Biochemical study of fibrinolytic protease from Euphausia superba possessing multifunctional serine protease activity

Background: Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated. Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated. Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4- (2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study. Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1 /mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data. Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill’s body was suggested.

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In SilicoIdentification of Potent PPAR-γAgonists from Traditional Chinese Medicine: A Bioactivity Prediction, Virtual Screening, and Molecular Dynamics Study

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2014/192452 ◽

2014 ◽

Vol 2014 ◽

pp. 1-19 ◽

Cited By ~ 6

Author(s):

Kuan-Chung Chen ◽

Calvin Yu-Chian Chen

Keyword(s):

Molecular Dynamics ◽

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Virtual Screening ◽

Prediction Models ◽

Protein Complexes ◽

Md Simulations ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptors ◽

Key Residues

The peroxisome proliferator-activated receptors (PPARs) related to regulation of lipid metabolism, inflammation, cell proliferation, differentiation, and glucose homeostasis by controlling the related ligand-dependent transcription of networks of genes. They are used to be served as therapeutic targets against metabolic disorder, such as obesity, dyslipidemia, and diabetes; especially, PPAR-γis the most extensively investigated isoform for the treatment of dyslipidemic type 2 diabetes. In this study, we filter compounds of traditional Chinese medicine (TCM) using bioactivities predicted by three distinct prediction models before the virtual screening. For the top candidates, the molecular dynamics (MD) simulations were also utilized to investigate the stability of interactions between ligand and PPAR-γprotein. The top two TCM candidates, 5-hydroxy-L-tryptophan and abrine, have an indole ring and carboxyl group to form the H-bonds with the key residues of PPAR-γprotein, such as residues Ser289 and Lys367. The secondary amine group of abrine also stabilized an H-bond with residue Ser289. From the figures of root mean square fluctuations (RMSFs), the key residues were stabilized in protein complexes with 5-Hydroxy-L-tryptophan and abrine as control. Hence, we propose 5-hydroxy-L-tryptophan and abrine as potential lead compounds for further study in drug development process with the PPAR-γprotein.

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Conformational Change of H64 and Substrate Transportation: Insight Into a Full Picture of Enzymatic Hydration of CO2 by Carbonic Anhydrase

Frontiers in Chemistry ◽

10.3389/fchem.2021.706959 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuzhuang Fu ◽

Fangfang Fan ◽

Yuwei Zhang ◽

Binju Wang ◽

Zexing Cao

Keyword(s):

Free Energy ◽

Carbonic Anhydrase ◽

Proton Transfer ◽

Energy Barrier ◽

Conformational Transition ◽

Md Simulations ◽

Nucleophilic Attack ◽

Free Energy Barrier ◽

Full Picture ◽

Key Residues

The enzymatic hydration of CO2 into HCO3− by carbonic anhydrase (CA) is highly efficient and environment-friendly measure for CO2 sequestration. Here extensive MM MD and QM/MM MD simulations were used to explore the whole enzymatic process, and a full picture of the enzymatic hydration of CO2 by CA was achieved. Prior to CO2 hydration, the proton transfer from the water molecule (WT1) to H64 is the rate-limiting step with the free energy barrier of 10.4 kcal/mol, which leads to the ready state with the Zn-bound OH−. The nucleophilic attack of OH− on CO2 produces HCO3− with the free energy barrier of 4.4 kcal/mol and the free energy release of about 8.0 kcal/mol. Q92 as the key residue manipulates both CO2 transportation to the active site and release of HCO3−. The unprotonated H64 in CA prefers in an inward orientation, while the outward conformation is favorable energetically for its protonated counterpart. The conformational transition of H64 between inward and outward correlates with its protonation state, which is mediated by the proton transfer and the product release. The whole enzymatic cycle has the free energy span of 10.4 kcal/mol for the initial proton transfer step and the free energy change of −6.5 kcal/mol. The mechanistic details provide a comprehensive understanding of the entire reversible conversion of CO2 into bicarbonate and roles of key residues in chemical and nonchemical steps for the enzymatic hydration of CO2.

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Key Interacting Residues Between RBD of SARS-CoV-2 and ACE2 Receptor: Combination of Molecular Dynamic Simulation and Density Functional Calculation

10.26434/chemrxiv.14582484.v1 ◽

2021 ◽

Author(s):

Bahaa Jawad ◽

Puja Adhikari ◽

Rudolf Podgornik ◽

Wai-Yim Ching

Keyword(s):

