Catalytic Triad Residues Research Articles

Molecular characterization of transesterification activity of novel lipase family I.1.

Lipase’s thermostability and organic solvent tolerance are two crucial properties that enable it to function as a biocatalyst. The present study examined the characteristics of two recombinant thermostable lipases (Lk2, Lk3) based on transesterification activity. Conversion of C12-C18 methyl ester with paranitrophenol was investigated in various organic solvent. Both lipases exhibited activity on difference carbon chain length (C12 - C18, C18:1, C18:2) of substrates. The activity of Lk2 was higher in each of substrate compared with that of Lk3. Experimental findings showed that the best substrates for Lk2 and Lk3 are C18:1 and C18:2 respectively, in agreement with the computational analysis. The activity of both enzymes prefers on nonpolar solvent. On nonpolar solvent the enzymes are able to keep its native folding shown by the value of radius gyration, solvent–enzyme interaction and orientation of triad catalytic residues. Lk3 appeared to be more thermostable, with maximum activity at 55°C. The presence of Fe3+ increased the activity of Lk2 and Lk3. However, the activity of both enzymes were dramatically decreased by the present of Ca2+ despite of the enzymes belong to family I.1 lipase known as calcium dependent enzyme. Molecular analysis on His loop of Lk2 and Lk3 on the present of Ca2+ showed that there were shifting on the orientation of catalytic triad residues. All the data suggest that Lk2 and Lk3 are novel lipase on the family I.1 and both lipase available as a biocatalyst candidate.

Structural and biochemical analysis of the GDSL-family esterase CJ0610C from Campylobacter jejuni

GDSL domain-containing proteins generally hydrolyze esters or lipids and play critical roles in diverse biological and industrial processes. GDSL hydrolases use catalytic triad and oxyanion hole residues from conserved blocks I, II, III, and V to drive the esterase reaction. However, GDSL hydrolases exhibit large deviations in sequence, structure, and substrate specificity, requiring the characterization of each GDSL hydrolase to reveal its catalytic mechanism. We identified a GDSL protein (CJ0610C) from pathogenic Campylobacter jejuni and assessed its biochemical and structural features. CJ0610C displayed esterase activity for p-nitrophenyl acetate and preferred short chain esters and alkaline pH. The C-terminal two-thirds of CJ0610C corresponding to the GDSL domain forms a three-layered α/β/α fold as a core structure in which a five-stranded β-sheet is sandwiched by α-helices. In the CJ0610C structure, conserved catalytic triad and oxyanion hole residues that are indispensable for esterase activity are found in blocks I, III, and V. However, CJ0610C lacks the conserved block-II glycine residue and instead employs a unique asparagine residue as another oxyanion hole residue. Moreover, our structural analysis suggests that substrate binding is mediated by a CJ0610C-specific pocket, which is surrounded by hydrophobic residues and occluded at one end by a positively charged arginine residue.

Consensus docking and MM-PBSA computations identify putative furin protease inhibitors for developing potential therapeutics against COVID-19.

