Catalytic Groove Research Articles

Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

Structural insights into curdlan degradation via a glycoside hydrolase containing a disruptive carbohydrate-binding module

BackgroundDegradation via enzymatic processes for the production of valuable β-1,3-glucooligosaccharides (GOS) from curdlan has attracted considerable interest. CBM6E functions as a curdlan-specific β-1,3-endoglucanase, composed of a glycoside hydrolase family 128 (GH128) module and a carbohydrate-binding module (CBM) derived from family CBM6.ResultsCrystallographic analyses were conducted to comprehend the substrate specificity mechanism of CBM6E. This unveiled structures of both apo CBM6E and its GOS-complexed form. The GH128 and CBM6 modules constitute a cohesive unit, binding nine glucoside moieties within the catalytic groove in a singular helical conformation. By extending the substrate-binding groove, we engineered CBM6E variants with heightened hydrolytic activities, generating diverse GOS profiles from curdlan. Molecular docking, followed by mutation validation, unveiled the cooperative recognition of triple-helical β-1,3-glucan by the GH128 and CBM6 modules, along with the identification of a novel sugar-binding residue situated within the CBM6 module. Interestingly, supplementing the CBM6 module into curdlan gel disrupted the gel’s network structure, enhancing the hydrolysis of curdlan by specific β-1,3-glucanases.ConclusionsThis study offers new insights into the recognition mechanism of glycoside hydrolases toward triple-helical β-1,3-glucans, presenting an effective method to enhance endoglucanase activity and manipulate its product profile. Furthermore, it discovered a CBM module capable of disrupting the quaternary structures of curdlan, thereby boosting the hydrolytic activity of curdlan gel when co-incubated with β-1,3-glucanases. These findings hold relevance for developing future enzyme and CBM cocktails useful in GOS production from curdlan degradation.

Structural and functional analysis of SpGlu64A: a novel glycoside hydrolase family 64 laminaripentaose-producing β-1,3-glucanase from Streptomyces pratensis.

Laminaripentaose (L5)-producing β-1,3-glucanases can preferentially cleave the triple-helix curdlan into β-1,3-glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, β-1,3-glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a β-1,3-glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 °C, and exhibited strict substrate specificity toward curdlan (83.1 U·mg-1). It efficiently hydrolyzed curdlan to produce L5 as the end product. The overall structure of SpGlu64A consisted of a barrel domain and a mixed (α/β) domain, which formed an unusually wide groove with a crescent-like structure. In the two complex structures (SpGlu64A-L3 and SpGlu64A-L4), two oligosaccharide chains were captured and the triple-helical structure was relatively compatible with the wide groove, which suggested the possibility of binding to the triple-helical β-1,3-glucan. A catalytic framework (β6-β9-β10) and the steric hindrance formed by the side chains of residues Y161, N163, and H393 in the catalytic groove were predicted to complete the exotype-like cleavage manner. On the basis of the structure, a fusion protein with the CBM56 domain (SpGlu64A-CBM) and a mutant (Y161F; by site-directed mutation) were obtained, with 1.2- and 1.7-fold increases in specific activity, respectively. Moreover, the combined expression of SpGlu64A-CBM and -Y161F improved the enzyme activity by 2.63-fold. The study will notonly be helpful in understanding the reaction mechanism of β-1,3-glucanases but will also provide a basis for further enzyme engineering.

Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM

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 1H/13C/15N 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.

Site-directed mutagenesis of family GH10 Aspergillus fumigatus xylanase A and the interaction with Oryza sativa xylanase inhibitor protein

