Tyr 118-Mediated Electron Transfer is Key to the Chlorite Decomposition in Heme-Dependent Chlorite Dismutase.

Chlorite dismutase (Cld) is a crucial enzyme that catalyzes the decomposition of chlorite ions into chloride ions (Cl-) and molecular oxygen (O2). Despite playing an important role in the detoxification of toxic chlorite ions, the mechanism of cleavage of the Cl-O bond by Cld remains highly debatable. The present study highlights the mechanism of such Cl-O bond cleavage in Cld using sophisticated computational tools such as hybrid quantum mechanical/molecular mechanical calculations and long-time scale molecular dynamics simulations. Here, we show that Cld forms a high spin ferric hexacoordinated substrate adduct in the presence of a chlorite ion, which subsequently reduces to a ferrous state. Our study shows a stepwise pathway with the homolytic cleavage of the Cl-O bond that produces a high spin Fe(III)-OH species and a diradicaloid species formed by the combination of a chlorine-based ClO• radical and a protein-based tyrosine118• radical. The findings provide significant insights into Cl-O bond cleavage and O2 formation which shows a crucial role of the tyrosine118 during the electron transfer process.

Identification of a novel inhibitor of SARS-CoV-2 main protease: an in silico, biochemical, and cell-based approach.

The recurrent nature of coronavirus outbreaks, severity of the COVID-19 pandemic, rapid emergence of novel variants, and concerns over the effectiveness of existing vaccines against novel variants have highlighted the need to develop therapeutic interventions. Targeted efforts to identify inhibitors of crucial viral proteins are the preferred strategy. In this study, we screened FDA-approved and natural product libraries using in silico approach for potential hits against the SARS-CoV-2 main protease (Mpro) and experimentally validated their potency using in vitro biochemical and cell-based assays. Seven potential hits were identified through in silico screening and were subsequently evaluated in SARS-CoV-2-based cell-free assays, followed by testing in the HCoV-229E-based culture system. Of the tested compounds, 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1-isopropyl-1H-benzofuro[3,2-b]pyrazolo[4,3-e]pyridin-3(2H)-one (PubChem CID:71755304, hereafter referred to as STL522228) exhibited significant antiviral activity. Subsequently, its potential as a novel COVID therapeutic molecule was validated in the SARS-CoV-2-culture system, where STL522228 demonstrated superior antiviral activity (EC50 = 0.44 µm) compared to Remdesivir (EC50 = 0.62 µm). Based on these findings, we report the strong anti-coronavirus activity of STL522228, and propose that it as a promising pan-coronavirus Mpro inhibitor for further experimental and preclinical validation.

Esterase Specific Fluorescent Probe: Mechanistic Understanding Using QM/MM Calculation and Cell States Discrimination.

Esterases enzymes regulate the body's homeostasis by catalyzing the hydrolysis of various esters. These are also involved in protein metabolism, detoxification, and signal transmission. Most importantly, esterase plays a significant role in cell viability and cytotoxicity assays. Hence, developing an efficient chemical probe is essential for monitoring the esterase activity. Several fluorescent probes for esterase have also been reported targeting cytosol and lysosomes. However, the ability to create efficient probes is constrained due to a lack of understanding of the esterase's active site for hydrolyzing the substrate. In addition, the fluorescent turn-on may limit efficient monitoring. Herein, we have developed a unique fluorescent probe, PM-OAc, to monitor mitochondrial esterase enzyme activity ratiometrically. This probe exhibited a bathochromic wavelength shift with esterase enzyme in alkaline pH (pH∼8.0) due to an intramolecular charge transfer (ICT) process. The phenomenon is well supported by TD-DFT calculation. Moreover, the substrate (PM-OAc) binding at the active site of esterase and its catalytic mechanism to hydrolyze the ester bond are elucidated by molecular dynamics (MD) simulation and QM/MM (Quantum mechanics/molecular mechanics) calculations, respectively. Fluorescent image-based analysis of the cellular environment reveals that our probe can distinguish between live and dead cells based on esterase enzyme activity.

