Role Of Active Site Residues Research Articles

Biochemical, kinetic, and structural characterization of a Bacillus tequilensis nitroreductase.

Nitroreductases (NRs) are NAD(P)H-dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell-imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized. Within this group of enzymes, only few are capable of amine synthesis, which is a fundamental chemical transformation for the pharmaceutical, agricultural, and textile industries. Herein, we provide a comprehensive description of a recently discovered NR from Bacillus tequilensis, named BtNR. This enzyme has previously been demonstrated to have the capability to fully convert nitro aromatic and heterocyclic compounds to their respective primary amines. In this study, we determined its biochemical, kinetic and structural properties, including its apparent melting temperature (Tm) of 59 °C, broad pH activity range (from pH 3 to 10) and a notably low redox potential (-236 ± 1 mV) in comparison to other well-known NRs. We also determined its steady-state and pre-steady-state kinetic parameters, which are consistent with other NRs. Additionally, we elucidated the crystal structure of BtNR, which resembles the well-characterized Escherichia coli oxygen-insensitive NAD(P)H nitroreductase (NfsB), and investigated the substrate binding in its active site through docking and molecular dynamics studies with four nitro aromatic substrates. Guided by these structural analyses, we probed the functional roles of active site residues by site-directed mutagenesis. Our findings provide valuable insights into the biochemical and structural properties of BtNR, as well as its potential applications in biotechnology.

Modeling of a near-attack conformation of oxime in phosphorylated acetylcholinesterase via a reactivation product, a phosphorylated oxime

At the present, only four antidotes are in use in therapy for poisoning by organophosphorus compounds: 2-PAM, HI-6, obidoxime and trimedoxime. Numerous compounds have been designed and synthetized to be more effective reactivators than those currently in use. Many of those new compounds fail at the enzyme level because interactions formed within the AChE active site are not favourable ones that lead to a successful reactivation. The approach in which the modeling of a phosphorylated oxime (POX), a product of successful reactivation in the AChE active site, may be a way to better understand the role of active site residues during the process of formation of the Michaelis type of complex between an enzyme and oxime. After reactivation, a change in phosphorus stereochemistry occurs leading to a different spatial arrangement of attached substituents, now including an oxime. To study interactions between the AChE oxyanion hole and a phosphorylated oxime, an S203G mutant was used to avoid the steric hindrance caused by the catalytic serine. In this way, the POX could be positioned close to the oxyanion hole. In the final step, the oxime without a phosphoester moiety was transferred into the phosphorylated AChE and molecular dynamics was used to test the stability of the near-attack conformation of the oxime near the phosphorylated serine.

The Roles of Coenzyme A Binding Pocket Residues in Short and Medium Chain Acyl-CoA Synthetases.

Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg193, Arg528, and Arg586 of Methanothermobacter thermautotrophicus Acs (AcsMt), are predicted to form important interactions with the 5'- and 3'-phosphate groups of CoA. Kinetic characterization of AcsMt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and catalysis. The predicted CoA binding site of Methanosarcina acetivorans Macs (MacsMa) is structurally more closely related to that of 4-chlorobenzoate:coenzyme A ligase (CBAL) than Acs. Alteration of MacsMa residues Tyr460, Arg490, Tyr525, and Tyr527, which correspond to CoA binding pocket residues in CBAL, strongly affected CoA binding and catalysis without substantially affecting acyl-adenylate formation. Both enzymes discriminate between 3'-dephospho-CoA and CoA, indicating interaction between the enzyme and the 3'-phosphate group is important. Alteration of MacsMa residues Lys461 and Lys519, located at positions equivalent to AcsMt Arg528 and Arg586, respectively, had only a moderate effect on CoA binding and catalysis. Overall, our results indicate the active site architecture in AcsMt and MacsMa differs even though these enzymes catalyze mechanistically similar reactions. The significance of this study is that we have delineated the active site architecture with respect to CoA binding and catalysis in this important enzyme superfamily.