In this study three alanine mutants (V99A, D145A and V99A/D145A) of family GH10 Aspergillus fumigatus xylanase A (AfXylA10) were engineered and characterized. These variants exhibited molecular mass of approximately 52.0 kDa and optimum temperature at 53 °C. The optimum pH of V99A/D145A was pH 4.0, and that of V99A and D145A was 5.0. Three variants were competitively inhibited by Oryza sativa xylanase inhibitor protein, rePOsXIP in a typical dose-dependent manner, with Ki of 214.8, 170.3 and 126.2 nM, respectively. Mutant V99A showed lower sensitivity to rePOsXIP and lower activity compared to reAfXylA10. However, D145A obtained higher activity and similar sensitivity to rePOsXIP. The underlying interaction mechanism was delved by molecular dynamics (MD) simulations and computational analysis. Binding free energy of V99A-OsXIP, D145A-OsXIP and V99A/D145A-OsXIP systems, calculated using the MM/PBSA method, were −19.7 ± 9.4, −26.9 ± 6.0, and −45.4 ± 9.8 kcal/mol, respectively. Representational snapshot of these systems from MD simulations showed that the α7-helix of OsXIP entered the active tunnel of mutant xylanases, which would hinder xylan from entering the catalytic groove. Particularly, residue K239 in OsXIP's α7-helix formed stable hydrogen bonding with the acid-base catalytic site (E140) of these mutants. Laplacian of electron density values of E140xylanase- K239OsXIP in V99A-OsXIP, D145A-OsXIP, and V99A/D145A-OsXIP systems were 0.07545, 0.09155 and 0.09764 au., respectively. The study provided preliminary insight into how V99AfXylA10 and D145AfXylA10 residue influence enzymatic properties and the interaction with OsXIP. These findings might inform design of mutant xylanase with enhanced catalytic activity and reduced sensitivity to xylanase inhibitory proteins (XIs).

3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor.

The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme-inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1Cat) without a pro-region. High-resolution 3D structures of PvS1Cat, alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1' and P2' positions of the inhibitor, although P' residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.

Structural basis for the tRNA-dependent activation of the terminal complex of selenocysteine synthesis in humans.

O-Phosphoseryl-tRNASec selenium transferase (SepSecS) catalyzes the terminal step of selenocysteine (Sec) synthesis in archaea and eukaryotes. How the Sec synthetic machinery recognizes and discriminates tRNASec from the tRNA pool is essential to the integrity of the selenoproteome. Previously, we suggested that SepSecS adopts a competent conformation that is pre-ordered for catalysis. Herein, using high-resolution X-ray crystallography, we visualized tRNA-dependent conformational changes in human SepSecS that may be a prerequisite for achieving catalytic competency. We show that tRNASec binding organizes the active sites of the catalytic protomer, while stabilizing the N- and C-termini of the non-catalytic protomer. Binding of large anions to the catalytic groove may further optimize the catalytic site for substrate binding and catalysis. Our biochemical and mutational analyses demonstrate that productive SepSecS•tRNASec complex formation is enthalpically driven and primarily governed by electrostatic interactions between the acceptor-, TΨC-, and variable arms of tRNASec and helices α1 and α14 of SepSecS. The detailed visualization of the tRNA-dependent activation of SepSecS provides a structural basis for a revised model of the terminal reaction of Sec formation in archaea and eukaryotes.

YidC as a potential antibiotic target

The membrane insertase YidC, is an essential bacterial component and functions in the folding and insertion of many membrane proteins during their biogenesis. It is a multispanning protein in the inner (cytoplasmic) membrane of Escherichia coli that binds its substrates in the “greasy slide” through hydrophobic interaction. The hydrophilic part of the substrate transiently localizes in the groove of YidC before it is translocated into the periplasm. The groove, which is flanked by the greasy slide, is within the center of the membrane, and provides a promising target for inhibitors that would block the insertase function of YidC. In addition, since the greasy slide is available for the binding of various substrates, it could also provide a binding site for inhibitory molecules. In this review we discuss in detail the structure and the mechanism of how YidC interacts not only with its substrates, but also with its partner proteins, the SecYEG translocase and the SRP signal recognition particle. Insight into the substrate binding to the YidC catalytic groove is presented. We wind up the review with the idea that the hydrophilic groove would be a potential site for drug binding and the feasibility of YidC-targeted drug development.

Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs).

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism ofjuvenile hormoneIII (JH III) in insects. To study the role that JHEH plays in female Aedes aegypti JHEH 1, 2, and 3 complementary DNA (cDNAs) were cloned and sequenced. Northern blot analyses show that the three transcripts are expressed in the head thorax, the gut, the ovaries, and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds JHIII acid (JH IIIA) at the catalytic groove better than JH III. The cDNA of JHEH 1 and 2 are very similar indicating close relationship. Knocking down of jheh 1, 2, and 3 in adult female and larval Ae. aegypti using double-stranded RNA(dsRNA) did not affect egg development or caused adult mortality. Larvae that were fed bacterial cells expressing dsRNA against jheh 1, 2, and 3 grew normally. Treating blood-fed female Ae. aegypti with [12-3 H](10R) JH III and analyzing the metabolites by C18 reversed phase chromatography showed that JHEH preferred substrate is not JH III but JH IIIA. Genomic analysis of jheh 1, 2, and 3 indicate that jheh 1 and 2 are transcribed from a 1.53 kb DNA whereas jheh 3 is transcribed from a 10.9 kb DNA. All three genes are found on chromosome two at distinct locations. JHEH 2 was expressed in bacterial cells and purified by Ni affinity chromatography. Sequencing of the recombinant protein by MS/MS identified JHEH 2 as the expressed recombinant protein.

