scholarly journals Reshaping the binding channel of a novel GH113 family β-Mannanase from Paenibacillus cineris (PcMan113) for enhanced activity

2021 ◽  
Author(s):  
Dengyue Sun ◽  
Chao Li ◽  
Pengpeng Cui ◽  
Jie Zhang ◽  
Yaolin Zhou ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Efficiency ◽  
Md Simulations ◽  
Homology Model ◽  
Commercial Application ◽  
Structural Homology ◽  
Highly Active ◽  
Active Variant ◽  
Significant Health ◽  
Important Enzyme

Abstract Endo-β-mannanases are an important enzyme for degrading lignocellulosic biomass to generate mannan, which has significant health effects as a prebiotic that promotes the development of gut microbiota. Here, a novel endo-β-mannanase belonging to glycoside hydrolase (GH) family 113 from Paenibacillus cineris (PcMan113) was cloned, expressed and characterized, as one of only a few reported GH113 family β-mannanases. Compared to other functionally and structurally characterized GH113 mannanases, recombinant PcMan113 showed a broader substrate spectrum and a better performance. Based on a structural homology model, the highly active mutant PcMT3 (F110E/N246Y) was obtained, with 4.60- and 5.53-fold increases of enzyme activity (towards KG) and catalytic efficiency (kcat/Km, against M5) compared with the WT enzyme, respectively. Furthermore, molecular dynamics (MD) simulations were conducted to precisely explore the differences of catalytic activity between WT and PcMT3, which revealed that PcMT3 has a less flexible conformation, as well as an enlarged substrate binding channel with decreased steric hindrance and increased binding energy in substrate recognition. In conclusion, we obtained a highly active variant of PcMan113 with potential for commercial application in the manufacture of mannooligosaccharides.

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

scholarly journals The role of side chains in the interaction of new antitumor pyrimidoacridinetriones with DNA: molecular dynamics simulations.

2000 ◽  
Vol 47 (1) ◽  
pp. 47-57 ◽  
Author(s):  
J Mazerski ◽  
I Antonini ◽  
S Martelli
Keyword(s):  
Molecular Dynamics ◽  
Rational Design ◽  
Md Simulations ◽  
Ring System ◽  
Side Chain ◽  
Side Chains ◽  
Intercalation Complex ◽  
Highly Active ◽  
Simulation Techniques ◽  
Dynamics Simulations

Pyrimidoacridinetriones (PATs) are a new group of highly active antitumor compounds. It seems reasonable to assume that, like for some other acridine derivatives, intercalation into DNA is a necessary, however not a sufficient condition for antitumor activity of these compounds. Rational design of new compounds of this chemotype requires knowledge about the structure of the intercalation complex, as well as about interactions responsible for its stability. Computer simulation techniques such as molecular dynamics (MD) may provide valuable information about these problems. The results of MD simulations performed for three rationally selected PATs are presented in this paper. The compounds differ in the number and position of side chains. Each of the compounds was simulated in two systems: i) in water, and ii) in the intercalation complex with the dodecamer duplex d(GCGCGCGCGCGC)2. The orientation of the side chain in relation to the ring system is determined by the position of its attachment. Orientation of the ring system inside the intercalation cavity depends on the number and position of side chain(s). The conformations of the side chain(s) of all PATs studied in the intercalation complex were found to be very similar to those observed in water.

scholarly journals Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

2010 ◽  
Vol 76 (5) ◽  
pp. 1653-1660 ◽  
Author(s):  
Ponnandy Prabhu ◽  
Marimuthu Jeya ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Catalytic Efficiency ◽  
Homology Model ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
High Turnover ◽  
Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

scholarly journals Highly Active and Stable Large Catalase Isolated from a Hydrocarbon Degrading Aspergillus terreus MTCC 6324

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Preety Vatsyayan ◽  
Pranab Goswami
Keyword(s):  
Molecular Mass ◽  
Aspergillus Terreus ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Highly Active ◽  
Peptide Mass ◽  
Tandem Mass Spectroscopy ◽  
Isoelectric Ph ◽  
Alkaline Ph Stability ◽  
High Level

