scholarly journals Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

2012 ◽  
Vol 79 (5) ◽  
pp. 1555-1562 ◽  
Author(s):  
Leticia L. Torres ◽  
Ángel Cantero ◽  
Mercedes del Valle ◽  
Anabel Marina ◽  
Fernando López-Gallego ◽  
...  
Keyword(s):  
Thermus Thermophilus ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Penicillin Acylase ◽  
Penicillin G ◽  
Content Type ◽  
Marked Preference ◽  
Three Dimensional Models ◽  
Phenylalanine Residue

ABSTRACTA homologue of theEscherichia colipenicillin acylase is encoded in the genomes of several thermophiles, including in differentThermus thermophilusstrains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of theE. colipenicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved theKmfor penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.

scholarly journals Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

1999 ◽  
Vol 43 (3) ◽  
pp. 543-548 ◽  
Author(s):  
Sonia Trépanier ◽  
James R. Knox ◽  
Natalie Clairoux ◽  
François Sanschagrin ◽  
Roger C. Levesque ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Enterobacter Cloacae ◽  
Acid Group ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Class C ◽  
Three Dimensional Models ◽  
Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

scholarly journals Characterization and Dye Decolorization Potential of Two Laccases from the Marine-Derived Fungus Pestalotiopsis sp.

2019 ◽  
Vol 20 (8) ◽  
pp. 1864 ◽  
Author(s):  
Wikee ◽  
Hatton ◽  
Turbé-Doan ◽  
Mathieu ◽  
Daou ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dye Decolorization ◽  
Sea Salt ◽  
Reactive Black 5 ◽  
Surface Charges ◽  
Recombinant Enzymes ◽  
Charged Surfaces ◽  
Three Dimensional Models ◽  
Encoding Genes

: Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of PsLac1 and PsLac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4–6. Different substrate specificities were exhibited, and the lowest Km and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for PsLac1 and PsLac2, respectively. The industrially important dyes—Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5—were more efficiently decolorized by PsLac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and PsLac2 seemed more adapted to the presence of sea salt than PsLac1. The overall surface charges of the predicted PsLac three-dimensional models showed large negatively charged surfaces for PsLac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for PsLac1, as seen in proteins from terrestrial organisms.

Study on the structural stability evaluation of umbilical winch for ROV LARS

2018 ◽  
Vol 35 (1) ◽  
pp. 202-210
Author(s):  
Namsub Woo ◽  
Sangmok Han ◽  
Youngju Kim ◽  
Sunchul Huh ◽  
Hyunji Kim
Keyword(s):  
Structural Analysis ◽  
Dynamic Simulation ◽  
Structural Stability ◽  
Three Dimensional ◽  
High Technology ◽  
Design Methods ◽  
Stability Evaluation ◽  
Content Type ◽  
Dimensional Models ◽  
Three Dimensional Models

Purpose The purpose of this study is structural stability evaluation of umbilical winch. In accordance with the recent trend for developing natural resources, high-technology equipment on exploration ships is becoming more technologically advanced. One such piece of high-technology equipment is the umbilical winch. In this study, the umbilical winch is divided into two parts (drum and winch), where each is respectively designed with three dimensional models using CATIA, and dynamic simulation and structural analysis are performed using ANSYS. Design/methodology/approach In this paper, the winch is divided into two parts for finite element analysis, the drum and whole winch model, and the parts are designed as three-dimensional models except for some small parts, such as bolt holes. Dynamic simulation and structural analysis are then performed using ANSYS. The analysis results ensure the reliability of the design methods and will be used in the domestic localization of remote operated vehicle (ROV) launch and recovery systems (LARS). Findings The strain is identified from the results, but it is very small. Some stress is concentrated at the lower corner of the drum, but the maximum stress value is lower than the allowable stress; therefore, the structure has no impact on the strain and stress. Thus, it is determined that the designed structure is safe. The results ensure the reliability of the design methods and will be used in the domestic localization of ROV LARS. Originality/value Previous studies focus on the static and mechanic problems of the winch by considering winch and drum breakage in the umbilical winch system. However, ships have a nonlinear motion characteristic with six degrees of freedom according to the constant influence of the external environment. In addition, from a design perspective, the dynamic characteristics (e.g. the ship’s motions) are more important than the static characteristics. Thus, the authors focus on winch stability securement with variable loads, such as ships moving, wave disturbance and other such important environment conditions.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Alkaloids as Inhibitors of Malate Synthase from Paracoccidioides spp.: Receptor-Ligand Interaction-Based Virtual Screening and Molecular Docking Studies, Antifungal Activity, and the Adhesion Process