Heterologous expression of family GH11 Aspergillus niger xylanase B (AnXylB11) in Pichia pastoris and competitive inhibition by riceXIP: An experimental and simulation study

The family GH11 Aspergillus niger JL15 xylanase B (AnXylB11) was heterologously expressed in Pichia pastoris X33. The recombinant AnXylB11 (reAnXylB11) was secreted into the culture medium with a molecular weight of approximately 33.0 kDa. The optimal temperature and pH of reAnXylB11 were 40 ℃ and 5.0, respectively. reAnXylB11 released xylobiose (X2)-xylohexaose (X6) from beechwood xylan, with xylotriose (X3) as the primary product. The hydrolysates showed significant antioxidant activity. reAnXylB11 was also competitively inhibited by recombinant rice xylanase inhibitory protein (rePriceXIP), with an inhibition constant (Ki) of 106.9 nM. Molecular dynamics (MD) simulations, non-covalent interactions (NCI), and binding free energy calculation and decomposition were conducted to decipher the interactional features between riceXIP and AnXyB11. Representative conformation of riceXIP-AnXylB11 complex showed that a U-shaped long loop between α4 and β5 (K143-L152) of riceXIP was protruded into the catalytic groove and formed tight interaction with many key residues of AnXylB11. The binding free energy of riceXIP-AnXylB11 was calculated to be − 46.1 ± 10.5 kcal/mol, with Coulomb and van der Waals forces contributing the most. NCI analysis showed that the hydrogen bonding networks such as R148riceXIP-E98AnXyl11B, K143riceXIP-D138AnXyl11B and R148riceXIP-E189AnXyl11B provided terrific contributions to the interface interaction. The Laplacian of electron density values of atom pairs R148riceXIP@ 2HH1-E89AnXylB11@OE2 and N142riceXIP@ 1HD2-D138AnXylB11@OD1 were 0.12190 and 0.16009 a.u., respectively. Exploring the interactional features between xylanase and inhibitor protein may aid in constructing mutant xylanase that is insensitive to xylanase inhibitory proteins (XIs).

Cloning and Characterization of Drosophila melanogaster Juvenile Hormone Epoxide Hydrolases (JHEH) and Their Promoters

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that the three transcripts are expressed in the head thorax, the gut, the ovaries and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds juvenile hormone III acid (JH IIIA) at the catalytic groove better than JH III. Analyses of the three JHEH promoters and expressing short promoter sequences behind a reporter gene (lacZ) in D. melanogaster cell culture identified a JHEH 3 promoter sequence (626 bp) that is 10- and 25-fold more active than the most active promoter sequences of JHEH 2 and JHEH 1, respectively. A transcription factor (TF) Sp1 that is involved in the activation of JHEH 3 promoter sequence was identified. Knocking down Sp1 using dsRNA inhibited the transcriptional activity of this promoter in transfected D. melanogaster cells and JH III and 20HE downregulated the JHEH 3 promoter. On the other hand, JH IIIA and farnesoic acid did not affect the promoter, indicating that JH IIIA is JHEH’s preferred substrate. A transgenic D. melanogaster expressing a highly activated JHEH 3 promoter behind a lacZ reporter gene showed promoter transcriptional activity in many D. melanogaster tissues.

Structure-Function Insights into the Fungal Endo-Chitinase Chit33 Depict its Mechanism on Chitinous Material.

Chitin is the most widespread amino renewable carbohydrate polymer in nature and the second most abundant polysaccharide. Therefore, chitin and chitinolytic enzymes are becoming more importance for biotechnological applications in food, health and agricultural fields, the design of effective enzymes being a paramount issue. We report the crystal structure of the plant-type endo-chitinase Chit33 from Trichoderma harzianum and its D165A/E167A-Chit33-(NAG)4 complex, which showed an extended catalytic cleft with six binding subsites lined with many polar interactions. The major trait of Chit33 is the location of the non-conserved Asp117 and Arg274 acting as a clamp, fixing the distorted conformation of the sugar at subsite –1 and the bent shape of the substrate, which occupies the full catalytic groove. Relevant residues were selected for mutagenesis experiments, the variants being biochemically characterized through their hydrolytic activity against colloidal chitin and other polymeric substrates with different molecular weights and deacetylation percentages. The mutant S118Y stands out, showing a superior performance in all the substrates tested, as well as detectable transglycosylation capacity, with this variant providing a promising platform for generation of novel Chit33 variants with adjusted performance by further design of rational mutants’. The putative role of Tyr in binding was extrapolated from molecular dynamics simulation.

