scholarly journals Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of a tRNA-modifying enzyme, archaeal tRNA-guanine transglycosylase

2015 ◽  
Vol 21 (1) ◽  
pp. 41-52 ◽  
Author(s):  
Yuichiro Nomura ◽  
Satoshi Ohno ◽  
Kazuya Nishikawa ◽  
Takashi Yokogawa
Keyword(s):  
Tertiary Structure ◽  
Catalytic Efficiency ◽  
The Stability

scholarly journals Further structural and functional properties of mini‒lipoxygenase, an active fragment of soybean lipoxygenase-1

2004 ◽  
Vol 18 (2) ◽  
pp. 331-338 ◽  
Author(s):  
Mauro Maccarrone ◽  
Almerinda Di Venere ◽  
Guus van Zadelhoff ◽  
Giampiero Mei ◽  
Gerrit Veldink ◽  
...  
Keyword(s):  
Functional Properties ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Catalytic Efficiency ◽  
Soybean Lipoxygenase ◽  
Binding Studies ◽  
Irreversible Inhibitor ◽  
Structural And Functional Properties ◽  
The Stability ◽  
Induced Changes

Lipoxygenases (Loxs) form a homologous family of non-heme, non-sulfur iron containing lipid-peroxidizing enzymes, which catalyze the dioxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives. Soybean lipoxygenase-1 (Lox-1) is widely used as a prototype for studying the structural and functional properties of lipoxygenases. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed “mini-Lox”, which shows enhanced catalytic efficiency and higher membrane binding ability than the native enzyme (M. Maccarrone, M.L. Salucci, G. van Zadelhoff, F. Malatesta, G. Veldink, J.F.G. Vliegenthart and A. Finazzi-Agrò,Biochemistry40(2001), 6819–6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride (GdHCl) and under high pressure by fluorescence and circular dichroism spectroscopy. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition. Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-8 anilinonaphtalene sulphonic acid binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by fluorescence techniques. In particular, eicosatetraynoic acid (ETYA), an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of ETYA. All these findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible and therefore less stable, that on the other hand is probably causing its higher catalytic efficiency.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

scholarly journals LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

2019 ◽  
Vol 47 (12) ◽  
pp. 6369-6385
Author(s):  
Jia-Yi Fan ◽  
Qian Huang ◽  
Quan-Quan Ji ◽  
En-Duo Wang
Keyword(s):  
Tertiary Structure ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Type I ◽  
Type Ii ◽  
Specific Domain ◽  
Recognition Mechanism ◽  
Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

para-Sulphonato-calix[n]arene capped silver nanoparticles challenge the catalytic efficiency and the stability of a novel human gut serine protease inhibitor

2019 ◽  
Vol 55 (61) ◽  
pp. 8935-8938 ◽  
Author(s):  
Nizar Akermi ◽  
Hela Mkaouar ◽  
Aicha Kriaa ◽  
Amin Jablaoui ◽  
Souha Soussou ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Gut Microbiota ◽  
Protease Inhibitor ◽  
Serine Protease ◽  
Catalytic Efficiency ◽  
Serine Protease Inhibitor ◽  
Human Gut ◽  
Pancreatic Elastase ◽  
Human Gut Microbiota ◽  
The Stability

Eubacterium saburreum serpin from human gut microbiota inhibits the pancreatic elastase associated with pancreatitis, inhibition is strongly increased by para-sulphonato-calix[8]arene silver nanoparticles.

scholarly journals Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles

2004 ◽  
Vol 378 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
M. Soledad CELEJ ◽  
Mariana G. D'ANDREA ◽  
Patricia T. CAMPANA ◽  
Gerardo D. FIDELIO ◽  
M. Lucia BIANCONI
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Enzyme Stability ◽  
Catalytic Efficiency ◽  
Tryptophan Fluorescence ◽  
Enzyme Activation ◽  
Hexadecyltrimethylammonium Bromide ◽  
Negative Band ◽  
Cationic Micelles ◽  
Α Helix

The catalytic behaviour of α-CT (α-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme–micelle interaction leads to an increase in both the Vmax and the affinity for the substrate p-nitrophenyl acetate, indicating higher catalytic efficiency for bound α-CT. The bell-shaped profile of α-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in α-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the α-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in α-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of α-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased α-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm−1, corresponding to the α-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the β-sheet content in micelle-bound α-CT. Our data suggest that the higher catalytic efficiency of micelle-bound α-CT results from significant conformational changes.

