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

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2370 ◽  
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.

scholarly journals Analysis of mutations leading to para-aminosalicylic acid resistance in Mycobacterium tuberculosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bharati Pandey ◽  
Sonam Grover ◽  
Jagdeep Kaur ◽  
Abhinav Grover
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Acid Resistance ◽  
Principal Component ◽  
Md Simulations ◽  
Missense Mutations ◽  
Aminosalicylic Acid ◽  
Folate Pathway ◽  
Key Enzyme ◽  
Key Residues

Abstract Thymidylate synthase A (ThyA) is the key enzyme involved in the folate pathway in Mycobacterium tuberculosis. Mutation of key residues of ThyA enzyme which are involved in interaction with substrate 2′-deoxyuridine-5′-monophosphate (dUMP), cofactor 5,10-methylenetetrahydrofolate (MTHF), and catalytic site have caused para-aminosalicylic acid (PAS) resistance in TB patients. Focusing on R127L, L143P, C146R, L172P, A182P, and V261G mutations, including wild-type, we performed long molecular dynamics (MD) simulations in explicit solvent to investigate the molecular principles underlying PAS resistance due to missense mutations. We found that these mutations lead to (i) extensive changes in the dUMP and MTHF binding sites, (ii) weak interaction of ThyA enzyme with dUMP and MTHF by inducing conformational changes in the structure, (iii) loss of the hydrogen bond and other atomic interactions and (iv) enhanced movement of protein atoms indicated by principal component analysis (PCA). In this study, MD simulations framework has provided considerable insight into mutation induced conformational changes in the ThyA enzyme of Mycobacterium.

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

2020 ◽  
Vol 27 ◽  
Author(s):  
Guo-Ying Qian ◽  
Gyutae Lim ◽  
Shang-Jun Yin ◽  
Jun-Mo Yang ◽  
Jinhyuk Lee ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Fluorescence Spectra ◽  
Biochemical Study ◽  
Euphausia Superba ◽  
Md Simulations ◽  
Time Interval ◽  
Serine Residue ◽  
Catalytic Function ◽  
Serine Protease Activity ◽  
Key Residues

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.

scholarly journals Insight into Inhibitor Binding in the Eukaryotic Proteasome: Computations of the 20S CP

2018 ◽  
Vol 19 (12) ◽  
pp. 3858
Author(s):  
Milan Hodošček ◽  
Nadia Elghobashi-Meinhardt
Keyword(s):  
Electrostatic Interactions ◽  
Active Sites ◽  
Sampling Period ◽  
Md Simulations ◽  
Structural Features ◽  
Relaxation Dynamics ◽  
Inhibitor Binding ◽  
Computational Analyses ◽  
Insight Into ◽  
Proteolytic Chamber

A combination of molecular dynamics (MD) simulations and computational analyses uncovers structural features that may influence substrate passage and exposure to the active sites within the proteolytic chamber of the 20S proteasome core particle (CP). MD simulations of the CP reveal relaxation dynamics in which the CP slowly contracts over the 54 ns sampling period. MD simulations of the SyringolinA (SylA) inhibitor within the proteolytic B 1 ring chamber of the CP indicate that favorable van der Waals and electrostatic interactions account for the predominant association of the inhibitor with the walls of the proteolytic chamber. The time scale required for the inhibitor to travel from the center of the proteolytic chamber to the chamber wall is on the order of 4 ns, accompanied by an average energetic stabilization of approximately −20 kcal/mol.

High-synconformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures ofShewanella oneidensisMR-1 uridine phosphorylase in the free form and in complex with uridine

2014 ◽  
Vol 70 (12) ◽  
pp. 3310-3319 ◽  
Author(s):  
Tatyana N. Safonova ◽  
Sergey N. Mikhailov ◽  
Vladimir P. Veiko ◽  
Nadezhda N. Mordkovich ◽  
Valentin A. Manuvera ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Sites ◽  
Shewanella Oneidensis ◽  
Free Form ◽  
Uridine Phosphorylase ◽  
Glycerol Molecule ◽  
Closed Conformation ◽  
Pyrimidine Salvage ◽  
Key Enzyme ◽  
Crystallization Solution

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP fromShewanella oneidensisMR-1 (SoUP) was performed inEscherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-synconformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.

scholarly journals A network of phosphatidylinositol 4,5-bisphosphate binding sites regulates gating of the Ca2+-activated Cl− channel ANO1 (TMEM16A)

2019 ◽  
Vol 116 (40) ◽  
pp. 19952-19962 ◽  
Author(s):  
Kuai Yu ◽  
Tao Jiang ◽  
YuanYuan Cui ◽  
Emad Tajkhorshid ◽  
H. Criss Hartzell
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Neuronal Excitability ◽  
Computational Study ◽  
Channel Gating ◽  
Md Simulations ◽  
Fluid Secretion ◽  
Hydroxyl Groups ◽  
Binding Modes ◽  
Cytoplasmic Extension