2015 ◽  
Vol 59 (9) ◽  
pp. 5581-5594 ◽  
Author(s):  
Fausto Guimaraes Costa ◽  
Benedito Rodrigues da Silva Neto ◽  
Ricardo Lemes Gonçalves ◽  
Roosevelt Alves da Silva ◽  
Cecília Maria Alves de Oliveira ◽  
...  
Keyword(s):  
3D Structure ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Docking Studies ◽  
Malate Synthase ◽  
Human Pathogens ◽  
Ligand Interaction ◽  
Content Type ◽  
Extracellular Matrix Components ◽  
Paracoccidioides Spp

ABSTRACTParacoccidioidesis the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase ofParacoccidioidesspecies (PbMLS). The three-dimensional (3D) structure ofPbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds withPbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket ofPbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket ofPbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinantPbMLS (PbMLSr) activity,Paracoccidioidesspecies growth, and adhesion of the fungus andPbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated.

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

scholarly journals Substrate Specificity of the Bacillus licheniformis Lyxose Isomerase YdaE and Its Application inIn VitroCatalysis for Bioproduction of Lyxose and Glucose by Two-Step Isomerization

2011 ◽  
Vol 77 (10) ◽  
pp. 3343-3350 ◽  
Author(s):  
Darshan H. Patel ◽  
Seung Gon Wi ◽  
Seong-Gene Lee ◽  
Dae-Seok Lee ◽  
Youn-ho Song ◽  
...  
Keyword(s):  
Bacillus Licheniformis ◽  
Thermus Thermophilus ◽  
Xylose Isomerase ◽  
Catalytic Efficiency ◽  
Sugar Production ◽  
Uncharacterized Protein ◽  
Easy Method ◽  
Content Type ◽  
Substrate Specificities

ABSTRACTEnzymatic processes are useful for industrially important sugar production, andin vitrotwo-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded byydaEofBacillus licheniformiswas found to have broad substrate specificities and has shown high catalytic efficiency ond-lyxose, suggesting that the enzyme isd-lyxose isomerase.Escherichia coliBL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn2+. The apparentKmvalues ford-lyxose andd-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (kcat/Km) for lyxose (3.2 ± 0.1 mM−1s−1) was higher than that ford-mannose (1.6 mM−1s−1). The purified protein was applied to the bioproduction ofd-lyxose andd-glucose fromd-xylose andd-mannose, respectively, along with the thermostable xylose isomerase ofThermus thermophilusHB08. From an initial concentration of 10 mMd-lyxose andd-mannose, 3.7 mM and 3.8 mMd-lyxose andd-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method forin vitrocatalysis and can be applied to industrial production.

scholarly journals Comparative enzymology of 11β-hydroxysteroid dehydrogenase type 1 from six species

2005 ◽  
Vol 35 (1) ◽  
pp. 89-101 ◽  
Author(s):  
Spyridon Arampatzis ◽  
Bert Kadereit ◽  
Daniela Schuster ◽  
Zoltan Balazs ◽  
Roberto A S Schweizer ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Animal Experiments ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Binding Pocket ◽  
Substrate Affinity ◽  
Drug Candidates ◽  
Species Specific