A hydrocarbon degrading Aspergillus terreus MTCC 6324 produces a high level of extremely active and stable cellular large catalase (CAT) during growth on n-hexadecane to combat the oxidative stress caused by the hydrocarbon degrading metabolic machinery inside the cell. A 160-fold purification with specific activity of around 66 × 105 U mg−1 protein was achieved. The native protein molecular mass was 368 ± 5 kDa with subunit molecular mass of nearly 90 kDa, which indicates that the native CAT protein is a homotetramer. The isoelectric pH (pI) of the purified CAT was 4.2. BLAST aligned peptide mass fragments of CAT protein showed its highest similarity with the catalase B protein from other fungal sources. CAT was active in a broad range of pH 4 to 12 and temperature 25°C to 90°C. The catalytic efficiency (Kcat/Km) of 4.7 × 108 M−1 s−1 within the studied substrate range and alkaline pH stability (half-life, t1/2 at pH 12~15 months) of CAT are considerably higher than most of the extensively studied catalases from different sources. The storage stability (t1/2) of CAT at physiological pH 7.5 and 4°C was nearly 30 months. The haem was identified as haem b by electrospray ionization tandem mass spectroscopy (ESI-MS/MS).

scholarly journals Structural basis for stereospecific inhibition of ASCT2 from rational design

2020 ◽  
Author(s):  
Rachel-Ann A. Garibsingh ◽  
Elias Ndaru ◽  
Alisa A. Garaeva ◽  
Massimiliano Bonomi ◽  
Dirk J. Slotboom ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Rational Design ◽  
Md Simulations ◽  
Homology Model ◽  
Structural Basis ◽  
Protein Targets ◽  
Peripheral Tissues ◽  
Ligand Discovery ◽  
Multiple Conformations

ABSTRACTASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. ASCT2 is upregulated in cancer, where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides an emerging strategy for cancer therapy. Here, guided by a homology model of ASCT2 in an outward-facing conformation, we rationally designed novel inhibitors exploiting stereospecific pockets in the substrate binding site. A cryo-EM structure of ASCT2 in complex with inhibitor (Lc-BPE) validated our predictions and was subsequently refined based on computational analysis. The final structures, combined with MD simulations, show that the inhibitor samples multiple conformations in the ASCT2 binding site. Our results demonstrate the utility of combining computational modeling and cryo-EM for SLC ligand discovery, and a viable strategy for structure determination of druggable conformational states for challenging membrane protein targets.

scholarly journals Mesoporous bimetallic Fe/Co as highly active heterogeneous Fenton catalyst for the degradation of tetracycline hydrochlorides

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Junchao Li ◽  
Xuhua Li ◽  
Jindong Han ◽  
Fansheng Meng ◽  
Jinyuan Jiang ◽  
...  
Keyword(s):  
Bimetallic Catalyst ◽  
Removal Rate ◽  
Catalytic Efficiency ◽  
Nitrogen Adsorption ◽  
Catalytic Performance ◽  
Operating Parameters ◽  
Co Catalyst ◽  
Highly Active ◽  
Fenton Catalyst ◽  
Potential Applications

Abstract Mesoporous bimetallic Fe/Co was prepared as a Fenton-like catalyst to degrade the tetracycline hydrochlorides (TC). The nanocasting strategy with KIT-6 as a hard template was carried out to synthesize the mesoporous bimetallic catalyst. The mesoporous bimetallic Fe/Co catalyst was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), nitrogen adsorption-desorption isotherms, and Brunauer-Emmett-Teller (BET) method. The results showed that the catalyst has significant nanofeatures; the surface area, pore size, and particle size were 113.8 m2g−1, 4 nm, and 10 nm, respectively. In addition, the effects of the operating parameters, such as the iron-to-cobalt ratio, pH, H2O2, and initial TC concentrations on its catalytic performance were investigated. The best operating parameters were as follows: iron-to-cobalt ratio = 2:1 to 1:1, pH = 5–9, H2O2: 30 mmol, initial TC less than 30 mg/L. Furthermore, the mesoporous bimetallic Fe/Co showed a good performance for degrading TC, achieving a removal rate of 86% of TC after 3 h under the reaction conditions of H2O2 = 30 mmol, mesoporous bimetallic Fe/Co = 0.6 g/L, TC = 30 mg/L, pH = 7.0, and temperature = 25.5 °C. The mesoporous bimetallic Fe/Co catalyst shows good stability and reusability. This work demonstrated that mesoporous bimetallic Fe/Co has excellent catalytic efficiency, smaller amounts of leached ions, and wider pH range, which enhance its potential applications.

