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Dye-decolorizing peroxidases (DyPs), a type of heme-containing oxidoreductase enzymes, catalyze the peroxide-dependent oxidation of various industrial dyes as well as lignin and lignin model compounds. In our previous work, we have recently reported the crystal structures of class A-type DyP from Bacillus subtilis at pH 7.0 (BsDyP7), exposing the location of three binding sites for small substrates and high redox-potential substrates. The biochemical studies revealed the optimum acidic pH for enzyme activity. In the present study, the crystal structure of BsDyP at acidic pH (BsDyP4) reveals two-monomer units stabilized by intermolecular salt bridges and a hydrogen bond network in a homo-dimeric unit. Based on the monomeric structural comparison of BsDyP4 and BsDyP7, minor differences were observed in the loop regions, that is, LI (Ala64-Gln71), LII (Glu96-Lys108), LIII (Pro117-Leu124), and LIV (Leu295-Asp303). Despite these differences, BsDyP4 adopts similar heme architecture as well as three substrate-binding sites to BsDyP7. In BsDyP4, a shift in Asp187, heme pocket residue discloses the plausible reason for optimal acidic pH for BsDyP activity. This study provides insight into the structural changes in BsDyP at acidic pH, where BsDyP is biologically active.

Bacterioferritin comigratory protein family 1 Cys peroxiredoxin from Candidatus Liberibacter asiaticus (CLaBCP) is an important antioxidant defense protein that participates in the reduction of ROS, free radicals, and peroxides. In the present study, we report the biochemical studies and in silico screening of potent antibacterial molecules against CLaBCP. The CLaBCP showed enzymatic activity with the Km value 54.43, 94.34, 120.6 µM, and Vmax of 59.37, 69.37, 70.0 µM min−1 for H2O2, TBHP, CHP respectively. The residual peroxidase activity of CLaBCP was analyzed at different ranges of pH and temperatures. The CLaBCP showed structural changes and unfolding in the presence of its substrates and guanidinium chloride by CD and fluorescence. The structure-based drug design method was employed to screen and identify the more efficient molecule against CLaBCP. The validated CLaBCP model was used for the virtual screening of potent antibacterial molecules. The docking was performed at CLaBCP active site to evaluate the binding energy of the top five molecules (LAS 34150849, BDE 33184869, LAS 51497689, BDE 33672484, and LAS 34150966). All identified molecule has a higher binding affinity than adenanthin analyzed by molecular docking. Molecular dynamics studies such as RMSD, Rg, SASA, and PCA results showed that the CLaBCP inhibitor(s) complex is more stable than the CLaBCP-adenanthin complex. MMPBSA results suggested that the identified molecule could form a lower energy CLaBCP-inhibiter(s) complex than the CLaBCP-adenanthin complex. The screened molecules may pave the route for the development of potent antibacterial molecules against CLa. Communicated by Ramaswamy H. Sarma

Lipid nanodiscs have become the standard reconstitution system for structural and biochemical studies of membrane proteins, especially using single particle cryo-EM. We find that reconstitution of the pentameric ligand-gated ion channel (pLGIC),Erwinialigand-gated ion channel (ELIC), in different nanodisc scaffolds (MSP1E3D1, SMA, saposin, spMSP1D1) produces distinct apo and agonist-bound structures. In the presence of agonist, different nanodiscs scaffolds produce concerted conformational changes associated with activation in ELIC, with larger nanodiscs showing more activated conformations. The effect of different nanodisc scaffolds on ELIC structure extends to the extracellular domain and agonist binding site. Molecular dynamic simulations of ELIC in small and large nanodiscs suggest that the impact of the nanodisc on ELIC structure is influenced by nanodisc size. Overall, the results indicate that the nanodisc profoundly affects the structure of a pLGIC, and suggest that larger circularized nanodiscs may be advantageous to approximate a lipid membrane environment.

