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1.
Four positively-charged residues, namely βLys-155, βArg-182, βArg-246, and αArg-376 have been identified as Pi binding residues
in Escherichia coli ATP synthase. They form a triangular Pi binding site in catalytic site βE where substrate Pi initially binds for ATP synthesis
in oxidative phosphorylation. Positive electrostatic charge in the vicinity of βArg-246 is shown to be one important component
of Pi binding. 相似文献
2.
Extensive studies suggest direct links between cholesteryl ester transfer protein (CETP), high-density lipoproteins-cholesterol
level and cardiovascular diseases. Many therapeutic approaches are aimed at the CETP. A series of N, N-disubstituted-trifluoro-3-amino-2-propanol analogues are among the most highly potent and selective inhibitors of CETP described
to date. For in-depth investigation into the structural and chemical features responsible for exploring the binding pocket
of these compounds, as well as for the binding recognition mechanism concerned, we performed a series of automated molecular
docking operations. Moreover, the docking results were quite robust as further validated by molecular dynamics. The docking
results reveal that the binding site mainly consists of two hydrophobic regions (P1 and P2 site) which are able to accommodate
the lipophilic arms of the compounds investigated. Val421 in P1 site and Met194 in P2 site could be considered to be two important
residues in forming the two hydrophobic regions. The presence of residues Phe197 and Phe463 in P2 site may be responsible
for the binding recognition through π-π stacking interactions. The hydrophobic 3-phenoxy substituent may be important in creating
the preferable inhibitive capability for increasing the binding potency. The hydrophobic character of the tetrafluoroethoxybenzyl
group at position 3 displays better hydrophobicity than a shorter hydrophobic substituent. An interaction model of CETP-inhibitors
is derived that can be successfully used to explain the different biologic activities of these inhibitors. It is anticipated
that the findings reported here may provide very useful information or clues for designing effective drugs for the therapeutic
treatment of CETP-related cardiovascular diseases. 相似文献
3.
Nirmal K. Prasad Vaibhav Vindal Vikash Kumar Ashish Kabra Navneet Phogat Manoj Kumar 《Journal of molecular modeling》2011,17(3):533-541
Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants
and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In
lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl
CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits
a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed
based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand
the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved
in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other
substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic
reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures
of other enzymes as well. 相似文献
4.
5.
The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498–amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 × 104 U/l) as determined with ABTS (2,2′-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the
enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed. 相似文献
6.
The arginine repressor (ArgR) of Escherichia coli binds to six L-arginine molecules that act as its co-repressor in order to bind to DNA. The binding of L-arginine molecules as well as its structural analogues is compared by means of computational docking. A grid-based energy evaluation method combined with a Monte Carlo simulated annealing process was used in the automated docking. For all ligands, the docking procedure proposed more than one binding site in the C-terminal domain of ArgR (ArgRc). Interaction patterns of ArgRc with L-arginine were also observed for L-canavanine and L-citrulline. L-lysine and L-homoarginine, on the other hand, were shown to bind poorly at the binding site. Figure A general overview of the sites found from docking the various ligands into ArgRc ( grey ribbons). Red coloured sticks: residues in binding site H that was selected for docking 相似文献
7.
V. Mohanasrinivasan A. Mohanapriya Swaroop Potdar Sourav Chatterji Srinath Konne Sweta Kumari S. Merlyn Keziah C. Subathra Devi 《生物学前沿》2017,12(3):219-225
Background
Nattokinase (NK) is a serine protease enzyme of the subtilisin family. It exhibits a strong fibrinolytic activity. The fibrinolytic enzymes from Bacillus sp. have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process including plasmin activation.Methods
In the present study, VIT garden soil was collected and subjected to isolation process in order to screen for the NK production. Screening for NK enzyme was performed by radial caseinolytic assay. The production of NK enzyme was done in two different production medium for comparative studies. The NK enzyme was purified by gel permeation chromatography. The activity of the purified NK was checked by clot lysis and casein digestion assay. To investigate the structural basis of NK and fibrinogen interaction and also to identify the best binding mode, molecular dynamics and docking studies were performed.Results
Based on the morphological and biochemical characterization, the isolate was identified as Bacillus sp. The overall purification fold of NK was about 3 with the specific activity of 664U/mg and 9.9% yield. Homogeneity of the purified enzyme was analyzed and confirmed by the single band obtained in SDS-PAGE. Molecular weight of the purified protease was estimated as 25 kDa. Purified NK enzyme exhibited 97% of effective clot lysis activity. The NK was docked in to the knob region of the fibrinogen at its binding site using Dock server. A total of 26 residues of fibrinogen and 29 residues of NK constitute the interface region. However, 9 residues of fibrinogen (THR238, MET264, LYS266, ARG275, THR277, ALA279, ASN308, MET310, and LYS321) and 8 residues of NK (GLY61, SER63, THR99, PHE189, LEU209, TYR217, ASN218, and MET222) are involved in intact binding.Conclusions
A significant amount of NK enzyme was obtained from Bacillus sp. The docking analysis revealed that the NK and fibrinogen adopt an extended binding pattern and interacts with the crucial residues to exhibit their activity.8.