The coronavirus disease 2019 (COVID-19) is a pandemic that has severely posed substantial health challenges and claimed millions of lives. Though vaccines have been produced to stem the spread of this disease, the death rate remains high since drugs used for treatment have therapeutic challenges. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes the disease, has a slew of potential therapeutic targets. Among them is the furin protease, which has a cleavage site on the virus’s spike protein. The cleavage site facilitates the entry of the virus into human cells via cell–cell fusion. This critical involvement of furin in the disease pathogenicity has made it a viable therapeutic strategy against the virus. This study employs the consensus docking approach using HYBRID and AutoDock Vina to virtually screen a pre-filtered library of 3942 natural product compounds of African origin against the human furin protease (PDB: 4RYD). Twenty of these compounds were selected as hits after meeting molecular docking cut-off of − 7 kcal.mol−1, pose alignment inspection, and having favorable furin-ligand interactions. An area under the curve (AUC) value of 0.72 was computed from the receiver operator characteristic (ROC) curve, and Boltzmann-enhanced discrimination of the ROC curve (BEDROC) value of 0.65 showed that AutoDock Vina was a reasonable tool for selecting actives for this target. Seven of these hits were proposed as potential leads having had bonding interactions with catalytic triad residues Ser368, His194, and Asp153, and other essential residues in the active site with plausible binding free energies between − 189 and − 95 kJ/mol from the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations as well as favorable ADME/Tox properties. The molecules were also predicted as antiviral, anti-inflammatory, membrane permeability inhibitors, RNA synthesis inhibitors, cytoprotective, and hepatoprotective with probable activity (Pa) above 0.5 and probable inactivity values below 0.1. Some of them also have anti-influenza activity. Influenza virus has many similarities with SARS-CoV-2 in their mode of entry into human cells as both are facilitated by the furin protease. Pinobanksin 3-(E)-caffeate, one of the potential leads is a propolis compound. Propolis compounds have shown inhibitory effects against ACE2, TMPRSS2, and PAK1 signaling pathways of SARS-CoV-2 in previous studies. Likewise, quercitrin is structurally similar to isoquercetin, which is currently in clinical trials as possible medication for COVID-19.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11224-022-02056-1.

Elucidation of the Conformational Transition of Oligopeptidase B by an Integrative Approach Based on the Combination of X-ray, SAXS, and Essential Dynamics Sampling Simulation

Oligopeptidase B (OPB) is the least studied group from the prolyl oligopeptidase family. OPBs are found in bacteria and parasitic protozoa and represent pathogenesis factors of the corresponding infections. OPBs consist of two domains connected by a hinge region and have the characteristics of conformational dynamics, which include two types of movements: the bridging/separation of α/β-hydrolase catalytic and β-propeller-regulatory domains and the movement of a loop carrying catalytic histidine, which regulates an assembly/disassembly of the catalytic triad. In this work, an elucidation of the interdomain dynamics of OPB from Serratia proteamaculans (SpOPB) with and without modification of the hinge region was performed using a combination of X-ray diffraction analysis and small-angle X-ray scattering, which was complemented with an essential dynamics sampling (EDS) simulation. The first crystal structure of catalytically deficient SpOPB (SpOPBS532A) with an intact hinge sequence is reported. Similarly to SpOPB with modified hinges, SpOPBS532A was crystallized in the presence of spermine and adopted an intermediate conformation in the crystal lattice. Despite the similarity of the crystal structures, a difference in the catalytic triad residue arrangement was detected, which explained the inhibitory effect of the hinge modification. The SpOPBS532A structure reconstituted to the wild-type form was used as a starting point to the classical MD followed by EDS simulation, which allowed us to simulate the domain separation and the transition of the enzyme from the intermediate to open conformation. The obtained open state model was in good agreement with the experimental SAXS data.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

SUMMARYSerine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

In silico analysis of nitrilase-3 protein from Corynebacterium glutamicum for bioremediation of nitrile herbicides

BackgroundThe nitrile compounds are produced either naturally or synthetically and are highly used in many manufacturing industries such as pharmaceuticals, pesticides, chemicals, and polymers. However, the extensive use and accumulation of these nitrile compounds have caused severe environmental pollution. Nitrilated herbicides are one such toxic substance that will persist in the soil for a long time. Therefore, effective measures must be taken to avoid its pollution to the environment. A variety of nitrile-converting bacterial species have the ability to convert these toxic substances into less toxic ones by using enzymatic processes. Among the bacterial groups, actinobacteria family members show good degradation capacity on these pollutants. The soil-dwelling Gram-positive industrial microbe Corynebacterium glutamicum is one such family member and its nitrile-degradation pathway is not well studied yet. In order to understand the effectiveness of using C. glutamicum for the degradation of such nitrile herbicides, an in silico approach has been done. In this perspective, this work focus on the structural analysis and molecular docking studies of C. glutamicum with nitrilated herbicides such as dichlobenil, bromoxynil, and chloroxynil. ResultsThe bioinformatics analysis using different tools and software helped to confirm that the genome of C. glutamicum ATCC 13032 species have genes (cg 3093) codes for carbon-nitrogen hydrolase enzyme, which specifically act on non-peptide bond present in the nitrile compounds. The conserved domain analysis indicated that this protein sequence was nitrilase-3 and comes under the nitrilase superfamily. The multiple sequence alignment analysis confirmed that the conserved catalytic triad residues were 40E, 115K, and 151C, and the existence of nitrilase-3 protein in the genome of Corynebacterium sp. was evaluated by a phylogenetic tree. The analysis of physico-chemical properties revealed that alanine is the most abounded amino acid (10.20%) in the nitrilase-3 protein, and these properties influence the substrate specificity of aliphatic and aromatic nitrile compounds. The homology modelled protein showed better affinity towards nitrile herbicides such as 2,6-dichlorobenzamide (BAM) and 3,5-dichloro-4-hydroxy-benzamide (CIAM) with the affinity value of − 5.8 and − 5.7 kcal/mol respectively. ConclusionsThe in silico studies manifested that C. glutamicum ATCC 13032 is one of the promising strains for the bioremediation of nitrilated herbicides contaminated soil.