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The recent pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) is a viral respiratory disease that has been spread all over the globe. Therefore, it is an urgent requirement to identify and develop drugs for this contagious infection. The papain-like protease (PLpro) of SARS-CoV-2 performs critical functions in virus replication and immune evasion, making it an enticing therapeutic target. SARS-CoV-2 and SARS-CoV PLpro proteases have significant similarities, and an inhibitor discovered for SARS-CoV PLpro is an exciting first step toward therapeutic development. Here, a set of antiviral molecules were screened at the catalytic and S-binding allosteric sites of papain-like protease (PLpro). Molecular docking results suggested that five molecules (44560613, 136277567, S5652, SC75741, and S3833) had good binding affinities at both sites of PLpro. Molecular dynamics analysis like root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), and hydrogen bond results showed that identified molecules with PLpro tend to form stable PLpro-inhibitor(s) complexes. Molecular Mechanics/Position-Boltzmann Surface Area (MMPBSA) analysis confirmed that antiviral molecules bound PLpro complex had lower energy (-184.72 ± 7.81 to -215.67 ± 6.73 kJ/mol) complexes. Noticeably, computational approaches revealed promising antivirals candidates for PLpro, which may be further tested by biochemical and cell-based assays to assess their potential for SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

FmtA is a novel esterase that shares the penicillin-binding protein (PBP) core structural folding but found to hydrolyze the removal of d-Ala from teichoic acids. Molecular docking, dynamics, and MM-GBSA of FmtA and its variants S127A, K130A, Y211A, D213A, and K130AY211A, in the presence or absence of wall teichoic acid (WTA), suggest that active site residues S127, K130, Y211, D213, N343, and G344 play a role in substrate binding. Quantum mechanics (QM)/molecular mechanics (MM) calculations reveal that during WTA catalysis, K130 deprotonates S127, and the nucleophilic S127 attacks the carbonyl carbon of d-Ala bound to WTA. The tetrahedral intermediate (TI) complex is stabilized by hydrogen bonding to the oxyanion holes. The TI complex displays a high energy gap and collapses to an energetically favorable acyl-enzyme complex.

In S. aureus, lipophilic membrane (LLM) protein is a methicillin resistance factor and is an essential role in peptidoglycan metabolism. The virtual screening of antibacterial molecules against the model of LLM was performed to identify the potent antibacterial molecules. Molecular docking results of pharmacokinetic filtered molecules illustrated that five molecules had higher binding affinities than tunicamycin (TUM) and were stabled via non-covalent interactions (hydrogen bond and hydrophobic interactions) at the active site of LLM. Further, molecular dynamics results revealed that binding of identified antibacterial molecules with LLM resulted in stable LLM-inhibitor(s) complexes. Molecular Mechanics/Position-Boltzmann Surface Area (MMPBSA) analysis showed that LLM-inhibitor(s) complexes had high binding affinities in the range of −213.49 ± 2.24 to −227.42 ± 3.05 kJ/mol. The amino acid residues decomposition analysis confirmed that identified antibacterial molecules bound at the active site (Asn148, Leu149, Asp151, Asp208, His269, His271, and His272) of LLM. Noticeably, the current study found five antibacterial molecules (BDE 27575101, BDE 33638168, BDE 33672484, LAS 51502073, and BDE 25098678) were highly potent than TUM and even than earlier reported molecules. Therefore, here reported antibacterial molecules may be used directly or developed to inhibit LLM of S. aureus. Communicated by Ramaswamy H. Sarma

The detrimental effect of coronavirus disease 2019 (COVID-19) pandemic has manifested itself as a global crisis. Currently, no specific treatment options are available for COVID-19, so therapeutic interventions to tackle the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection must be urgently established. Therefore, cohesive and multidimensional efforts are required to identify new therapies or investigate the efficacy of small molecules and existing drugs against SARS-CoV-2. Since the RNA-dependent RNA Polymerase (RdRP) of SARS-CoV-2 is a promising therapeutic target, this study addresses the identification of antiviral molecules that can specifically target SARS-CoV-2 RdRP. The computational approach of drug development was used to screen the antiviral molecules from two antiviral libraries (Life Chemicals [LC] and ASINEX) against RdRP. Here, we report six antiviral molecules (F3407-4105, F6523-2250, F6559-0746 from LC and BDG 33693278, BDG 33693315, LAS 34156196 from ASINEX), which show substantial interactions with key amino acid residues of the active site of SARS-CoV-2 RdRP and exhibit higher binding affinity (>7.5 kcalmol(−1)) than Galidesivir, an Food and Drug Administration-approved inhibitor of the same. Further, molecular dynamics simulation and Molecular Mechanics Poisson-Boltzmann Surface Area results confirmed that identified molecules with RdRP formed higher stable RdRP-inhibitor(s) complex than RdRP-Galidesvir complex. Our findings suggest that these molecules could be potential inhibitors of SARS-CoV-2 RdRP. However, further in vitro and preclinical experiments would be required to validate these potential inhibitors of SARS-CoV-2 protein.