Density Functional ◽

Vaccine Development ◽

Hydrophobic Interactions ◽

Binding Free Energy ◽

Tight Binding ◽

Md Simulations ◽

Functional Theory ◽

Generalized Born ◽

The Density Functional Theory ◽

Key Residues

The spike protein of SARS-CoV-2 binds to ACE2 receptor via its receptor-binding domain (RBD), with the RBD-ACE2 complex presenting an essential molecular target for vaccine development to stall the virus infection proliferation. The computational analysis at molecular, amino acid (AA) and atomic levels have been performed systematically to identify the key interacting AAs in the formation of the RBD-ACE2 complex, including the MD simulations with molecular mechanics generalized Born surface area (MM-GBSA) method to predict binding free energy (BFE) and to determine the actual interacting AAs, as well as two ab initio quantum chemical protocols based on the density functional theory (DFT) implementation. Based on MD results, Q493, Y505, Q498, N501, T500, N487, Y449, F486, K417, Y489, F456, Y495, and L455 have been identified as hotspots in RBD, while those in ACE2 are K353, K31, D30, D355, H34, D38, Q24, T27, Y83, Y41, E35, and E37. Both the electrostatic and hydrophobic interactions are the main driving force to form the AA-AA binding pairs. We confirm that Q493, N501, F486, K417, and F456 in RBD are the key residues responsible for the tight binding of SARS-CoV-2 with ACE2 compared to SARS-CoV. The DFT results reveal that N487, Q493, Y449, T500, G496, G446 and G502 in RBD form pairs via specific hydrogen bonding with Q24, H34, E35, D38, Y41, Q42 and K353 in ACE2.

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Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011322 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3734-3745

Author(s):

Chia-Yu Kang ◽

I-Hsiu Huang ◽

Chi-Chi Chou ◽

Tsai-Yu Wu ◽

Jyun-Cyuan Chang ◽

...

Keyword(s):

Cell Wall ◽

Catalytic Activity ◽

Clostridium Difficile ◽

Substrate Specificity ◽

Surface Proteins ◽

Site Directed Mutagenesis ◽

Serine Residue ◽

Peptidoglycan Cell Wall ◽

Attractive Therapeutic Target ◽

Key Residues

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus. The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB–substrate complexes. Furthermore, we also demonstrated that residues 163–168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

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Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase

Journal of Biological Chemistry ◽

10.1074/jbc.m116.744912 ◽

2016 ◽

Vol 291 (37) ◽

pp. 19401-19412 ◽

Cited By ~ 16

Author(s):

Xuewu Sui ◽

Jianye Zhang ◽

Marcin Golczak ◽

Krzysztof Palczewski ◽

Philip D. Kiser

Keyword(s):

Metal Coordination ◽

Carotenoid Cleavage Dioxygenase ◽

Catalytic Function ◽

Key Residues

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Computational Investigation of Bisphosphate Inhibitors of 3-Deoxy-d-manno-octulosonate 8-phosphate Synthase

Molecules ◽

10.3390/molecules24132370 ◽

2019 ◽

Vol 24 (13) ◽

pp. 2370 ◽

Cited By ~ 3

Author(s):

Jéssica de Oliveira Araújo ◽

Alberto Monteiro dos Santos ◽

Jerônimo Lameira ◽

Cláudio Nahum Alves ◽

Anderson Henrique Lima

Keyword(s):

Active Sites ◽

Antimicrobial Agents ◽

Md Simulations ◽

Hydroxyl Groups ◽

Ligand Complex ◽

Effective Interactions ◽

Key Enzyme ◽

The Many ◽

Key Residues ◽

Computational Molecular Modeling

The synthase, 3-deoxy-d-manno-octulosonate 8-phosphate (KDO8P), is a key enzyme for the lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria and a potential target for developing new antimicrobial agents. In this study, computational molecular modeling methods were used to determine the complete structure of the KDO8P synthase from Neisseria meningitidis and to investigate the molecular mechanism of its inhibition by three bisphosphate inhibitors: BPH1, BPH2, and BPH3. Our results showed that BPH1 presented a protein–ligand complex with the highest affinity, which is in agreement with experimental data. Furthermore, molecular dynamics (MD) simulations showed that BPH1 is more active due to the many effective interactions, most of which are derived from its phosphoenolpyruvate moiety. Conversely, BPH2 exhibited few hydrogen interactions during the MD simulations with key residues located at the active sites of the KDO8P synthase. In addition, we hydroxylated BPH2 to create the hypothetical molecule named BPH3, to investigate the influence of the hydroxyl groups on the affinity of the bisphosphate inhibitors toward the KDO8P synthase. Overall, we discuss the main interactions between the KDO8P synthase and the bisphosphate inhibitors that are potential starting points for the design of new molecules with significant antibiotic activities.

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Insight into Structural Characteristics of Protein-Substrate Interaction in Pimaricin Thioesterase

International Journal of Molecular Sciences ◽

10.3390/ijms20040877 ◽

2019 ◽

Vol 20 (4) ◽

pp. 877 ◽

Cited By ~ 2

Author(s):

Shuobing Fan ◽

Rufan Wang ◽

Chen Li ◽

Linquan Bai ◽

Yi-Lei Zhao ◽

...