Structure and Function Insight of the α-Glucosidase QsGH13 From Qipengyuania seohaensis sp. SW-135.

The α-glucosidases play indispensable roles in the metabolic mechanism of organism, prevention, and treatment of the disease, and sugar hydrolysis, and are widely used in chemical synthesis, clinical diagnosis, and other fields. However, improving their catalytic efficiency and production to meet commercial demand remains a huge challenge. Here we detected a novel GH13 family α-glucosidase, QsGH13, from the deep-sea bacterium Qipengyuania seohaensis sp. SW-135. QsGH13 is highly substrate specific and only hydrolyzes sugars containing alpha-1,4 glucoside bonds. For example, its enzymatic activity for p-nitrophenyl-α-D-glucopyranoside was 25.41 U/mg, and the Km value was 0.2952 ± 0.0322 mM. The biochemical results showed that the optimum temperature of QsGH13 is 45°C, the optimum pH is 10.0, and it has excellent biological characteristics such as alkali resistance and salt resistance. The crystal structure of QsGH13 was resolved with a resolution of 2.2 Å, where QsGH13 is composed of a typical TIM barrel catalytic domain A, a loop-rich domain B, and a conserved domain C. QsGH13 crystal belonged to the monoclinic space group P212121, with unit-cell parameters a = 58.816 Å, b = 129.920 Å, c = 161.307 Å, α = γ = β = 90°, which contains two monomers per asymmetric unit. The β → α loop 4 of QsGH13 was located above catalytic pocket. Typical catalytic triad residues Glu202, Asp266, and Glu329 were found in QsGH13. The biochemical properties and structural analysis of QsGH13 have greatly improved our understanding of the catalytic mechanism of GH13 family. This study provides new ideas to broaden the application of α-glucosidase in alcohol fermentation, glycolysis, and other industries.

Critical residues for proteolysis activity of maize chlorotic dwarf virus (MCDV) 3C-like protease and comparison of activity of orthologous waikavirus proteases

Maize chlorotic dwarf virus (MCDV) encodes a 3C-like protease that cleaves the N-terminal polyprotein (R78) as previously demonstrated. Here, we examined amino acid residues required for catalytic activity of the protease, including those in the predicted catalytic triad, amino acid residues H2667, D2704, and C2798, as well as H2817 hypothesized to be important in substrate binding. These and other residues were targeted for mutagenesis and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that abolished catalytic activity. Mutations that altered the predicted catalytic triad residues and H2817 disrupted MCDV-S protease activity, as did mutagenesis of a conserved tyrosine residue, Y2774. The protease activity and R78 cleavage of orthologs from divergent MCDV isolates MCDV-Tn and MCDV-M1, and other waikavirus species including rice tungro spherical virus (RTSV) and bellflower vein chlorosis virus (BVCV) were also examined.