Treatment of C. difficile infection is one of the most difficult biomedical challenges. To develop novel antibacterials, researchers have been targeting
bacterial molecular functions that are essential for its growth. The methionyl tRNA synthetase (MetRS) is strictly required
for protein biosynthesis and success was reported in developing antibacterials to inhibit this enzyme. The present study was
aimed at building and analyzing a homology model for C. difficile MetRS in the context of drug design. A homology model of C. difficile MetRS was constructed using Molecular Operating Environment (MOE) software. A. aeolicus MetRS was the main template while the query zinc binding domain was modeled using T. thermophilus MetRS. The model has been assessed and compared to its main template (Ramachandran, ERRAT and ProSA). The active site of
the query protein has been predicted from its sequence using a detailed conservation analysis (ClustalW2). Using MOE software,
suitable ligands were docked in the constructed model, including a C. difficile MetRS inhibitor REP3123 and the enzyme natural substrate, and the key active site residues and interactions were identified.
These docking studies have validated the active site conformation in the constructed model and identified binding interactions. 相似文献
9.
Neisseria meningitidis, a gram negative bacterium, is the leading cause of bacterial meningitis and severe sepsis. Neisseria meningitidis genome contains 2,160 predicted coding regions including 1,000 hypothetical genes. Re-annotation of N. meningitidis hypothetical proteins identified nine putative peptidases. Among them, the NMB1620 protein was annotated as LD-carboxypeptidase
involved in peptidoglycan recycling. Structural bioinformatics studies of NMB1620 protein using homology modeling and ligand
docking were carried out. Structural comparison of substrate binding site of LD-carboxypeptidase was performed based on binding
of tetrapeptide substrate ‘l-alanyl-d-glutamyl-meso-diaminopimelyl-d-alanine’. Inspection of different subsite-forming residues showed changeability in the S1 subsite across different bacterial
species. This variability was predicted to provide a structural basis to S1-subsite for accommodating different amino acid
residues at P1 position of the tetrapeptide substrate ‘l-alanyl-d-glutamyl-meso-diaminopimelyl-d-alanine’. 相似文献
10.
Background
A tannic acid-inducible and mycoviral-regulated laccase3 (lac 3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae. 相似文献11.
Kumarasamy Murugesan In-Hee Yang Young-Mo Kim Jong-Rok Jeon Yoon-Seok Chang 《Applied microbiology and biotechnology》2009,82(2):341-350
In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural
redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus
Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l−1) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly
(p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone,
p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic
mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass
spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition
of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that
of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence
reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the
natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization
of MG. 相似文献
12.
Design and discovery of new potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), equally active against both the wild-type and mutant strains, is urgently needed. In this
study, a computer-aided molecular design approach that involved ab initio molecular orbital and density functional theory
calculations, along with molecular electrostatic potential analysis, and molecular docking studies was employed to design
15 1H-imidazole-2,4-diamine derivatives as potential inhibitors of PfDHFR enzyme. Visual inspection of the binding modes of
the compounds demonstrated that they all interact, via H-bond interactions, with key amino acid residues (Asp54, Ileu/Leu164,
Asn/Ser108 and Ile14) similar to those of WR99210 (3) in the active site of the enzymes used in the study. These interactions are known to be essential for enzyme inhibition.
These compounds showed better or comparable binding affinities to that of the bound ligand (WR99210). In silico toxicity predictions,
carried out using TOPKAT software, also indicated that the compounds are non-toxic. 相似文献
13.
G Schmidt U Krings M Nimtz RG Berger 《World journal of microbiology & biotechnology》2012,28(4):1623-1632
A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size
exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular
mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1
exhibited low K
m
values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437
k
cat/k
m (s−1 mM−1). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating
a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of
stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence
of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The
deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability
towards metal ions and bipolar compounds. 相似文献
14.
Summary The present paper studies the production of laccase by Trametes hirsuta immobilized into alginate beads in an airlift bioreactor. In order to enhance laccase production fresh ammonium chloride was added, which led to the production, of high laccase activities (around 1000 U l−1). The bioreactor operated for 40 days without operational problems and the bioparticles maintained their shape throughout fermentation. Dye decolorization was performed at bioreactor scale operating in the batch mode. High decolorization percentages were obtained in a short time (96% for indigo carmine and 69% for phenol red in 24 h), indicating the suitability of this process for application to synthetic dye decolorization. On the other hand, in vitro decolorization of several industrial azo dyes by crude laccase produced in the above bioreactor was also performed. It was found that some of the dyes needed the addition of 1-hydroxybenzotriazole for their decolorization. 相似文献
15.