A novel neutral thermophilic β-mannanase from Malbranchea cinnamomea for controllable production of partially hydrolyzed konjac powder.

Partially hydrolyzed konjac powder (PHKP) can be used to increase the daily intake of dietary fibers of consumers. To produce PHKP by enzymatic hydrolysis, a novel β-mannanase gene (McMan5B) from Malbranchea cinnamomea was expressed in Pichia pastoris. It showed a low identity of less than 52% with other GH family 5 β-mannanases. Through high cell density fermentation, the highest β-mannanase activity of 42200 U mL-1 was obtained. McMan5B showed the maximal activity at pH 7.5 and 75°C, respectively. It exhibited excellent pH stability and thermostability. Due to the different residues (Phe214, Pro253, and His328) in catalytic groove and the change of β2-α2 loop, McMan5B showed unique hydrolysis property as compared to other β-mannanases. The enzyme was employed to hydrolyze konjac powder for controllable production of PHKP with a weight-average molecular weight of 22000Da (average degree of polymerization 136). Furthermore, the influence of PHKP (1.0%-4.0%) on the qualities of steamed bread was evaluated. The steamed bread adding 3.0% PHKP had the maximum specific volume and the minimum hardness, which showed 11.0% increment and 25.4% decrement as compared to the control, respectively. Thus, a suitable β-mannanase for PHKP controllable production and a fiber supplement for steamed bread preparation were provided in this study. KEY POINTS: • A novel β-mannanase gene (McMan5B) was cloned from Malbranchea cinnamomea and expressed in Pichia pastoris at high level. • McMan5B hydrolyzed konjac powder to yield partially hydrolyzed konjac powder (PHKP) instead of manno-oligosaccharides. • PHKP showed more positive effect on the quality of steamed bread than many other dietary fibers including konjac powder.

Allosteric control of Ubp6 and the proteasome via a bidirectional switch

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome.

Validation and molecular analysis of β-1,3-GLU2 SNP marker associated with resistance to Colletotrichum gloeosporioides in mango (Mangifera indica L.)

Anthracnose caused by Colletotrichum gloeosporioides is one of the most destructive diseases of mango worldwide. Breeding for genetic resistance to anthracnose is necessary to improve the yield and fruit quality of mango. Here, we identified and analyzed the molecular basis of a SNP within the pathogenesis-related β-1,3-glucanase 2 (β-1,3-GLU2) gene putatively linked with resistance to anthracnose. This SNP designated ‘21881933’ causes a missense mutation (I196V) in the highly conserved glycosyl hydrolase family 17 (GH17) domain—a structural unit involved in physiologically important processes in plants, notably in response to biotic and abiotic stresses, activation of phytohormones, lignification, and cell wall remodeling. Structural analysis suggested that the I196V mutation results in unfavorable conformational changes in the enzyme's (β/α)8 TIM-barrel motif and catalytic groove. In phenotyping and AS-PCR assays, we found that the two alleles of SNP 21881933 are significantly correlated to anthracnose resistance. Mango genotypes with the β-1,3-GLU2 allele ‘G’ showed significantly higher disease severity post-inoculation while those with β-1,3-GLU2 allele ‘A’ showed phenotypic resistance to the disease. These results indicate that β-1,3-GLU2 is involved in the primary defense response of mango against C. gloeosporioides and that SNP 21881933 enhances the activities of β-1,3-glucanases. To our knowledge, this is the first report describing the molecular basis of anthracnose resistance in mango involving β-1,3-glucanases. Furthermore, we developed a marker that can discriminate β-1,3-GLU2 SNP alleles using a simple and cost-effective allele-specific PCR assay. Our findings support the utilization of this marker for marker-assisted selection (MAS) and genotyping of resistant mangoes as early as the seedling stage. This will help improve mango breeding efficiency and significantly reduce the expenses in field inputs, maintenance, and evaluation of material over the years.