Relaxed complex scheme and molecular dynamics simulation suggests small molecule inhibitor of human TMPRSS2 for combating COVID-19

As the coronavirus disease 19 (COVID-19) pandemic continues to pose a health and economic crisis worldwide, the quest for drugs and/or vaccines against the virus continues. The human transmembrane protease serine 2 (TMPRSS2) has attracted attention as a target for drug discovery, as inhibition of its catalytic reaction would result in the inactivation of the proteolytic cleavage of the SARS-CoV-2 S protein. As a result, the inactivation prevents viral cell entry to the host’s cell. In this work, we screened and identified two potent molecules that interact and inhibit the catalytic reaction by using computational approaches. Two docking screening experiments were performed utilizing the crystal structure and holo ensemble structure obtained from molecular dynamics in bound form. There is enhancement and sensitivity of docking results to the holo ensemble as compared to the crystal structure. Compound 1 demonstrated a similar inhibition value to nafamostat by interacting with catalytic triad residues His296 and Ser441, thereby disrupting the already established hydrogen bond interaction. The stability of the ligand–TMPRSS2 complexes was studied by molecular dynamics simulation, and the binding energy was re-scored by using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy. The obtained compounds may serve as an initial point toward the discovery of potent TMPRSS2 inhibitors upon further in vivo validation. Communicated by Ramaswamy H. Sarma

Human Prothrombin Variants with Modifications in the Active Site S4 Subpocket Demonstrate Resistance to Dabigatran

Chymotrypsin-like serine proteases are hallmarked by a protease domain comprising the catalytic triad residues His57, Asp102, and Ser195 (chymotrypsinogen numbering) situated in the active site cleft. While the catalytic triad in conjunction with the oxyanion hole residues regulate substrate cleavage, the active site subpockets (S1-4) control substrate recognition and binding. The high structural homology of the serine protease domains allows for analogous strategies in drug design, which is underscored by the direct oral anticoagulants (DOACs) for the prophylactic management of stroke in atrial fibrillation and prevention and treatment of venous thrombosis. DOACs inhibit coagulation serine proteases by reversibly engaging the active site with high affinity. To expand the repertoire of DOAC-specific reversal agents we have previously successfully modified the S4 active site subpocket of human factor Xa to prevent DOAC binding while preserving catalytic activity [Verhoef 2017 Nature Commun.]. To explore whether an analogous strategy can be applied to create DOAC resistance in the serine protease thrombin, specific substitutions or sequences in or around the dabigatran-binding S4 subsite derived from naturally occurring serine proteases or plasma proteins were introduced in prothrombin.A panel of prothrombin variants was generated and transfected into HEK293 cells to allow for stable protein expression. In some of the generated prothrombin variants comprising insertions in amino acid sequence 91-99 that is directly adjacent to the S4 subsite protein expression was severely impaired. This indicates that exchange of any surface-exposed serine protease or plasma protein region into the prothrombin protease domain is not necessarily compatible with protein expression. In contrast, exchange of the human prothrombin 91-99 sequence for that of human kallikrein 3 or targeted amino acid replacement of S4 subsite residue Ile174 resulted in prothrombin protein expression levels similar to wild-type prothrombin.Following expression, prothrombin variants were purified to homogeneity using the CaptureSelect tm affinity matrix that selects for fully gamma-carboxylated prothrombin. The specific prothrombin clotting activity analyses of the purified prothrombin variants KL3 (0.7 ± 0.2 U/mg), I174A (0.8 ± 0.2 U/mg), and I174F (0.8 ± 0.3 U/mg) demonstrated an overall ~10-fold reduced specific activity relative to wild-type prothrombin (7.5 ± 0.1 U/mg). As such, modification of the S4 subsite likely interferes with the binding and subsequent conversion of fibrinogen by thrombin. To determine whether the prothrombin variants supported tissue factor-initiated thrombin formation in human plasma, prothrombin-deficient plasma was supplemented with increasing plasma concentrations of prothrombin variant (90-180 ug/mL). Consistent with their reduced specific clotting activity, 180 ug/mL prothrombin variant was required to obtain substantial thrombin generation but with reduced thrombin generation parameters (peak thrombin, ETP) relative to supplementation with plasma concentrations of wild-type prothrombin (90 ug/mL). This calibrated automated thrombin generation assay set-up was used to assess the DOAC-resistance of the prothrombin variants. While thrombin formation reached half-maximum inhibition at 532 ± 58 nM dabigatran in wild-type prothrombin-supplemented plasma, addition of the prothrombin variants displayed a ~2-fold reduced sensitivity to dabigatran inhibition (IC50: 1186 ± 136 nM prothrombin-KL3; 851 ± 97 nM prothrombin-I174F; 772 ± 80 nM prothrombin-I174A). This demonstrates that the S4 subsite-modified prothrombin variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor.Collectively, our findings indicate that human prothrombin variants comprising a single point mutation at position Ile174 in the S4 subsite or at a region directly adjacent to the S4 subsite are able to generate thrombin in plasma inhibited by dabigatran. Hence, serine proteases with S4 subpocket modifications have the potential to bypass DOAC therapy and could provide a generic strategy in the development of novel DOAC-bypassing agents. [Display omitted] DisclosuresReitsma: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Verhoef: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company. Bos: VarmX B.V.: Research Funding; uniQure Biopharma B.V.: Research Funding.