ANO1 (TMEM16A) is a Ca2+-activated Cl− channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here, we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid–protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2. Three of these sites captured 85% of all ANO1–PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane–cytosol interface found 3 regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same 3 sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid side chains and phosphate/hydroxyl groups on PI(4,5)P2. Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl− ions. We propose a model consisting of a network of 3 PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.

scholarly journals Psoromic Acid, a Lichen-Derived Molecule, Inhibits the Replication of HSV-1 and HSV-2, and Inactivates HSV-1 DNA Polymerase: Shedding Light on Antiherpetic Properties

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2912 ◽  
Author(s):  
Sherif T. S. Hassan ◽  
Miroslava Šudomová ◽  
Kateřina Berchová-Bímová ◽  
Karel Šmejkal ◽  
Javier Echeverría
Keyword(s):  
Dna Polymerase ◽  
Active Sites ◽  
Inhibitory Action ◽  
Inhibitory Concentration ◽  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Standard Drug ◽  
Key Enzyme ◽  
Hsv 1

Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC50): 1.9 μM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC50: 2.6 μM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC50: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC50): 2.7 μM; SI: 114.8) compared with that of ACV (EC50: 2.8 μM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC50: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC50): 0.7 μM; inhibition constant (Ki): 0.3 μM) compared with reference drugs aphidicolin (IC50: 0.8 μM; Ki: 0.4 μM) and ACV triphosphate (ACV-TP) (IC50: 0.9 μM; Ki: 0.5 μM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.

scholarly journals Zr(IV), La(III), and Ce(IV) Chelates with 2-[(4-[(Z)-1-(2-Hydroxyphenyl)ethylidene]aminobutyl)-ethanimidoyl]phenol: Synthesis, Spectroscopic Characterization, and Antimicrobial Studies

2015 ◽  
Vol 2015 ◽  
pp. 1-15 ◽  
Author(s):  
M. M. El-ajaily ◽  
H. A. Abdullah ◽  
Ahmed Al-janga ◽  
E. E. Saad ◽  
A. A. Maihub
Keyword(s):  
Schiff Base ◽  
Spectral Data ◽  
Schiff Base Ligand ◽  
Active Sites ◽  
Antimicrobial Agents ◽  
Spectroscopic Characterization ◽  
Metal Chelates ◽  
Molar Ratio ◽  
Bacterial Strains ◽  
Activity Data

La(III), Zr(IV), and Ce(IV) chelates of 2-[(4-[(Z)-1-(2-hydroxyphenyl)ethylidene]aminobutyl)-ethanimidoyl]phenol were synthesized and characterized by using several physical techniques. The Schiff base was obtained by refluxing of o-hydroxyacetophenone with 1,4-butanediamine in 2 : 1 molar ratio. The CHN elemental analysis results showed the formation of the Schiff base and the chelates has been found to be in 1 : 1 [M : L] ratio. The molar conductance measurements revealed that all the chelates are nonelectrolytes. Structural elucidations of the ligand and its chelates were based on compatible analytical and spectroscopic evidences. The infrared spectral data revealed that the Schiff base coordinates to the metal ions through active sites which are –OH and –C=N groups. According to the electronic spectral data, an octahedral geometry was proposed for the chelates. The synthesized ligand and its metal chelates were screened for their antimicrobial activity against two Gram negative (Escherichia coli, Salmonella kentucky) and two Gram positive (Lactobacillus fermentum, Streptococcus faecalis) bacterial strains, unicellular fungi (Fusarium solani), and filamentous fungi (Aspergillus niger). The activity data showed that the metal chelates have antibacterial and antifungal activity more than the parent Schiff base ligand against one or more bacterial or fungi species. The results also indicated that the metal chelates are higher sensitive antimicrobial agents as compared to the Schiff base ligand.

scholarly journals Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Catherine P. Chia ◽  
Noriko Inoguchi ◽  
Kyle C. Varon ◽  
Bradley M. Bartholomai ◽  
Hideaki Moriyama
Keyword(s):  
Dictyostelium Discoideum ◽  
Active Sites ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Unicellular Eukaryote ◽  
Gene Encoding ◽  
Conserved Motifs ◽  
N Terminus ◽  
Key Enzyme ◽  
Green Fluorescent

Abstract Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

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

2014 ◽  
Vol 2014 ◽  
pp. 1-19 ◽  
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.

Photochemical mapping of the active site of myosin

1992 ◽  
Vol 336 (1276) ◽  
pp. 55-61 ◽  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Cross Linking ◽  
Detailed Structure ◽  
Atp Binding Site ◽  
Adenine Ring ◽  
Skeletal Myosin ◽  
Key Residues ◽  
Specific Labelling

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the γ-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.

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