Phytochemicals from Leucas zeylanica Targeting Main Protease of SARS-CoV-2: Chemical Profiles, Molecular Docking, and Molecular Dynamics Simulations.

Simple SummaryMolecular docking in conjunction with molecular dynamics simulation was accomplished as they extend an ample opportunity to screen plausible inhibitors of the main protease from Leucas zeylanica. The preferential phytochemicals were identified from L. zeylanica through gas chromatography–mass spectrometry (GC-MS). The pre-eminent three identified phytochemicals exhibited toxicity by no means during the scrutinization of ADME/T prominences. Moreover, pharmacologically distinguishing characteristics and the biological activity of the lead phytochemicals were satisfying as an antiviral drug contender. Additionally, the molecular dynamics simulation exhibited thermal stability and a stable binding affinity of the protein–compound complex that referred to the appreciable efficacy of lead optimization. Therefore, the preferable phytochemicals are worth substantial evaluation in the biological laboratory to recommend plausible antiviral drug candidates.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a contemporary coronavirus, has impacted global economic activity and has a high transmission rate. As a result of the virus’s severe medical effects, developing effective vaccinations is vital. Plant-derived metabolites have been discovered as potential SARS-CoV-2 inhibitors. The SARS-CoV-2 main protease (Mpro) is a target for therapeutic research because of its highly conserved protein sequence. Gas chromatography–mass spectrometry (GC-MS) and molecular docking were used to screen 34 compounds identified from Leucas zeylanica for potential inhibitory activity against the SARS-CoV-2 Mpro. In addition, prime molecular mechanics–generalized Born surface area (MM-GBSA) was used to screen the compound dataset using a molecular dynamics simulation. From molecular docking analysis, 26 compounds were capable of interaction with the SARS-CoV-2 Mpro, while three compounds, namely 11-oxa-dispiro[4.0.4.1]undecan-1-ol (−5.755 kcal/mol), azetidin-2-one 3,3-dimethyl-4-(1-aminoethyl) (−5.39 kcal/mol), and lorazepam, 2TMS derivative (−5.246 kcal/mol), exhibited the highest docking scores. These three ligands were assessed by MM-GBSA, which revealed that they bind with the necessary Mpro amino acids in the catalytic groove to cause protein inhibition, including Ser144, Cys145, and His41. The molecular dynamics simulation confirmed the complex rigidity and stability of the docked ligand–Mpro complexes based on the analysis of mean radical variations, root-mean-square fluctuations, solvent-accessible surface area, radius of gyration, and hydrogen bond formation. The study of the postmolecular dynamics confirmation also confirmed that lorazepam, 11-oxa-dispiro[4.0.4.1]undecan-1-ol, and azetidin-2-one-3, 3-dimethyl-4-(1-aminoethyl) interact with similar Mpro binding pockets. The results of our computerized drug design approach may assist in the fight against SARS-CoV-2.

Structure–function analysis of a new PL17 oligoalginate lyase from the marine bacterium Zobellia galactanivorans DsijT

Alginate is a major compound of brown macroalgae and as such an important carbon and energy source for heterotrophic marine bacteria. Despite the rather simple composition of alginate only comprising mannuronate and guluronate units, these bacteria feature complex alginolytic systems that can contain up to seven alginate lyases. This reflects the necessity of large enzyme systems for the complete degradation of the abundant substrate. Numerous alginate lyases have been characterized. They belong to different polysaccharide lyase (PL) families, but only one crystal structure of a family 17 (PL17) alginate lyase has been reported to date, namely Alg17c from the gammaproteobacterium Saccharophagus degradans. Biochemical and structural characterizations are helpful to link sequence profiles to function, evolution of functions and niche-specific characteristics. Here, we combined detailed biochemical and crystallographic analysis of AlyA3, a PL17 alginate lyase from the marine flavobacteria Zobellia galactanivorans DsijT, providing the first structure of a PL17 in the Bacteroidetes phylum. AlyA3 is exo-lytic and highly specific of mannuronate stretches. As part of an “alginate utilizing locus”, its activity is complementary to that of other characterized alginate lyases from the same bacterium. Structural comparison with Alg17c highlights a common mode of action for exo-lytic cleavage of the substrate, strengthening our understanding of the PL17 catalytic mechanism. We show that unlike Alg17c, AlyA3 contains an inserted flexible loop at the entrance to the catalytic groove, likely involved in substrate recognition, processivity and turn over.