Crystal structure of aspartyl dipeptidase from Xenopus laevis revealed ligand binding induced loop ordering and catalytic triad assembly.

Gene encoding aspartyl dipeptidase from Xenopus levies (PepExl) is upregulated by thyroid hormone and is proposed to play a significant role in resorption of tadpole tail during metamorphosis. However, the importance of peptidase activity for the resorption of the tail remain elusive. Here we report the crystal structures of first eukaryotic S51 peptidase, PepExl, in its ligand-free and Asp-bound states at 1.4 and 1.8Å resolutions, respectively. The active site is located at dimeric interface and the catalytic triad is found to be dissembled in ligand-free and assembled in Asp-bound state. Structural comparison and molecular dynamic simulations of ligand-free and Asp-bound states shows that distinct loop (loop-A) plays an important role in active site shielding, substrate binding and enzyme activation. This study illuminates the Asp-X dipeptide binding in PepExl is associated with ordering of the loop-A and assembly of residues of catalytic triad in active conformation for enzymatic activity.

In silico study on spice-derived antiviral phytochemicals against SARS-CoV-2 TMPRSS2 target

Corona Virus Disease (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a pandemic that has claimed so far over half a million human life across the globe. Researchers all over the world are exploring various molecules including phytochemicals to get a potential anti-COVID-19 drug. Certain phytochemicals present in some spices are claimed to possess antiviral, anti-bacterial, and anti-fungal properties. Hence, an in-silico study was done by selecting eighteen well reported antiviral phytochemicals from some spices commonly used in Indian kitchen viz. Curcuma longa (Turmeric), Nigella sativa (Black cumin), Piper nigrum (Black pepper), Trachyspermum ammi (Carom) and Zingiber officinale (Ginger) to find out whether they can prevent SARS-CoV-2 infection. Firstly, we predicted the structure of TMPRSS2 (transmembrane protease serine 2), a host protein that truncates spike protein of SARS-CoV-2 thereby facilitating its endocytosis, and then docked against its catalytic domain the selected phytochemicals and camostat (a well-known synthetic inhibitor of TMPRSS2). Thereafter, stability of seven best docked phytochemicals and camostat were scrutinized by Molecular Dynamic Simulation (MDS). MDS analysis indicated bisdemethoxycurcumin (BDMC), carvacrol and thymol as better inhibitors than the camostat due to their stable binding with TMPRSS2 in its oxyanion hole and inducing subtle modification in the spatial arrangement of the catalytic triad residues. Among these three phytochemicals, carvacrol appeared to be the best inhibitor, followed by BDMC, whereas thymol was least effective.