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), catalyzing the intracellular activation of cortisone to cortisol, is currently considered a promising target to treat patients with metabolic syndrome; hence, there is considerable interest in the development of selective inhibitors. For preclinical tests of such inhibitors, the characteristics of 11β-HSD1 from the commonly used species have to be known. Therefore, we determined differences in substrate affinity and inhibitor effects for 11β-HSD1 from six species. The differences in catalytic activities with cortisone and 11-dehydrocorticosterone were rather modest. Human, hamster and guinea-pig 11β-HSD1 displayed the highest catalytic efficiency in the oxoreduction of cortisone, while mouse and rat showed intermediate and dog the lowest activity. Murine 11β-HSD1 most efficiently reduced 11-dehydrocorticosterone, while the enzyme from dog showed lower activity than those from the other species. 7-Ketocholesterol (7KC) was stereospecifically converted to 7β-hydroxycholesterol by recombinant 11β-HSD1 from all species analyzed except hamster, which showed a slight preference for the formation of 7α-hydroxycholesterol. Importantly, guinea-pig and canine 11β-HSD1 displayed very low 7-oxoreductase activities. Furthermore, we demonstrate significant species-specific variability in the potency of various 11β-HSD1 inhibitors, including endogenous compounds, natural chemicals and pharmaceutical compounds. The results suggest significant differences in the three-dimensional organization of the hydrophobic substrate-binding pocket of 11β-HSD1, and they emphasize that species-specific variability must be considered in the interpretation of results obtained from different animal experiments. The assessment of such differences, by cell-based test systems, may help to choose the appropriate animal for safety and efficacy studies of novel potential drug candidates.

scholarly journals Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

2014 ◽  
Vol 80 (17) ◽  
pp. 5317-5329 ◽  
Author(s):  
Weijun Wang ◽  
Galina Mai-Gisondi ◽  
Peter J. Stogios ◽  
Amrit Kaur ◽  
Xiaohui Xu ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Complex Structure ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Content Type ◽  
Wheat Arabinoxylan ◽  
Debranching Enzymes ◽  
The Family ◽  
Multiple Binding ◽  
Hydrolysis Of

ABSTRACTXylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase fromStreptomyces thermoviolaceus(SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3l-Arafsubstituents from disubstituted Xylpresidues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2l-Arafsubstituents, confirming the ability of SthAbf62A to remove α-l-Arafresidues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylpsugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose andl-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigidl-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking thel-arabinose binding pocket.

Critical lifting simulation of heavy industrial construction in gaming environment

Facilities ◽  
2020 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Christoph Sydora ◽  
Zhen Lei ◽  
Ming Fung Francis Siu ◽  
SangHyeok Han ◽  
Ulrich Hermann
Keyword(s):  
Three Dimensional ◽  
Interactive Simulation ◽  
Industrial Construction ◽  
Content Type ◽  
Interactive Analysis ◽  
Lifting Capacity ◽  
Mobile Crane ◽  
Gaming Environment ◽  
Game Environment ◽  
Three Dimensional Models

Purpose Heavy industrial construction often relies on large mobile cranes to erect equipment and pre-assembled modules. Engineering calculations are required for the lifting analysis where lifting capacity is analyzed to ensure the feasibility of the lifting scenarios. Such engineering calculations are often presented in static formats, e.g. two-dimensional or three-dimensional models. However, it is difficult to help practitioners (e.g. lifting engineers, site crews and operators) understand the complexity of the lifting process and thus operational decisions are often made intuitively. Therefore, this paper aims to introduce a game-based simulation system to allow for interactive analysis of the lifting process to improve lifting efficiency and safety. Design/methodology/approach The proposed method treats the mobile crane as a robot with degree-of-freedoms, and the movements are simulated in the Unity game environment. The lifting capacity is calculated dynamically based on the lifting object weight, rigging weight and lifting radius. Findings Compared with the four-dimensional visualization, this development has added a dimension of real-time interactive simulation; this allows the users to understand the complexity and feasibility of the lifting process. Originality/value The developed prototype has been tested and validated using a real case study from a heavy industrial project with the possibility of generalizing crane lifting configurations.

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