Keyword(s):

Molecular Conformation ◽

Conformational Transition ◽

Hydrophobic Interactions ◽

Mutual Recognition ◽

Structural Characteristics ◽

Md Simulations ◽

Binding Energies ◽

Product Release ◽

Molecular Hydrogen Bond ◽

Key Residues

As a polyene antibiotic of great pharmaceutical significance, pimaricin has been extensively studied to enhance its productivity and effectiveness. In our previous studies, pre-reaction state (PRS) has been validated as one of the significant conformational categories before macrocyclization, and is critical to mutual recognition and catalytic preparation in thioesterase (TE)-catalyzed systems. In our study, molecular dynamics (MD) simulations were conducted on pimaricin TE-polyketide complex and PRS, as well as pre-organization state (POS), a molecular conformation possessing a pivotal intra-molecular hydrogen bond, were detected. Conformational transition between POS and PRS was observed in one of the simulations, and POS was calculated to be energetically more stable than PRS by 4.58 kcal/mol. The structural characteristics of PRS and POS-based hydrogen-bonding, and hydrophobic interactions were uncovered, and additional simulations were carried out to rationalize the functions of several key residues (Q29, M210, and R186). Binding energies, obtained from MM/PBSA calculations, were further decomposed to residues, in order to reveal their roles in product release. Our study advanced a comprehensive understanding of pimaricin TE-catalyzed macrocyclization from the perspectives of conformational change, protein-polyketide recognition, and product release, and provided potential residues for rational modification of pimaricin TE.

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Computational drug repurposing study elucidating simultaneous inhibition of entry and replication of novel corona virus by Grazoprevir

Scientific Reports ◽

10.1038/s41598-021-86712-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Santosh Kumar Behera ◽

Nazmina Vhora ◽

Darshan Contractor ◽

Amit Shard ◽

Dinesh Kumar ◽

...

Keyword(s):

Viral Entry ◽

Clinical Studies ◽

Drug Repurposing ◽

Md Simulations ◽

Angiotensin Converting Enzyme 2 ◽

Multiple Pathway ◽

Poisson Boltzmann ◽

Drug Receptor ◽

Transmembrane Serine Protease ◽

Key Residues

AbstractOutcomes of various clinical studies for the coronavirus disease 2019 (COVID-19) treatment indicated that the drug acts via inhibition of multiple pathways (targets) is likely to be more successful and promising. Keeping this hypothesis intact, the present study describes for the first-time, Grazoprevir, an FDA approved anti-viral drug primarily approved for Hepatitis C Virus (HCV), mediated multiple pathway control via synergistic inhibition of viral entry targeting host cell Angiotensin-Converting Enzyme 2 (ACE-2)/transmembrane serine protease 2 (TMPRSS2) and viral replication targeting RNA-dependent RNA polymerase (RdRP). Molecular modeling followed by in-depth structural analysis clearly demonstrated that Grazoprevir interacts with the key residues of these targets. Futher, Molecular Dynamics (MD) simulations showed stability and burial of key residues after the complex formation. Finally, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis identified the governing force of drug-receptor interactions and stability. Thus, we believe that Grazoprevir could be an effective therapeutics for the treatment of the COVID-19 pandemic with a promise of unlikely drug resistance owing to multiple inhibitions of eukaryotic and viral proteins, thus warrants further clinical studies.

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Key Interacting Residues Between RBD of SARS-CoV-2 and ACE2 Receptor: Combination of Molecular Dynamic Simulation and Density Functional Calculation

10.26434/chemrxiv.14582484 ◽

2021 ◽

Author(s):

Bahaa Jawad ◽

Puja Adhikari ◽

Rudolf Podgornik ◽

Wai-Yim Ching

Keyword(s):

Density Functional ◽

Vaccine Development ◽

Hydrophobic Interactions ◽

Binding Free Energy ◽

Tight Binding ◽

Md Simulations ◽

Functional Theory ◽

Generalized Born ◽

The Density Functional Theory ◽

Key Residues

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Studying of the complexes product of the nerve agent Soman with the Butyrylcholinesterase and Acetylcholinesterase Enzymes

Baghdad Science Journal ◽

10.21123/bsj.5.2.297-304 ◽

2008 ◽

Vol 5 (2) ◽

pp. 297-304

Author(s):

Baghdad Science Journal

Keyword(s):

Active Site ◽

Unknown Function ◽

Reaction Path ◽

Nerve Agent ◽

Serine Residue ◽

Catalytic Function ◽

Mechanism Of Inhibition ◽

3D Geometry ◽

Degradation Reaction ◽

Hydrolysis Of

Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase (AChE) involved in the hydrolysis of the neurotransimitter acetylcholine, and butyrylcholinesterase (BChE) of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge. Human BChE has attracted attention because it can hydrolyze toxic esters and nerve agents. Here we analyze the complexes of cholinesterase with soman by describing the 3D geometry of the complex, the active site, the changes happened through the inhibition and provide a description for the mechanism of inhibition. Soman undergoes degradation in the active site of the AChE and BChE. We calculate the energy of the products of the degradation reaction and suggest the reaction path. The product of the former reaction bind to serine residue in the active site and forming a stable bond and ends the catalytic function of the enzyme. This study has a useful role in the search of inhibitors to help in the treatment of Alzahimer's disease.

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