Investigating the Roles of Active Site Residues in Mycobacterium tuberculosis Indole-3-glycerol Phosphate Synthase, a Potential Target for Antitubercular Agents.

Mycobacterium tuberculosis drug resistance is emerging and new drug targets are needed. Tryptophan biosynthesis is necessary for M. tuberculosis replication and virulence. Indole-3-glycerol phosphate synthase (IGPS) catalyzes a step in M. tuberculosis tryptophan biosynthesis and has been suggested as a potential anti-infective target, but our understanding of this enzyme is limited. To aid in inhibitor design and gain a greater mechanistic picture of this enzyme, there is a need to understand the roles of active site amino acids in ligand binding and catalysis. In this work, we explored the roles of conserved active site amino acids Glu57, Lys59, Lys119, Glu168, and Glu219. Mutation of each to Ala results in loss of all detectable activity. The Glu57Gln, Lys59Arg, Lys119Arg, Glu168Gln, and Glu219Asp mutations result in large activity losses, while Glu219Gln has enhanced activity. Analysis of the enzymatic data yields the following main conclusions: (A) Lys119 is the likely catalytic acid in the CdRP ring closure step. (B) Glu168 stabilizes a charged reaction intermediate and may also be the catalytic base. (C) Glu57, Glu219, and Lys119 form a closely arranged triad in which Glu57 and Glu219 modulate the pKa of Lys119, and thus overall activity. This increased understanding of inter- and intramolecular interactions and demonstration of the highly coordinated nature of the M. tuberculosis IGPS active site provide new mechanistic information and guidance for future work with this potential new drug target.

Mapping roles of active site residues in the acceptor site of the PA3944 Gcn5-related N-acetyltransferase enzyme.

An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site.

Distinctive Formation of a DNA-Protein Cross-Link during the Repair of DNA Oxidative Damage: Insights into Human Disease from MD Simulations and QM/MM Calculations.

Reactive oxygen species damage DNA and result in health issues. The major damage product, 8-oxo-7,8-dihydroguanine (8oG), is repaired by human adenine DNA glycosylase homologue (MUTYH). Although MUTYH misfunction is associated with a genetic disorder called MUTYH-associated polyposis (MAP) and MUTYH is a potential target for cancer drugs, the catalytic mechanism required to develop disease treatments is debated in the literature. This study uses molecular dynamics simulations and quantum mechanics/molecular mechanics techniques initiated from DNA-protein complexes that represent different stages of the repair pathway to map the catalytic mechanism of the wild-type MUTYH bacterial homologue (MutY). This multipronged computational approach characterizes a DNA-protein cross-linking mechanism that is consistent with all previous experimental data and is a distinct pathway across the broad class of monofunctional glycosylase repair enzymes. In addition to clarifying how the cross-link is formed, accommodated by the enzyme, and hydrolyzed for product release, our calculations rationalize why cross-link formation is favored over immediate glycosidic bond hydrolysis, the accepted mechanism for all other monofunctional DNA glycosylases to date. Calculations on the Y126F mutant MutY highlight critical roles for active site residues throughout the reaction, while investigation of the N146S mutant rationalizes the connection between the analogous N224S MUTYH mutation and MAP. In addition to furthering our knowledge of the chemistry associated with a devastating disorder, the structural information gained about the distinctive MutY mechanism compared to other repair enzymes represents an important step for the development of specific and potent small-molecule inhibitors as cancer therapeutics.