One of the major constraints limiting the use of abundantly available lignocellulosic biomass as potential feedstock for alcohol industry is the lack of C

Klebsiella pneumonia is known to cause several nosocomial infections in immunocompromised patients. It has developed resistance against a broad range of presently available antibiotics, resulting in high mortality rates in patients and declared an urgent threat. Therefore, exploration of possible novel drug targets against this opportunistic bacteria needs to be undertaken. In the present study, we performed an extensive in-silico analysis for functional and structural annotation and characterized HP CP995_08280 from K. pneumonia as a drug target and aimed to identify potent drug candidates. The functional and structural studies using several bioinformatics tools and databases predicted that HP CP995_08280 is a cytosolic protein that belongs to the β-lactamase family and shares structural similarity with FmtA protein from Staphylococcus aureus (PDB ID: 5ZH8). The structure of HP CP995_08280 was successfully modeled followed by structure-based virtual screening, docking, molecular dynamics, and Molecular mechanic/Poisson-Boltzmann surface area (MMPBSA) were performed to identify the potential compounds. We have found five potent antibacterial molecules, namely BDD 24083171, BDD 24085737, BDE 25098678, BDE 33638819, and BDE 33672484, which exhibited high binding affinity (-7.5 kcal/mol) and were stabilized by hydrogen bonding and hydrophobic interactions with active site residues (Ser42, Lys45, Tyr126, and Asp128) of protein. Molecular dynamics and MMPBSA revealed that HP CP995_08280 - ligand(s) complexes were less dynamic and more stable than native HP CP995_08280. Hence, the present study may serve as a potential lead for developing inhibitors against drug-resistant Klebsiella pneumonia.

Huanglongbing (HLB) is a worldwide citrus plant disease-related to non-culturable and fastidious α-proteobacteria

One of the major constraints limiting the use of abundantly available lignocellulosic biomass as potential feedstock for alcohol industry is the lack of C6/C5 co-sugar fermenting yeast. The present study explores a mutant yeast Pichia kudriavzevii BGY1-γm as a potential strain for bioconversion of glucose/xylose sugars of green biomass into ethanol under batch fermentation. The mutant strain having higher alcohol dehydrogenase activity (11.31%) showed significantly higher ethanol concentration during co-fermentation of glucose/xylose sugars (14.2%) as compared to the native strain. Based on 99% sequence similarity of ADH encoding gene from the mutant with the gene sequences from other yeast strains, the ADH enzyme was identified as ADH-1 type. The study reveals first three-dimensional model of ADH-1 utilizing glucose/xylose sugars from P. kudriavzevii BGY1-γm (PkADH mutant). The refined and validated model of PkADH mutant was used for molecular docking against the substrate (acetaldehyde) and product (ethanol). Molecular docking results showed that substrate and product exhibited a binding affinity of −4.55 and −4.5 kcal/mol with PkADH mutant. Acetaldehyde and ethanol interacted at the active site of PkADH mutant via hydrogen bonds (Ser42, His69 and Asp163) and hydrophobic interactions (Cys40, Ser42, His69, Cys95, Trp123 and Asp163) to form the stable protein–ligand complex. Molecular dynamics analysis revealed that PkADH-mutant acetaldehyde and PkADH-mutant ethanol complexes were more stable than PkADH mutant. MMPBSA binding energy confirmed that binding of substrate and product results in the formation of a lower energy stable protein–ligand complex. Communicated by Ramaswamy H. Sarma

