtb). The enzymatic activity of Eis results in the acetylation of kanamycin, thereby impairing the drug's action. In this study, we expressed and purified recombinant Eis (rEis) to determine the enzymatic activity of Eis and its potential inhibitor. Glide-enhanced precision docking was used to perform molecular docking with chosen ligands. Quercetin was found to interact Eis with a maximum binding affinity of -8.379 kcal/mol as compared to other ligands. Quercetin shows a specific interaction between the positively charged amino acid arginine in Eis and the aromatic ring of quercetin through π-cation interaction. Further, the effect of rEis was studied on the antibiotic activity of kanamycin A in the presence and absence of quercetin. It was observed that the activity of rEis aminoglycoside acetyltransferase decreased with increasing quercetin concentration. The results from the disk diffusion assay confirmed that increasing the concentration of quercetin inhibits the rEis protein activity. In conclusion, quercetin may act as a potential Eis inhibitor., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump's structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump's assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarA ΔN19 ) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1 H, 13 C and 15 N NMR assignment of SarA ΔN19 /DNA complex which is the first step towards further structure and function analysis., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

NifA is a σ 54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein's central AAA + domain. In this work, we examine if an additional Cys pair that is part of a C(X) 5  C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues' redox state may directly influence the DNA binding domain's DNA binding affinity and/or alter the protein's oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The K d of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The K d of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner., (© 2023. The Author(s).)

Multidrug-resistant tuberculosis (MDR-TB) continues to spread worldwide and remains one of the leading causes of death among infectious diseases. The enoyl-acyl carrier protein reductase (InhA) belongs to FAS-II family and is essential for the formation of the Mycobacterium tuberculosis cell wall. Recent years, InhA direct inhibitors have been extensively studied to overcome MDR-TB. However, there are still no inhibitors that have entered clinical research. Here, the ensemble docking-based virtual screening along with biological assay were used to identify potent InhA direct inhibitors from Chembridge, Chemdiv, and Specs. Ultimately, 34 compounds were purchased and first assayed for the binding affinity, of which four compounds can bind InhA well with K D values ranging from 48.4 to 56.2 µM. Among them, compound 9,222,034 has the best inhibitory activity against InhA enzyme with an IC 50 value of 18.05 µM. In addition, the molecular dynamic simulation and binding free energy calculation indicate that the identified compounds bind to InhA with "extended" conformation. Residue energy decomposition shows that residues such as Tyr158, Met161, and Met191 have higher energy contributions in the binding of compounds. By analyzing the binding modes, we found that these compounds can bind to a hydrophobic sub-pocket formed by residues Tyr158, Phe149, Ile215, Leu218, etc., resulting in extensive van der Waals interactions. In summary, this study proposed an efficient strategy for discovering InhA direct inhibitors through ensemble docking-based virtual screening, and finally identified four active compounds with new skeletons, which can provide valuable information for the discovery and optimization of InhA direct inhibitors., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Xanthorhodopsin (XR) from Salinibacter ruber is a light-driven proton pump containing retinal and a light-harvesting carotenoid antenna salinixanthin. Previous structure-functional studies of XR were conducted using a protein isolated from the native host only due to the absence of heterologous expression in Escherichia coli. In this paper, we describe cell-free synthesis and incorporation in lipid-protein nanodiscs of the recombinant XR that demonstrated its principal compatibility with E. coli biosynthetic machinery. To produce XR in E. coli, three C-terminal deletion variants of this protein were constructed. In contrast to the full-length XR, their expression resulted in efficient synthesis in E. coli cells. However, cells producing recombinant XR variants bound retinal only upon growth in minimal medium, not in the rich one. The XR3 variant with deletion of ten C-terminal amino acid residues was obtained and characterized. Its absorption spectrum and photocycle kinetics were close to those reported for XR isolated from S. ruber membranes and bleached from salinixanthin. We have also constructed the first mutants of XR, H62M and D96N, and examined their properties., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A significant portion of the earth has a salty environment, and the literature on bacterial survival mechanisms in salty environments is limited. During molecular evolution, halophiles increase acidic amino acid residues on their protein surfaces which leads to a negatively charged surface potential that helps them to maintain the protein integrity and protect them from denaturation by competing with salt ions. Through protein family analysis, we have investigated the molecular-level adaptive features of DNA polymerase III's catalytic subunit (alpha) and its structure-function relationship. This study throws light on the novel understanding of halophilic bacterial replication and the molecular basis of salt adaptation. Comparisons of the amino acid contents and electronegativity of halophilic and mesophilic bacterial proteins revealed adaptations that allow halophilic bacteria to thrive in high salt concentrations. A significantly lower isoelectric point of halophilic bacterial proteins indicates the acidic nature. Also, an abundance of disordered regions in halophiles suggests the requirement of the salt ions that play a crucial role in their stable protein folding. Despite having similar topology, mesophilic and halophilic proteins, a set of very prominent molecular modifications was observed in the alpha subunit of halophiles., (© 2023. The Author(s), under exclusive licence to Springer Nature Japan KK, part of Springer Nature.)