Computational Mechanistic Study of l-Aspartate Oxidase by ONIOM Method.

l-Aspartate oxidase (Laspo) is responsible for the oxidation of l-aspartate into iminoaspartate using flavin as a cofactor. During this process flavin is reduced, and it can be reoxidized by either molecular oxygen or fumarate. The overall fold and the catalytic residues of Laspo are similar to succinate dehydrogenase and fumarate reductase. On the basis of deuterium kinetic isotope effects as well as other kinetic and structural data, it is proposed that the enzyme can catalyze the oxidation of l-aspartate through a mechanism similar to amino acid oxidases. It is suggested that a proton is removed from the α-amino group, while a hydride is transferred from C2 to flavin. It is also suggested that the hydride transfer is a rate-limiting step. However, there is still an ambiguity about the stepwise or concerted mechanism of hydride- and proton-transfer steps. In this study, we formulated some computational models to study the hydride-transfer mechanism using the crystal structure of Escherichia colil-aspartate oxidase in complexes with succinate. The calculations involved our own N-layered integrated molecular orbital and molecular mechanics method, and we evaluated the geometry and energetics of the hydride/proton-transfer processes while probing the roles of active site residues. Based on the calculations, it is concluded that proton- and hydride-transfer steps are decoupled, and a stepwise mechanism might be operative as opposed to the concerted one.

Functional significance of serine 13 in the active site of glutathione S-transferase F3 from Oryza sativa

Plant glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme that detoxifies various electrophilic compounds including herbicides and organic pollutants by catalyzing the formation of conjugates with reduced glutathione (GSH). Although the structure and function of the GST subunits in rice, an important food in Asia, are not well understood, they are crucial for herbicide development. To investigate the role of active site residues in rice Phi-class GSTF3 (OsGSTF3), evolutionarily conserved serine residues were replaced with alanine using site-directed mutagenesis to obtain the mutants S13A, S38A, S69A, and S169A. These four mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity using immobilized GSH affinity chromatography. Mutation of Ser13 to Ala resulted in substantial reductions in specific activities and kcat/Km values for the GSH-[1-chloro-2,4-dinitrobenzene (CDNB)] conjugation reaction. In contrast, mutations of Ser38, Ser69, and Ser169 to Ala had little effect on the activities and kinetic parameters. Additionally, the mutation of Ser13 to Ala significantly affected the KmGSH and I50 values of S-hexylglutathione and S-(2,4-dinitrophenyl)glutathione, which compete with GSH and the product of GSH-CDNB conjugation, respectively. A pH-log (kcat/KmCDNB) plot was used to estimate the pKa value of GSH in the enzyme-GSH complex of the wild-type enzyme, which was approximately 6.9. However, the pKa value of GSH in the enzyme-GSH complex of the S13A mutant was approximately 8.7, which was about 1.8 pK units higher than that of the wild-type enzyme. OsGSTF3 was also crystallized for crystallographic study, and the structure analyses revealed that Ser13 is located in the active site and that its side chain is in close proximity to the thiol group of glutathione bound in the enzyme. Based on these substitution effects on kinetic parameters, the dependence of kinetic parameters on the pH and 3-dimensional structure, it was suggested that Ser13 in rice OsGSTF3 is the residue responsible for catalytic activity by lowering the pKa of GSH in the enzyme-GSH complex and enhancing the nucleophilicity of the GSH thiol in the active site.

Mechanistic and evolutionary insights into alkaline phosphatase superfamily through structure-function studies on Sphingomonas alkaline phosphatase

Alkaline phosphatases (APs), represented by E. coli AP (ECAP), employ an arginine residue to stabilize the phosphoryl group in the active site; whereas, AP from Sphingomonas (SPAP) shows a unique combination of substrate-binding residues; Thr89, Asn110, Lys171, and Arg173. Although such combination has been observed only in SPAP, these residues are present separately in different members of the AP superfamily. Here, we establish the presence of two distinct classes of APs; ECAP-type and SPAP-type. Bioinformatic analyses show that SPAP-type of APs are widely distributed in the bacterial kingdom. The role of active site residues in the catalytic mechanism has been delineated through a set of crystal structures reported here. These structures, representing different stages of the reaction pathway provide wealth of information for the catalytic mechanism. Despite critical differences in the substrate binding residues, SPAP follows a mechanism similar to that of ECAP-type of APs. Structure-based phylogenetic analysis suggests that SPAP and ECAP may have diverged very early during the evolution from a common ancestor. Moreover, it is proposed that the SPAP-type of APs are fundamental members of the AP superfamily and are more closely related to other members of the superfamily as compared to the ECAP-type of APs.