In S. aureus, ribosome biogenesis GTP-binding (YsxC), a GTPase interacts with 50S subunit and 30S subunit of ribosome, and β’ subunit of RNA polymerase and played an important role in protein synthesis. For the identification of potent lead molecules, we have conducted pharmacophore modeling by consideration of pharmacophore features of GTP among YsxC-GTP complex. Virtual screening and molecular docking results displayed that five pharmacokinetic and ADMET filtered molecules—ZINC000006424138, ZINC000095502032, ZINC000225415132, ZINC000095475800, and ZINC000012990761—had higher binding affinities than GTP with YsxC. All the identified molecules shared similar pharmacophore features of GTP and were stabilized via hydrogen bonds and hydrophobic interactions with YsxC. Molecular dynamics analysis revealed that YsxC-inhibitor(s) complexes were lesser dynamics and higher stable than YsxC-GTP complex. Molecular Mechanics/Poisson-Boltzmann Surface Area (MMPBSA) results confirmed that identified molecules bound at the active site (Arg33, Ser34, Asn35, Val36, Lys38, Ser39, Thr40, Thr54, Ser55, Pro58, Lys60, Thr61, Thr144, Lys145, Ser178, and Ile179) of YsxC and formed the lower energy (–190.32 ± 3.46 to –217.03 ± 2.55 kJ/mol) complexes than YsxC-GTP (–157.16 ± 2.89 kJ/mol) complex. The identified molecules in this study can be further tested and utilized to design novel antimicrobial agents for S. aureus. Communicated by Ramaswamy H. Sarma

Staphylococcus aureus is resistant to β-lactam antibiotics and causes several skin diseases to life-threatening diseases. FmtA is found to be one of the main factors involved in methicillin resistance in S. aureus. FmtA exhibits an esterase activity that removes the D-Ala from teichoic acid. Teichoic acids played a significant role in cell wall synthesis, cell division, colonization, biofilm formation, virulence, antibiotic resistance, and pathogenesis. The virtual screening of drug molecules against the crystal structure of FmtA was performed and the binding affinities of top three molecules (ofloxacin, roflumilast, and furazolidone) were predicted using molecular docking. The presence of positive potential and electron affinity regions in screened drug molecules by DFT analysis illustrated that these molecules are reactive in nature. The protein-ligand complexes were subjected to molecular dynamics simulation. Molecular dynamics analysis such as RMSD, RMSF, Rg, SASA, PCA, and FEL results suggested that FmtA-drug(s) complexes are stable. MM-GBSA binding affinity and QM/MM results (ΔG, ΔH, and ΔS) revealed that active site residues (Ser127, Lys130, Tyr211, Asp213, and Asn343) of FmtA played an essential for the binding of the drug(s) to form a lower energy stable protein-ligand complexes. FmtAΔ42 was purified using cation exchange and gel filtration chromatography. Fluorescence spectroscopy and circular dichroism results showed that interactions of drugs with FmtAΔ42 affect the tertiary structure and increase the thermostability of the protein. The screened molecules need to be tested and could be further modified to develop the antimicrobial compounds against S. aureus.

The Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 virus has raised severe health problems in china and across the world as well. CoVs encode the nucleocapsid protein (N-protein), an essential RNA-binding protein that performs different roles throughout the virus replication cycle and forms the ribonucleoprotein complex with viral RNA using the N-terminal domain (NTD) of N-protein. Recent studies have shown that NTD-N-protein is a legitimate target for the development of antiviral drugs against human CoVs. Owing to the importance of NTD, the present study focuses on targeting the NTD-N-protein from SARS-CoV-2 to identify the potential compounds. The pharmacophore model has been developed based on the guanosine monophosphate (GMP), a RNA substrate and further pharmacophore-based virtual screening was performed against ZINC database. The screened compounds were filtered by analysing the in silico ADMET properties and drug-like properties. The pharmacokinetically screened compounds (ZINC000257324845, ZINC000005169973, and ZINC000009913056) were further scrutinized through computational approaches including molecular docking and molecular dynamics simulations and revealed that these compounds exhibited good binding affinity as compared to GMP and provide stability to their respective complex with the NTD. Our findings could disrupt the binding of viral RNA to NTD, which may inhibit the essential functions of NTD. These findings may further provide an impetus to develop the novel and potential inhibitor against SARS-CoV-2.

