scholarly journals Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination

Symmetry ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 461
Author(s):  
Fereniki Perperopoulou ◽  
Maria Fragoulaki ◽  
Anastassios C. Papageorgiou ◽  
Nikolaos E. Labrou
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Dna Recombination ◽  
Enzyme Reaction ◽  
Optical Biosensor ◽  
Enzyme Design ◽  
Ph Indicator ◽  
Environment Friendly ◽  
Subunit Interface ◽  
Molecular Modeling Studies

In the present work, DNA recombination of three homologous tau class glutathione transferases (GSTUs) allowed the creation of a library of tau class GmGSTUs. The library was activity screened for the identification of glutathione transferase (GST) variants with enhanced catalytic activity towards the herbicide alachlor (2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide). One enzyme variant (GmGSTsf) with improved catalytic activity and binding affinity for alachlor was identified and explored for the development of an optical biosensor for alachlor determination. Kinetics analysis and molecular modeling studies revealed a key mutation (Ile69Val) at the subunit interface (helix α3) that appeared to be responsible for the altered catalytic properties. The enzyme was immobilized directly on polyvinylidenefluoride membrane by crosslinking with glutaraldehyde and was placed on the inner surface of a plastic cuvette. The rate of pH changes observed as a result of the enzyme reaction was followed optometrically using a pH indicator. A calibration curve indicated that the linear concentration range for alachlor was 30–300 μM. The approach used in the present study can provide tools for the generation of novel enzymes for eco-efficient and environment-friendly analytical technologies. In addition, the outcome of this study gives an example for harnessing protein symmetry for enzyme design.

scholarly journals Cloning and heterologous expression of cDNA encoding class Alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic α,β-unsaturated carbonyl compounds

1992 ◽  
Vol 284 (2) ◽  
pp. 313-319 ◽  
Author(s):  
G Stenberg ◽  
M Ridderström ◽  
Å Engström ◽  
S E Pemble ◽  
B Mannervik
Keyword(s):  
Catalytic Activity ◽  
Cdna Clone ◽  
Carbonyl Compounds ◽  
Glutathione Transferase ◽  
Ethacrynic Acid ◽  
Glutathione Transferases ◽  
High Catalytic Activity ◽  
Reading Frame ◽  
Dna Fragment ◽  
Polymerase Chain

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha, beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

Physics-based enzyme design: Predicting binding affinity and catalytic activity

2014 ◽  
Vol 82 (12) ◽  
pp. 3397-3409 ◽  
Author(s):  
Sarah Sirin ◽  
David A. Pearlman ◽  
Woody Sherman
Keyword(s):  
Catalytic Activity ◽  
Binding Affinity ◽  
Enzyme Design

Structural Basis of the Suppressed Catalytic Activity of Wild-type Human Glutathione Transferase T1-1 Compared to its W234R Mutant

2006 ◽  
Vol 355 (1) ◽  
pp. 96-105 ◽  
Author(s):  
Kaspars Tars ◽  
Anna-Karin Larsson ◽  
Abeer Shokeer ◽  
Birgit Olin ◽  
Bengt Mannervik ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Structural Basis ◽  
Wild Type

scholarly journals Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

2000 ◽  
Vol 276 (8) ◽  
pp. 5432-5437 ◽  
Author(s):  
Anna Maria Caccuri ◽  
Giovanni Antonini ◽  
Philip G. Board ◽  
Jack Flanagan ◽  
Michael W. Parker ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Glutathione Transferases ◽  
Evolutionary Strategies

scholarly journals The intersubunit lock-and-key motif in human glutathione transferase A1-1: role of the key residues Met51 and Phe52 in function and dimer stability

2005 ◽  
Vol 393 (2) ◽  
pp. 523-528 ◽  
Author(s):  
Carla S. Alves ◽  
Diane C. Kuhnert ◽  
Yasien Sayed ◽  
Heini W. Dirr
Keyword(s):  
Glutathione Transferase ◽  
Tryptophan Fluorescence ◽  
Size Exclusion ◽  
Dimeric Structure ◽  
Subunit Interface ◽  
Exclusion Chromatography ◽  
Region Data ◽  
Glutathione S Transferases ◽  
Urea Unfolding ◽  
Key Residues