Computational Analysis of the Inhibition Mechanism of NOTUM by the ONIOM Method.

Notum is a member of serine hydrolyses that cleaves the palmitoleate moiety from Wingless-related integration site (Wnt) ligands. This enzyme plays crucial functions through modulating the Wnt signaling pathway. Inhibition of Notum carries therapeutic effects against a number of maladies including osteoporosis, cancer, and Alzheimer’s disease. Recently, a class of irreversible inhibitors based on esters of 4-(indolin-1-yl)-4-oxobutanoic acid have been reported. Using the crystal structures of enzyme-4-(indolin-1-yl)-4-oxobutanoate adduct and 4-(indolin-1-yl)-4-oxobutanoic acid-enzyme complex, we studied computationally the proposed inhibition mechanism using model systems based on the own n-layered integrated molecular orbital and molecular mechanics (ONIOM) method. In the first place, model systems were formulated to investigate the transesterification between the catalytic serine residue, Ser-232, and the methyl ester of 4-(indolin-1-yl)-4-oxobutanoate. In the second place, the hydrolysis mechanism of the resultant enzyme–inhibitor adduct was studied. The energetics of these steps were analyzed using a density functional theory functional in the ONIOM method. In addition, the roles of active-site residues during these steps were highlighted. It was found that the hydrolysis of the covalent adduct is highly endergonic corroborating the irreversible inhibition mechanism. These results will shed light not only on the inhibition mechanism but also on the catalytic mechanism.

Kinetic and Structural Properties of a Robust Bacterial L-Amino Acid Oxidase

L-Amino acid oxidase (LAAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme active on most proteinogenic L-amino acids, catalysing their conversion to α-keto acids by oxidative deamination of the substrate. For this oxidation reaction, molecular oxygen is used as the electron acceptor, generating hydrogen peroxide. LAAO can be used to detect L-amino acids, for the production of hydrogen peroxide as an oxidative agent or antimicrobial agent, and for the production of enantiopure amino acids from racemates. In this work, we characterised a previously reported LAAO from the bacterium Pseudoalteromonas luteoviolacea. The substrate scope and kinetic properties of the enzyme were determined, and the thermostability was evaluated. Additionally, we elucidated the crystal structure of this bacterial LAAO, enabling us to test the role of active site residues concerning their function in catalysis. The obtained insights and ease of expression of this thermostable LAAO provides a solid basis for the development of engineered LAAO variants tuned for biosensing and/or biocatalysis.

Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis.

Acid/base catalysis is an important catalytic strategy used by ribonucleases and ribozymes; however, understanding the number and identity of functional groups involved in proton transfer remains challenging. The proton inventory (PI) technique analyzes the dependence of the enzyme reaction rate on the ratio of D2O to H2O and can provide information about the number of exchangeable sites that produce isotope effects and their magnitude. The Gross-Butler (GB) equation is used to evaluate H/D fractionation factors from PI data typically collected under conditions (i.e., a "plateau" in the pH-rate profile) assuming minimal change in active site residue ionization. However, restricting PI analysis to these conditions is problematic for many ribonucleases, ribozymes, and their variants due to ambiguity in the roles of active site residues, the lack of a plateau within the accessible pL range, or cooperative interactions between active site functional groups undergoing ionization. Here, we extend the integration of species distributions for alternative enzyme states in noncooperative models of acid/base catalysis into the GB equation, first used by Bevilacqua and colleagues for the HDV ribozyme, to develop a general population-weighted GB equation that allows simulation and global fitting of the three-dimensional relationship of the D2O ratio (n) versus pL versus kn/k0. Simulations using the GPW-GB equation of PI results for RNase A, HDVrz, and VSrz illustrate that data obtained at multiple selected pL values across the pL-rate profile can assist in the planning and interpreting of solvent isotope effect experiments to distinguish alternative mechanistic models.

Computational Analysis of the Nicotine Oxidoreductase Mechanism by the ONIOM Method.