FmtA is a penicillin-recognizing protein (PRP) with novel hydrolytic activity toward the ester bond between d -Ala and the backbone of teichoic acids. Teichoic acids are polyol-phosphate polymers found in the Staphylococcus aureus cell wall, and they play important roles in antibiotic resistance and pathogenesis. Two of the PRPs conserved motifs, namely, SXXK and Y(S)XN, are involved in the hydrolysis by FmtA, but the catalytic mechanism remains elusive. Here we determined the crystal structure of FmtA. FmtA shares the core structure of PRPs: an all α-helical domain and α/β domain sandwiched together. However, it does not have the typical PRPs active-site cleft. Its active site is shallow, solvent-exposed, and enlarged. Furthermore, our mutagenesis and kinetic studies suggest that the SXXK and Y(S)XN motifs of FmtA offer all that is necessary for catalysis, and more: the active-site nucleophile (serine), the general base (lysine) required for the acylation step and the deacylation step, and an anchor (tyrosine) to hold the active-site serine, and possibly the substrate, in an optimum conformation for catalysis. Our study establishes that the FmtA esterase activity represents an expansion of the catalytic activity repertoire of the PRPs core structure, which typically displays peptidase activity. This finding points toward a novel mechanism of ester bond hydrolysis by a PRP. The structure of FmtA provides insights to the design of inhibitor molecules with the potential to serve as leads in the development of novel antibacterial chemotherapeutic agents.

Arginase, the binuclear metalloenzyme, is a potential target for therapeutic intervention in protozoan infections. Entamoeba histolytica infection causes amebiasis which is the second most common cause of protozoan-related human deaths after malaria. Here, we report the crystal structure of E. histolytica arginase (EhArg) in complex with two known inhibitors N

Integrated analytical and experimental research, conducted for exploring objective, global condition assessment and evaluation of bridge serviceability and safety, is summarized. A related terminology with definitions for critical terms is offered. Nondestructive and destructive field testing and structural identification of seven actual bridge test specimens enabled formulating a bridge condition assessment and reliability evaluation methodology. Field-calibrated analytical models, developed by structural identification, are studied to understand the real behavior and performance of actual bridges and to quantify relative contributions of different stiffness and resistance mechanisms. While our heuristic knowledge base has served us well, further rationalizing our bridge engineering practice is needed to achieve the optimum management of the highway transportation infrastructure necessary for continued advances in the next century.

Background/Aims: Short-chain fatty acids (SCFAs) provide energy for colonocytes and stimulate colonic fluid and electrolyte absorption. The impact of acute colitis on SCFA-stimulated Na + absorption and SCFA absorption was examined. Methods: Proximal colon from rabbits infected with Yersinia entercolitica , a pair-fed group, and controls was mounted in Ussing chambers, and Na + transport, short-circuit current, and tissue conductance were examined during a basal period and after stimulation with the SCFAs, butyrate, or propionate. Propionate transport and luminal SCFA concentrations were evaluated. Results: Butyrate and propionate stimulated electroneutral Na + absorption above basal levels in the control and pair-fed groups, as evidenced by significant increases in mucosal-to-serosal and net Na + fluxes with no change in serosal-to-mucosal flux, short-circuit current, or conductance. Butyrate-stimulated Na + absorption and propionate absorption were blocked by amiloride, an inhibitor of Na + -H + exchange. In the infected group, both butyrate and propionate failed to stimulate colonic Na + absorption above basal levels. Propionate absorption was inhibited, and epinephrine failed to stimulate Na + or propionate absorption. Luminal SCFA concentrations were increased in acute colitis. Conclusions: Inhibition of SCFA-stimulated Na + -H + exchange and SCFA absorption contribute to the diarrheal fluid loses observed in acute colitis and may reduce colonocyte energy supply.

We describe a new technique, Reactive Plasma Beam Deposition, for growing a‐Silicon films at higher temperatures. The technique uses a reactive beam of H atoms and ions, produced using an Electron Cyclotron Resonance (ECR) plasma source, to decompose SiH4 and deposit films and devices. We find that the quality of the films depends critically upon the flux of H radicals reaching the substrate. Control over this flux by allows us to grow high quality films even at temperatures of 400–450C. We find that these films are more stable than comparable quality films deposited by glow discharge at lower temperatures, presumably due to a better local microstructure created by higher growth temperatures. The films have very good electronic properties. We have also made preliminary p‐i‐n superstrate devices in these materials. The quantum efficiency data indicate that the hole mu‐Tau products in these materials are quite good, 3–4×10−8 cm2/sec.