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation., (© 2023. The Author(s).)

N-geranyl-N΄-(2-adamantyl)ethane-1,2-diamine (SQ109) is a tuberculosis drug that has high potency against Mycobacterium tuberculosis (Mtb) and may function by blocking cell wall biosynthesis. After the crystal structure of MmpL3 from Mycobacterium smegmatis in complex with SQ109 became available, it was suggested that SQ109 inhibits Mmpl3 mycolic acid transporter. Here, we showed using molecular dynamics (MD) simulations that the binding profile of nine SQ109 analogs with inhibitory potency against Mtb and alkyl or aryl adducts at C-2 or C-1 adamantyl carbon to MmpL3 was consistent with the X-ray structure of MmpL3 - SQ109 complex. We showed that rotation of SQ109 around carbon-carbon bond in the monoprotonated ethylenediamine unit favors two gauche conformations as minima in water and lipophilic solvent using DFT calculations as well as inside the transporter's binding area using MD simulations. The binding assays in micelles suggested that the binding affinity of the SQ109 analogs was increased for the larger, more hydrophobic adducts, which was consistent with our results from MD simulations of the SQ109 analogues suggesting that sizeable C-2 adamantyl adducts of SQ109 can fill a lipophilic region between Y257, Y646, F260 and F649 in MmpL3. This was confirmed quantitatively by our calculations of the relative binding free energies using the thermodynamic integration coupled with MD simulations method with a mean assigned error of 0.74 kcal mol -1 compared to the experimental values., (© 2023. The Author(s).)

Orange carotenoid protein (OCP) is a photoactive carotenoprotein involved in photoprotection of cyanobacteria, which uses a keto-catorenoid as a chromophore. When it absorbs blue-green light, it converts from an inactive OCP O orange form to an activated OCP R red form, the latter being able to bind the light-harvesting complexes facilitating thermal dissipation of the excess of absorbed light energy. Several research groups have focused their attention on the photoactivation mechanism, characterized by several steps, involving both carotenoid photophysics and protein conformational changes. Among the used techniques, time-resolved IR spectroscopy have the advantage of providing simultaneously information on both the chromophore and the protein, giving thereby the possibility to explore links between carotenoid dynamics and protein dynamics, leading to a better understanding of the mechanism. However, an appropriate interpretation of data requires previous assignment of marker IR bands, for both the carotenoid and the protein. To date, some assignments have concerned specific α-helices of the OCP backbone, but no specific marker band for the carotenoid was identified on solid ground. This paper provides evidence for the assignment of putative marker bands for three carotenoids bound in three different OCPs: 3'-hydroxyechineone (3'-hECN), echinenone (ECN), canthaxanthin (CAN). Light-induced FTIR difference spectra were recorded in H 2 O and D 2 O and compared with spectra of isolated carotenoids. The use of DFT calculations allowed to propose a description for the vibrations responsible of several IR bands. Interestingly, most bands are located at the same wavenumber for the three kinds of OCPs suggesting that the conformation of the three carotenoids is the same in the red and in the orange form. These results are discussed in the framework of recent time-resolved IR studies on OCP., (© 2023. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)