Nicotine oxidoreductase (NicA2) is a monoamine oxidase (MAO)-based flavoenzyme that catalyzes the oxidation of S-nicotine into N-methylmyosmine. Due to its nanomolar binding affinity toward nicotine, it is seen as an ideal candidate for the treatment of nicotine addiction. Based on the crystal structure of the substrate-bound enzyme, hydrophobic interactions mainly govern the binding of the substrate in the active site through Trp108, Trp364, Trp427, and Leu217 residues. In addition, Tyr308 forms H-bonding with the pyridyl nitrogen of the substrate. Experimental and computational studies support the hydride transfer mechanism for MAO-based enzymes. In this mechanism, a hydride ion transfers from the substrate to the flavin cofactor. In this study, computational models involving the ONIOM method were formulated to study the hydride transfer mechanism based on the crystal structure of the enzyme–substrate complex. The geometry and energetics of the hydride transfer mechanism were analyzed, and the roles of active site residues were highlighted.

Mechanistic study of L-6-hydroxynicotine oxidase by DFT and ONIOM methods.

L-6-Hydroxynicotine oxidase (LHNO) is a member of monoamine oxidase (MAO) family and catalyzes conversion of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during bacterial degradation of nicotine. Recent studies indicated that the enzyme catalyzes oxidation of carbon-nitrogen bond instead of previously proposed carbon-carbon bond. Based on kinetics and mutagenesis studies, Asn166, Tyr311, and Lys287 as well as an active site water molecule have roles in the catalysis of the enzyme. A number of studies including experimental and computational methods support hydride transfer mechanism in MAO family as a common mechanism in which a hydride ion transfer from amine substrate to flavin cofactor is the rate-limiting step. In this study, we formulated computational models to study the hydride transfer mechanism using crystal structure of enzyme-substrate complex. The calculations involved ONIOM and DFT methods, and we evaluated the geometry and energetics of the hydride transfer process while probing the roles of active site residues. Based on the calculations involving hydride, radical, and polar mechanisms, it was concluded that hydride transfer mechanism is the only viable mechanism for LHNO.

Elucidating the Molecular Basis of Avibactam-Mediated Inhibition of Class A β-Lactamases.

Disseminating antibiotic resistance rendered by bacteria against the widely used β-lactam antibiotics is a serious concern for public health care. The development of inhibitors for drug-resistant β-lactamase enzymes is vital to combat this rapidly escalating problem. Recently, the U.S. Food and Drug Administration approved a non-β-lactam inhibitor called avibactam for the treatment of complicated intra-abdominal and urinary tract infections caused by drug-resistant Gram-negative bacteria. This work sheds light on the molecular origin of the inhibitory effect of avibactam against the drug-resistant CTX-M variant of class A β-lactamases. In particular, we probed the structural evolution, dynamics features, and energetics along the acylation and deacylation reaction pathways through enhanced sampling molecular dynamics methods and free-energy calculations. We scrutinized the roles of active site residues, the nature of the carbamoyl linkage formed in the inhibitor-enzyme covalent intermediate, and other structural features of the inhibitor molecule. By unraveling the reasons behind the inhibition of all the deacylation routes, we can explain various experimental structural and kinetics data, and propose a way to design new inhibitors based on the β-lactam framework.

Computational mechanistic study of human liver glycerol 3‐phosphate dehydrogenase using ONIOM method

AbstractHuman liver glycerol 3‐phosphate dehydrogenase catalyzes transfer of a hydride anion from NADH to dihydroxyacetone phosphate (DHAP) forming L‐glycerol 3‐phosphate and NAD+ in a single step reaction. It is proposed that a hydride ion is transferred from NADH to the carbonyl carbon of DHAP through a general acid catalysis in which the carbonyl oxygen of DHAP is protonated by a nearby acidic residue. Based on the crystal structure of enzyme, it was suggested that one of the active site lysine residues (Lys120/Lys204) might act as general acid for the protonation of the carbonyl oxygen at DHAP. In this study, we formulated a number of computational systems to study the hydride transfer mechanism including main active site amino acid side chains, NADH cofactor, and DHAP. The calculations involved ONIOM method consisting of DFT and molecular mechanics (MM). We evaluated the energetics of the hydride transfer process in different model systems while probing the roles of active site residues, Lys120/Lys204/Asp260. Based on calculations, protonated Asp260 has more favorable energetics to act as the general acid catalysis as compared to Lys120/204 residues.

Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

Understanding the role of active site residues in CotB2 catalysis using a cluster model.

Terpene cyclases are responsible for the initial cyclization cascade in the multistep synthesis of a large number of terpenes. CotB2 is a diterpene cyclase from Streptomyces melanosporofaciens, which catalyzes the formation of cycloocta-9-en-7-ol, a precursor to the next-generation anti-inflammatory drug cyclooctatin. In this work, we present evidence for the significant role of the active site's residues in CotB2 on the reaction energetics using quantum mechanical calculations in an active site cluster model. The results revealed the significant effect of the active site residues on the relative electronic energy of the intermediates and transition state structures with respect to gas phase data. A detailed understanding of the role of the enzyme environment on the CotB2 reaction cascade can provide important information towards a biosynthetic strategy for cyclooctatin and the biomanufacturing of related terpene structures.

Bioactive molecules from eucalyptus essential oil as potential inhibitors of COVID 19 corona virus infection by molecular docking studies

Across the world, COVID-19, a positive single-stranded RNA virus member of corona virus family, is spreading its tentacles due to lack of drugs at present causing 15% mortality worldwide due to cough, fever and respiratory problems cause more than. Due to its vital role in polyprotein processing necessary for coronavirus reproduction, the main viral proteinase (Mpro/3CLpro) has recently been regarded as a suitable target for drug design against SARS infection. The present in silico docking study was designed to evaluate the effect of potent 12 bioactive molecules present in essential oils of eucalyptus plant leaves. In the present study, molecular docking studies were conducted using a 1-click dock. Docked complex analysis was carried out using the Biovia Discovery studio, Chimera and VMD tools. Active sites were obtained from CAST P analysis. The calculated parameters such as RMSD, docking score indicated effective binding of bioactive essential oil molecules, especially Jensenone, to COVID-19 proteinase. Active site prediction further validated the role of active site residues in ligand binding. Molecular interactions results indicated that Mpro/eucalyptol complexes form hydrophobic interactions, hydrogen bond interactions and strong ionic interactions. Therefore, eucalyptus essential oil bioactive compounds may represent potential treatment potential to act as COVID-19 Mpro inhibitor. However, further research is necessary to investigate their potential medicinal use.

Function of active site residues in the dUTPase from Epstein‐Barr Virus

Epstein‐Barr Virus, which causes infectious mononucleosis, contributes to many diseases such as Burkitt lymphoma and other cancers. The viral genome encodes a dUTPase, an enzyme that catalyzes the reaction of dUTP + H2O to dUMP + PPi + H+. This is an intermediate reaction involved in the formation of the thymidine nucleotide. The dUTPase found in humans is a trimeric enzyme containing three active sites while the viral enzyme is monomeric. Due to variation in the structures of the human and viral proteins, there may be differences in each enzyme's catalytic activity. Our results demonstrate the importance of a metal cofactor for activity of the viral enzyme. The role of active site residues in substrate binding and the catalysis of dUTP to dUMP is being examined by site‐directed mutagenesis. Characterization of the EBV dUTPase may facilitate the discovery of inhibitors. Inhibition of dUTPase decreases the formation of thymidine nucleotides creating a high dUTP/dTTP ratio. Uracil becomes incorporated into DNA in place of thymine which causes mutations that may be detrimental to cell survival. Selectively inhibiting the dUTPase from Epstein‐Barr Virus provides a mechanism to target cells infected with the virus without damaging uninfected cells.Support or Funding InformationUWL Undergraduate Research and Creativity GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.