The dimeric structure of certain cytosolic GSTs (glutathione S-transferases) is stabilized by a hydrophobic lock-and-key motif at their subunit interface. In hGSTA1-1 (human class Alpha GST with two type-1 subunits), the key consists of two residues, Met51 and Phe52, that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)–HPLC, far-UV CD and tryptophan fluorescence of the M51A and M51A/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51A mutant retained 80% of wild-type activity, the activity of the M51A/F52S was markedly diminished, indicating the importance of Phe52 in maintaining the correct conformation at the active site. The M51A and M51A/F52S mutations altered the binding of ANS (8-anilinonaphthalene-l-sulphonic acid) at the H-site by destabilizing helix 9 in the C-terminal region. Data from urea unfolding studies show that the dimer is destabilized by both mutations and that the dimer dissociates to aggregation-prone monomers at low urea concentrations before global unfolding. Although not essential for the assembly of the dimeric structure of hGSTA1-1, both Met51 and Phe52 in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.

scholarly journals The contribution of the C-terminal sequence to the catalytic activity of GST2, a human Alpha-class glutathione transferase

1991 ◽  
Vol 275 (1) ◽  
pp. 171-174 ◽  
Author(s):  
P G Board ◽  
B Mannervik
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Affinity Purification ◽  
Cumene Hydroperoxide ◽  
Plasmid Vector ◽  
Competitive Inhibitor ◽  
Terminal Segment ◽  
Truncated Form ◽  
C Terminus

A plasmid vector was constructed that encodes the expression in Escherichia coli of a truncated form of GST2, a human Alpha-class glutathione transferase. The truncated enzyme, GST2del210, has 12 residues deleted from the C-terminus and has the last two residues of the new C-terminal mutated from aspartic acid and glutamic acid to histidine and glycine respectively. GST2del210 has substantially diminished specific activity with either 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide as substrate. The affinity of the truncated enzyme for a GSH-agarose matrix was also diminished, but sufficient interaction remained to enable affinity purification. Inhibition of GST2del210 by bromosulphophthalein was not altered. In contrast, this truncated form was not inhibited by S-pentylglutathione, a competitive inhibitor of the wild-type GST2 isoenzyme. The results show that the C-terminal segment of the Alpha-class glutathione transferases may form a component of the hydrophobic substrate-binding site. In contrast, this region appears not to be directly involved in GSH binding and is not absolutely essential for catalytic activity.

scholarly journals Contribution of Arginine 13 to the Catalytic Activity of Human Class Pi Glutathione Transferase P1-1

2010 ◽  
Vol 31 (9) ◽  
pp. 2497-2502 ◽  
Author(s):  
Ji-Na Kong ◽  
Dong-Hyeon Jo ◽  
Hyun-Dong Do ◽  
Jin-Ju Lee ◽  
Kwang-Hoon Kong
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Human Class

scholarly journals Cu-metal organic frameworks (Cu-MOF) as an environment-friendly and economical catalyst for one pot synthesis of tacrine derivatives

RSC Advances ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 1995-2003 ◽  
Author(s):  
Hoda Mollabagher ◽  
Salman Taheri ◽  
Mohammad majid Mojtahedi ◽  
SeyedAmirhossein Seyedmousavi
Keyword(s):  
Catalytic Activity ◽  
Metal Organic Frameworks ◽  
One Pot ◽  
Environment Friendly ◽  
One Pot Synthesis ◽  
Metal Organic ◽  
The One

The present work describes the catalytic activity of Cu-MOF for the one-pot synthesis of tacrine derivatives via a four-component reaction of 2-hydroxynaphthalene-1,4-dione, aldehydes, malononitrile and cycloketones in the presence of AlCl3.

Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination

2019 ◽  
Vol 1863 (3) ◽  
pp. 565-576 ◽  
Author(s):  
Evangelia G. Chronopoulou ◽  
Dimitrios Vlachakis ◽  
Anastassios C. Papageorgiou ◽  
Farid S. Ataya ◽  
Nikolaos E. Labrou
Keyword(s):  
Glutathione Transferase ◽  
Optical Biosensor ◽  
Design And Application
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