Sun J Peng RH Xiong AS Tian Y Zhao W Xu H Liu DT Chen JM Yao QH 《Molecular biology reports》2012,39(4):3807-3814
Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides, polycyclic aromatic hydrocarbons
as well as a very wide range of other compounds with high redox potential. Based on the bias of genetic codons between fungus
and yeast, we synthesized a laccase gene GlLCCI, originated from Ganoderma lucidum using optimized codons and a PCR-based two-step DNA synthesis method. The recombinant laccase, GlLCCI was successfully over-expressed in yeast, Pichia pastoris, with an alcohol oxidase1 promoter. The recombinant GlLCCI has a molecular mass of approximately 58 kDa. The K
m values of GlLCCI for 2-2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122 mM,
respectively. The V
max of GlLCCI for both substrates was 3,024 and 82.13 μM mg−1 min−1. When ABTS was used as a substrate, the enzyme had an optimal temperature of approximately 55°C. The enzyme was detected
over pH values from 2 to 8. The enzyme was strongly activated by K+, Na+, Cu2+ and mannitol. Six amino acids (alanine, histidine, glycine, arginine, aspartate and phenylalanine) increased the catalytic
ability of the enzyme. The activity of laccase was obviously inhibited by Fe2+, Fe3+, sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62 mg l−1 of azo dye methyl orange (MO) in cultural medium. With a high MO degradation ability, GlLCCI may have potential in the treatment
of industrial effluent containing azo dye MO. 相似文献
16.
M. V. Titova E. A. Berkovich O. V. Reshetnyak I. E. Kulichenko A. V. Oreshnikov A. M. Nosov 《Applied Biochemistry and Microbiology》2011,47(1):87-92
Peculiarities of respiration of cells cultures producing biologically active compounds (isoprenoids and alkaloids) were investigated
in order to optimize productivity of culture growth and biosynthesis. It had been revealed that studied cells cultures of
Dioscorea deltoidea Wall (producer of furistanol glycosides), Stephania glabra (Roxb.) Miers (producer of stepharin alkaloid) and Polyscias filicifolia Bailey (complex of biologically active agents) differ both in joint respiration activity and in ratio between cytochrome
and cyanide-resistant respiration, while changes of rate of total oxygen consumption and activity of alternative oxidase during
growth were found to be individual for every investigated culture. Maximum rate of oxygen consumption for cells of D. deltoidea and S. glabra was marked in the period preceding active synthesis of secondary metabolites (lag phase for D. deltoidea and exponential phase for S. glabra). The revealed trends can be used for further monitoring and regulation of growth and biosynthesis of secondary metabolites
in producing cell cultures during deep cultivation. 相似文献
17.
Hye-Jung Kim Soo-Jin Yeom Kwangsoo Kim Sangkee Rhee Dooil Kim Deok-Kun Oh 《Biotechnology letters》2010,32(2):261-268
d-Psicose 3-epimerase from Agrobacterium tumefacience catalyzes the conversion of d-fructose to d-psicose. According to mutational analysis, the ring at position 112, the negative charge at position 156, and the positive
charge at position 215 were essential components for enzyme activity and for binding fructose and psicose. The surface contact
area and distance to the bound substrate by molecular modeling suggest that the positive charge of Arg215 was involved in
stabilization of cis-endiol intermediate. The distances between the catalytic residues (Glu150 and Glu244) and Mn2+ are critical to the catalysis, and the negative charges of the metal-binding residues are important for interaction with
metal ion. The kinetic parameters of the D183E and H209A mutants for metal-binding residues with substrate and the near-UV
circular dichroism spectra indicate that the metal ion bound to Asp183 and His209 is involved not only in catalysis but also
in substrate binding. 相似文献
18.
19.
Thymidylate synthase (TS) of Plasmodium dihydrofolate reductase-thymidylate synthase (DHFR-TS) functions as a homodimeric enzyme with two active sites located near
the subunit interface. The dimerization is essential for catalysis, since the active site of each subunit contains amino acid
residues contributed from the other TS domain. In P. falciparum DHFR-TS, it has been shown that the active sites require Cys-490 from one domain and Arg-470 donated from the other domain.
Mutants of these two series can complement one another giving rise to active enzyme. Here, the potential to form cross-species
heterodimers between P. falciparum and P. vivax TS has been explored. Formation of cross-species heterodimer was tested by co-transformation of TS-inactive Cys-490 mutants
of P. falciparum or P. vivax with corresponding TS-inactive Arg-486 mutants of P. vivax or P. falciparum into thymidine-requiring Escherichia coli. Active heterodimers were detected by subunit complementation and 6-[3H]-FdUMP binding assays. All combinations of the mutants tested, except for (Pf)R470A+(Pv)C506Y, were able to form catalytically
active cross-species heterodimers. The single active site formed by (Pf)R470D+(Pv)C506Y and (Pv)R486D+(Pf)C490A pairs of cross-species
heterodimers has k
cat and K
m values similar to those of intra-species heterodimers of P. falciparum and P. vivax. This is the first report to demonstrate that the TS subunit interface between Plasmodium species is sufficiently conserved to allow formation of fully active cross-species heterodimer. 相似文献
20.
A. R. Abyanova A. M. Chulkin E. A. Vavilova T. V. Fedorova D. S. Loginov O. V. Koroleva S. V. Benevolensky 《Applied Biochemistry and Microbiology》2010,46(3):313-317
A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta 072 accase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches
3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency of heterologous secretion.
The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072. 相似文献