Unraveling the interaction of an opium poppy alkaloid noscapine ionic liquid with human hemoglobin: Biophysical and computational studies

Apart from a wide range of applications, nowadays, Ionic liquids (ILs) are showing their major importance in the field of pharmaceuticals for the development of effective therapeutics. Along with them, proteins are the major targets of a drug and a little alteration in their conformation can lead them to act differently. In the present study, a step has been taken towards the binding of a noscapine (Nos) based ionic liquid [Pip-Nos]OTf with human hemoglobin (Hb), as the blood plasma has a special role in the transport and targeting of a drug. Noscapine, is a phthalideisoquinoline alkaloid, derived from an opium poppy (Papaver Somniferum) having various biological properties. UV–Vis absorption spectroscopy, steady state fluorescence spectroscopy, circular dichroism (CD) and computational studies have been used to study the molecular interaction of [Pip-Nos]OTf with human hemoglobin. UV–Vis spectra showed an increase in absorption intensity with a considerable red (bathochromic) shift on continuous addition of [Pip-Nos]OTf. A static quenching was observed with the formation of ground state complex in fluorescence spectroscopy. CD analysis showed no significant changes in the secondary structure of Hb on the addition of [Pip-Nos]OTf. It was found to bind strongly with hemoglobin in its catalytic groove with a high molecular docking score of −265.47 kJ/mol. Besides, molecular dynamics simulation studies delineate the stable and prolonged binding of [Pip-Nos]OTf to hemoglobin with minimal deviations in deformations plot and covariance matrix statistics. These favorable outcomes of stable binding of [Pip-Nos]OTf with hemoglobin, also depicted certain common spectroscopic and in silico features to the previously reported interaction of this ionic liquid with BSA and conclusively, showed its promising candidature as cancer therapeutics.

Virtual screening and molecular dynamics simulation study of plant-derived compounds to identify potential inhibitors of main protease from SARS-CoV-2.

The new coronavirus (SARS-CoV-2) halts the world economy and caused unbearable medical emergency due to high transmission rate and also no effective vaccine and drugs has been developed which brought the world pandemic situations. The main protease (Mpro) of SARS-CoV-2 may act as an effective target for drug development due to the conservation level. Herein, we have employed a rigorous literature review pipeline to enlist 3063 compounds from more than 200 plants from the Asian region. Therefore, the virtual screening procedure helps us to shortlist the total compounds into 19 based on their better binding energy. Moreover, the Prime MM-GBSA procedure screened the compound dataset further where curcumin, gartanin and robinetin had a score of (−59.439, −52.421 and − 47.544) kcal/mol, respectively. The top three ligands based on binding energy and MM-GBSA scores have most of the binding in the catalytic groove Cys145, His41, Met165, required for the target protein inhibition. The molecular dynamics simulation study confirms the docked complex rigidity and stability by exploring root mean square deviations, root mean square fluctuations, solvent accessible surface area, radius of gyration and hydrogen bond analysis from simulation trajectories. The post-molecular dynamics analysis also confirms the interactions of the curcumin, gartanin and robinetin in the similar binding pockets. Our computational drug designing approach may contribute to the development of drugs against SARS-CoV-2.

Selection and mutational analyses of the substrate interacting residues of a chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) to improve transglycosylation

Transglycosylation (TG) by Enterobacter cloacae subsp. cloacae chitinase 2 (EcChi2) has been deciphered by site-directed mutagenesis. EcChi2 originally displayed feeble TG with chitin oligomer with a degree of polymerization (DP4), for a short duration. Based on the 3D modelling and molecular docking analyses, we altered the substrate interactions at the substrate-binding cleft, catalytic center, and catalytic groove of EcChi2 by mutational approach to improve TG. The mutation of W166A and T277A increased TG by EcChi2 and also affected its catalytic efficiency on the polymeric substrates. Whereas, R171A had a drastically decreased hydrolytic activity but, retained TG activity. In the increased hydrolytic activity of the T277A, altered interactions with the substrates played an indirect role in the catalysis. Mutation of the central Asp, in the conserved DxDxE motif, to Ala (D314A) and Asn (D314N) conversion yielded DP5-DP8 TG products. The quantifiable TG products (DP5 and DP6) increased to 8% (D314A) and 7% (D314N), resulting in a hyper-transglycosylating mutant. Mutation of W276A and W398A resulted in the loss of TG activity, indicating that the aromatic residues (W276 and W398) at +1 and +2 subsites are essential for the TG activity of EcChi2.