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library

A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short-chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17mM and 1910s-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Structural modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking revealed the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

Catalytically active peptides affected by self-assembly and residues order

Amphiphilic peptides that induce catalysis are interesting alternatives to natural enzymes thanks to robustness of their synthesis and the ability to induce certain types of conformations by specific motifs of amino acid sequences. Various studies aimed at mimicking the activity of serine proteases by designed peptides. Here we demonstrate that the order by which the catalytic triad residues are positioned along amphiphilic β-strands influences both assembly structures and catalytic activity. A set of three β-sheet amphiphilic peptides, decorated with different orders of the catalytic triad amino acids, Glu, His and Ser along the strands were evaluated for their catalytic hydrolysis efficiency of p-nitrophenyl acetate (pNPA) substrate. Among the three peptides, Ac-Cys-Phe-Glu-Phe-Ser-Phe-His-Phe-Pro-NH2 (ESH) achieved the greatest catalytic efficiency with a value of 0.19 M−1 s−1, at peptide concentration of 250 μM. This study sheds light on an overlooked factor in designing catalytic amphiphilic assemblies whereby charged residues that make up the active sites, are in fact engaged in intermolecular stabilizing interactions that in turn may hamper their catalytic action.

Discovery of Kasugamycin as a Potent Inhibitor of Glycoside Hydrolase Family 18 Chitinases.

Kasugamycin, a well-known aminoglycoside antibiotic, has been used widely in agriculture and medicine to combat microbial pathogens by binding the ribosome to inhibit translation. Here, kasugamycin was discovered to be a competitive inhibitor of glycoside hydrolase family 18 (GH18) chitinases from three different organisms (bacterium, insect and human). Results from tryptophan fluorescence spectroscopy and molecular docking revealed that kasugamycin binds to the substrate-binding clefts in a similar mode as the substrate. An electrostatic interaction between the amino group of kasugamycin and the carboxyl group of a conserved aspartate in GH18 chitinase (one of the catalytic triad residues) was found to be vital for the inhibitory activity. This paper not only reports new molecular targets of kasugamycin, but also expands our thinking about GH inhibitor design by using a scaffold unrelated to the substrate.

Acyldepsipeptide activated ClpP1P2 macromolecule of Leptospira, an ideal Achilles’ heel to hamper the cell survival and deregulate ClpP proteolytic activity

Antibiotic acyldepsipeptide (ADEP) targets the bacterial ClpP serine protease and can inhibit the growth of numerous bacterial species by activating/dysregulating the protease activity within the cell. The spirochete Leptospira interrogans harbors two ClpP isoforms (LepClpP1 and LepClpP2). Supplementation of ADEP in the Leptospira growth medium resulted in the inhibition of bacterial growth. The ADEP mediated activation of the LepClpP mixture was dependent on the time allowed for the self-assembly of LepClpP1 and LepClpP2. The dynamic light scattering of the LepClpP mixture in the presence of the ADEP indicated a conformational transformation of the LepClpP machinery. Serine 98, a catalytic triad residue of the LepClpP1 in the LepClpP1P2 heterocomplex, was critical for the ADEP mediated activation. The computational prototype of the LepClpP1P2 structure suggested that the hydrophobic pockets wherein the ADEPs or the physiological chaperone ClpX predominantly dock are exclusively present in the LepClpP2 heptamer. Using the ADEP as a tool, this investigation provides an insight into the molecular function of the LepClpP1P2 in a coalition with its ATPase chaperone LepClpX. The shreds of the evidence illustrated in this investigation verify that ADEP1 possesses the ability to control the LepClpP system in an unconventional approach than the other organisms.

Escherichia coli FabG 3-ketoacyl-ACP reductase proteins lacking the assigned catalytic triad residues are active enzymes