Deciphering the immunogenic T-cell epitopes from spike protein of SARS-CoV-2 concerning the diverse population of India.

Scientists are rigorously looking for an efficient vaccine against the current pandemic due to the SARS-CoV-2 virus. The reverse vaccinology approach may provide us with significant therapeutic leads in this direction and further determination of T-cell/B-cell response to antigen. In the present study, we conducted a population coverage analysis referring to the diverse Indian population. From the Immune epitope database (IEDB), HLA- distribution analysis was performed to find the most promiscuous T-cell epitope out of In silico determined epitope of Spike protein from SARS-CoV-2. Epitopes were selected based on their binding affinity with the maximum number of HLA alleles belonging to the highest population coverage rate values for the chosen geographical area in India. 404 cleavage sites within the 1288 amino acids sequence of spike glycoprotein were determined by NetChop proteasomal cleavage prediction suggesting the presence of adequate sites in the protein sequence for cleaving into appropriate epitopes. For population coverage analysis, 179 selected epitopes present the projected population coverage up to 97.45% with 56.16 average hit and 15.07 pc90. 54 epitopes are found with the highest coverage among the Indian population and highly conserved within the given spike RBD domain sequence. Among all the predicted epitopes, 9-mer TRFASVYAW and RFDNPVLPF along with 12-mer LLAGTITSGWTF and VSQPFLMDLEGK epitopes are observed as the best due to their decent docking score and best binding affinity to corresponding HLA alleles during MD simulations. Outcomes from this study could be critical to design a vaccine against SARS-CoV-2 for a different set of populations within the country.Communicated by Ramaswamy H. Sarma.

Computations reveal a crucial role of an aromatic dyad in the catalytic function of plant cytochrome P450 mint superfamily.

Enzymes are highly specific for their native functions, however with advances in bioengineering tools such as directed evolution, several enzymes are being repurposed for the secondary function of contemporary significance(Khersonsky and Tawfik, 2010 [1]). Due to the functional versatility, the Cytochrome P450 (CYP450) superfamily has become the ideal scaffold for such bioengineering. In the current study, using MD (molecular dynamics) simulations and hybrid QM/MM (Quantum mechanics/molecular mechanics) calculations, we have studied the mechanism of spontaneous emergence of a secondary function due to a single site mutation in two plant CYP450 enzymes from the mint family. The MD simulations of WT (wild type) CYP71D18 and CYP71D13 enzymes and their variants show a crucial gating mechanism by aromatic dyad formed by Phe121 and Phe363 which regulates the substrate recognition. The QM/MM calculations reveal that the hydroxylation reactions at C3 and C6 positions in WT CYP71D18 and CYP71D13 enzymes as well as their variants follow a hydrogen atom transfer (HAT) followed by a single electron transfer (SET) mechanism, which is different from the typical rebound mechanism shown by most of the CYP450 enzymes.

The Performance of Different Water Models on the Structure and Function of Cytochrome P450 Enzymes.

Modeling approaches and modern simulations to investigate the biomolecular structure and function rely on various methods. Since water molecules play a crucial role in all sorts of chemistry, the accurate modeling of water molecules is vital for such simulations. In cytochrome P450 (CYP450), in particular, water molecules play a key role in forming active oxidant that ultimately performs oxidation and metabolism. In the present study, we have highlighted the behavior of the three most widely used water models─TIP3P, SPC/E, and OPC─for three different CYP450 enzymes─CYP450BM3, CYP450OleT, and CYP450BSß─during MD simulations and QM/MM calculations. We studied the various properties, such as RMSD, RMSF, H-bond, water occupancy, and hydrogen atom transfer (HAT), using QM/MM calculations and compared them for all three water models. Our study shows that the stabilities of the enzyme complexes are well maintained in all three water models. However, the OPC water model performs well for the polar active sites, that is, in CYP450OleT and CYP450BSß, while the TIP3P water model is superior for the hydrophobic site, such as CYP450BM3.