scholarly journals Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Canfang Niu ◽  
Peilong Yang ◽  
Huiying Luo ◽  
Huoqing Huang ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Side Chains ◽  
Cleavage Sites ◽  
Corn Meal ◽  
Feed Enzymes ◽  
The Stability ◽  
Phytate Content

Abstract Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.

scholarly journals Effects of osmolytes and macromolecular crowders on stable GAAA tetraloops and their preference for a CG closing base pair

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4236
Author(s):  
Kaethe N. Leonard ◽  
Joshua M. Blose
Keyword(s):  
Secondary Structure ◽  
Base Pair ◽  
Structural Changes ◽  
Tertiary Structure ◽  
Vis Spectroscopy ◽  
Rna Tertiary Structure ◽  
The Stability ◽  
And Function ◽  
Peg 8000

Osmolytes and macromolecular crowders have the potential to influence the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in vivo. To further understand the cellular function of RNA we observed the effects of a model osmolyte, polyethylene glycol (PEG) 200, and a model macromolecular crowding agent, PEG 8000, on the GAAA tetraloop motif. GAAA tetraloops are conserved, stable tetraloops, and are critical participants in RNA tertiary structure. They also have a thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing GAAA loops was monitored using UV-Vis spectroscopy in the presence and absence of PEG 200 or PEG 8000. Both of the cosolutes tested influenced the thermodynamic preference for a CG base pair by destabilizing the loop with a CG closing base pair relative to the loop with a GC closing base pair. This result also extended to a related DNA triloop, which provides further evidence that the interactions between the loop and closing base pair are identical for the d(GCA) triloop and the GAAA tetraloop. Our results suggest that in the presence of model PEG molecules, loops with a GC closing base pair may retain some preferential interactions with the cosolutes that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes could influence how neutral cosolutes tune the stability and function of secondary structure motifs in vivo.

scholarly journals Structural Analysis of Avian Encephalomyelitis Virus Polyprotein for Development of Multi Epitopes Vaccine Using Immunoinformatics Approach

2021 ◽  
Vol 15 (1) ◽  
pp. 262-278
Author(s):  
Fatima Khalid Elhassan ◽  
Yassir A. Almofti ◽  
Khoubieb Ali Abd-elrahman ◽  
Mashair AA Nouri ◽  
Elsideeq EM Eltilib
Keyword(s):  
T Cells ◽  
Disulfide Bonds ◽  
Tertiary Structure ◽  
High Mobility ◽  
Dynamics Simulation ◽  
High Morbidity ◽  
E Coli ◽  
Encephalomyelitis Virus ◽  
The Stability ◽  
Chimeric Vaccine

Avian Encephalomyelitis (AE) is the disease caused by avian encephalomyelitis virus (AEV). The disease mainly affects young birds nervous system worldwide causing high morbidity and variable mortality rate in chicks and noticed egg dropping and hatchability in mature hens. Vaccination is the only way to control AEV infection since there is no treatment yet to the avian encephalomyelitis. This study aimed to use immunoinformatics approaches to predict multi epitopes vaccine from the AEV polyprotein that could elicit both B and T cells. The vaccine construct comprises 482 amino acids obtained from epitopes predicted against B and T cells by IEDB server, adjuvant, linkers and 6-His-tag. The chimeric vaccine was potentially antigenic and nonallergic and demonstrated thermostability and hydrophilicity in protparam server. The solubility of the vaccine was measured in comparison to E. coli proteins. The stability was also assessed by disulfide bonds engineering to reduce the high mobility regions in the designed vaccine. Furthermore molecular dynamics simulation further strengthen stability of the predicted vaccine. Tertiary structure of the vaccine construct after prediction, refinement was used for molecular docking with chicken alleles BF2*2101 and BF2*0401 and the docking process demonstrated favourable binding energy score of -337.47 kcal/mol and -326.87 kcal/mol, respectively. Molecular cloning demonstrated the potential clonability of the chimeric vaccine in pET28a(+) vector. This could guarantee the efficient translation and expression of the vaccine within suitable expression vector.