The FabG 3-ketoacyl-acyl carrier protein (ACP) reductase of Escherichia coli has long been thought to be a classical member of the short-chain alcohol dehydrogenase/reductase (SDR) family. FabG catalyzes the essential 3-ketoacyl-ACP reduction step in the FAS II fatty acid synthesis pathway. Site-directed mutagenesis studies of several other SDR enzymes has identified three highly conserved amino acid residues, Ser, Tyr, and Lys, as the catalytic triad. Structural analyses of E. coli FabG suggested the triad S138-Y151-K155 to form a catalytically competent active site. To test this hypothesis, we constructed a series of E. coli FabG mutants and tested their 3-ketoacyl-ACP reductase activities both in vivo and in vitro. Our data show that plasmid-borne FabG mutants, including the double and triple mutants, restored growth of E. coli and Salmonella enterica fabG temperature-sensitive mutant strains under nonpermissive conditions. In vitro assays demonstrated that all of the purified FabG mutant proteins maintained fatty acid synthetic ability, although the activities of the single mutant proteins were 20% to 50% lower than that of wildtype FabG. The S138A, Y151F, and K155A residue substitutions were confirmed by tandem mass spectral sequencing of peptides that spanned all three residues. We conclude that FabG is not a classical short-chain alcohol dehydrogenase/reductase, suggesting that an alternative mode of 3-ketoacyl-ACP reduction awaits discovery.

Biochemical characterization and molecular docking analysis of novel esterases from Sphingobium chungbukense DJ77

We identified three novel microbial esterase (Est1, Est2, and Est3) from Sphingobium chungbukense DJ77. Multiple sequence alignment showed the Est1 and Est3 have distinct motifs, such as tetrapeptide motif HGGG, a pentapeptide sequence motif GXSXG, and catalytic triad residues Ser-Asp-His, indicating that the identified enzymes belong to family IV esterases. Interestingly, Est1 exhibited strong activity toward classical esterase substrates, p-nitrophenyl ester of short-chain fatty acids and long-chain. However, Est3 did not exhibit any activity despite having high sequence similarity and sharing the identical catalytic active residues with Est1. Est3 only showed hydrolytic degradation activity to polycaprolactone (PCL). MOE-docking prediction also provided the parameters consisting of binding energy, molecular docking score, and molecular distance between substrate and catalytic nucleophilic residue, serine. The engineered mutEst3 has hydrolytic activity for a variety of esters ranging from p-nitrophenyl esters to PCL. In the present study, we demonstrated that MOE-docking simulation provides a valuable insight for facilitating biocatalytic performance.

Molecular docking of potential SARS-CoV-2 papain-like protease inhibitors

SARS-CoV-2 papain-like protease is considered as an important potential target for anti-SARS-CoV-2 drug discovery due to its crucial roles in viral spread and innate immunity. Here, we have utilized an in silico molecular docking approach to identify the possible inhibitors of the SARS-CoV-2 papain-like protease, by screening 21 antiviral, antifungal and anticancer compounds. Among them, Neobavaisoflavone has the highest binding energy for SARS-CoV-2 papain-like protease. These molecules could bind near the SARS-CoV-2 papain-like protease crucial catalytic triad, ubiquitination and ISGylation residues: Trp106, Asn109, Cys111, Met208, Lys232, Pro247, Tyr268, Gln269, His272, Asp286 and Thr301. Because blocking the papain-like protease is an important strategy in fighting against viruses, these compounds might be promising candidates for therapeutic intervention against COVID-19.

Design, synthesis, and evaluation of different scaffold derivatives against NS2B-NS3 protease of dengue virus

The number of deaths or critical health issues is a threat in the infection caused by Dengue virus, which complicates the situation, as only symptomatic treatment is the current solution. In this regard we have targeted the dengue protease NS2B-NS3 that is responsible for the replication. The series was designed with the help of molecular modeling approach using docking protocols. The series comprised of different scaffolds viz. cinnamic acid analogs (CA1–CA11), chalcone (C1–C10) and their molecular hybrids (Lik1–Lik10), analogs of benzimidazole (BZ1-BZ5), mercaptobenzimidazole (BS1-BS4), and phenylsulfanylmethylbenzimidazole (PS1-PS4). Virtual screening of various natural phytoconstituents was employed to determine the interactions of designed analogs with the residues of catalytic triad in the active site of NS2B-NS3. We have further synthesized the selected leads. The synthesized analogs were evaluated for the cytotoxicity and NS2B-NS3 protease inhibition activity and compared with known anti-dengue natural phytoconstituent quercetin as the standard. CA2, BZ1, and BS2 were found to be more potent and efficacious than the standard quercetin as evident from the protease inhibition assay.