scholarly journals Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Laura Plaza-Vinuesa ◽  
Oswaldo Hernandez-Hernandez ◽  
F. Javier Moreno ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Glycoside Hydrolase ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Structural Features ◽  
Biochemical Properties ◽  
Glycoside Hydrolase Family ◽  
Reaction Conditions ◽  
Genes Encoding ◽  
Hydrolysis Of

Abstract Background α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. Results In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. Conclusions The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

scholarly journals Highly Active Hydrogenation Catalysts Based on Pd Nanoparticles Dispersed along Hierarchical Porous Silica Covered with Polydopamine as Interfacial Glue

Catalysts ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 449
Author(s):  
Miguel Ródenas ◽  
Jamal El Haskouri ◽  
José Vicente Ros-Lis ◽  
M. Dolores Marcos ◽  
Pedro Amorós ◽  
...  
Keyword(s):  
Porous Silica ◽  
Catalytic Efficiency ◽  
Pd Nanoparticles ◽  
Hydrogenation Catalysts ◽  
Highly Active ◽  
Hierarchical Porous ◽  
Similar Materials ◽  
Comprehensive Comparison ◽  
Palladium Content

New catalysts based on Pd(0) nanoparticles (Pd NPs) on a bimodal porous silica of the UVM-7/polydopamine (PDA) support have been synthesized following two preparative strategies based on the sequential or joint incorporation of two components of the composite (Pd and PDA). We analyzed the role played by the PDA as ‘interfacial glue’ between the silica scaffold and the Pd NPs. The catalysts were tested for the hydrogenation of 4-nitrophenol using (NEt4)BH4 as the hydrogenating agent. In addition to the palladium content, the characterization of the catalysts at the micro and nanoscale has highlighted the importance of different parameters, such as the size and dispersion of the Pd NPs, as well as their accessibility to the substrate (greater or lesser depending on their entrapment level in the PDA) on the catalytic efficiency. Staged sequential synthesis has led to better catalytic results. The most active Pd(0) centers seem to be Pd NPs of less than 1 nm on the PDA surface. The efficiency of the catalysts obtained is superior to that of similar materials without PDA. A comprehensive comparison has been made with other catalysts based on Pd NPs in a wide variety of supports. The TOF values achieved are among the best described in the literature.

scholarly journals Experimental accuracy in protein structure refinement via molecular dynamics simulations

2018 ◽  
Vol 115 (52) ◽  
pp. 13276-13281 ◽  
Author(s):  
Lim Heo ◽  
Michael Feig
Keyword(s):  
Molecular Dynamics ◽  
Protein Structure ◽  
Structure Prediction ◽  
Structure Refinement ◽  
Energy Landscape ◽  
Md Simulations ◽  
Homology Model ◽  
Experimental Accuracy ◽  
Markov State Modeling ◽  
Homology Models

Refinement is the last step in protein structure prediction pipelines to convert approximate homology models to experimental accuracy. Protocols based on molecular dynamics (MD) simulations have shown promise, but current methods are limited to moderate levels of consistent refinement. To explore the energy landscape between homology models and native structures and analyze the challenges of MD-based refinement, eight test cases were studied via extensive simulations followed by Markov state modeling. In all cases, native states were found very close to the experimental structures and at the lowest free energies, but refinement was hindered by a rough energy landscape. Transitions from the homology model to the native states require the crossing of significant kinetic barriers on at least microsecond time scales. A significant energetic driving force toward the native state was lacking until its immediate vicinity, and there was significant sampling of off-pathway states competing for productive refinement. The role of recent force field improvements is discussed and transition paths are analyzed in detail to inform which key transitions have to be overcome to achieve successful refinement.

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