Preparation of Pt/TiO2/Graphene/Polyethylene Sheets via a Facile Molding Process for Azo Dye Electrodegradation

2020 ◽  
Vol 20 (5) ◽  
pp. 3287-3294 ◽  
Author(s):  
Qian Zhang ◽  
Chang Yao ◽  
Jun-Ming Hong ◽  
Chang-Tang Chang
Keyword(s):  
Electrochemical Impedance ◽  
Removal Rate ◽  
Catalytic Efficiency ◽  
Metal Electrode ◽  
High Price ◽  
Molding Process ◽  
Electrode Coating ◽  
Transmission Electron ◽  
The Stability ◽  
High Removal Rate

As the characterizations of electrode are meaningful for electric catalytic efficiency and mechanism, the improvement of electrode have raised considerable public concern in recent decades. However, the metal electrode have the drawbacks of high price and easy for toxicity, nano electrode restricted by difficulties for electrode coating, possibility of agglomeration, and abscission during reactions. Focus on those defects, the proposed study is going to establish a useful technique for polymer combined nano-electrode preparation. The morphology, functional groups, and other characterization of the Pt/TiO2/graphene particles and organic composite nano Pt/TiO2/graphene sheets were analyzed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), electrochemical impedance spectroscopy (EIS). To identify the stability of self-prepared electrodes, parameters such as catalysts dosage, current density and pH will be analyzed by using RBK5 as target pollutions. The results shows that after treatment for 50 min under optimized conditions (20 mA, 1 g/L NaCl), the degradation rate of acetaminophen almost reached 100%. After five times recycle, the self-prepared electrode could still maintained a high removal rate of 90%.

scholarly journals Designing and Analyzing the Structure of DT-STXB Fusion Protein as an Anti-tumor Agent: An in Silico Approach

2019 ◽  
Vol 14 (4) ◽  
pp. 305-312 ◽  
Author(s):  
Zeynab Mohseni Moghadam ◽  
Raheleh Halabian ◽  
Hamid Sedighian ◽  
Elham Behzadi ◽  
Jafar Amani ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Fusion Protein ◽  
Shiga Toxin ◽  
In Silico ◽  
Tertiary Structure ◽  
Chimeric Protein ◽  
Human Tumors ◽  
Energy Calculation ◽  
Mrna Structure ◽  
The Stability

Background & Objective: A main contest in chemotherapy is to obtain regulator above the biodistribution of cytotoxic drugs. The utmost promising strategy comprises of drugs coupled with a tumor-targeting bearer that results in wide cytotoxic activity and particular delivery. The B-subunit of Shiga toxin (STxB) is nontoxic and possesses low immunogenicity that exactly binds to the globotriaosylceramide (Gb3/CD77). Gb3/CD77 extremely expresses on a number of human tumors such as pancreatic, colon, and breast cancer and acts as a functional receptor for Shiga toxin (STx). Then, this toxin can be applied to target Gb3-positive human tumors. In this study, we evaluated DT390-STXB chimeric protein as a new anti-tumor candidate via genetically fusing the DT390 fragment of DT538 (Native diphtheria toxin) to STxB. Methods: This study intended to investigate the DT390- STxB fusion protein structure in silico. Considering the Escherichia coli codon usage, the genomic construct was designed. The properties and the structure of the protein were determined by an in silico technique. The mRNA structure and the physicochemical characteristics, construction, and the stability of the designed chimeric protein were analyzed using computational and bioinformatics tools and servers. Hence, the GOR4 and I-TASSER online web servers were used to predict the secondary and tertiary structures of the designed protein. Results: The results demonstrated that codon adaptation index (CAI) of dt390-stxB chimeric gene raised from 0.6 in the wild type to 0.9 in the chimeric optimized gene. The mfold data revealed that the dt390-stxB mRNA was completely stable to be translated effectively in the novel host. The normal activity of the fusion protein determined by considering the secondary and tertiary structure of each construct. Energy calculation data indicated that the thermodynamic ensemble for mRNA structure was -427.40 kJ/mol. The stability index (SI) of DT390-STxB was 36.95, which is quite appropriate to preserve the stability of the construct. Ultimately, the DT390-STxB was classified as a steady fusion protein according to the Ramachandran plot. Conclusion: Our results showed that DT390-STXB was a stable chimeric protein and it can be recruited as a candidate of novel anti-tumor agents for the development of breast cancer treatment.

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