Double Mutation at the Subunit Interface of Glutathione Transferase rGSTM1-1 Results in a Stable, Folded Monomer†

Biochemistry ◽  
2006 ◽  
Vol 45 (7) ◽  
pp. 2267-2273 ◽  
Author(s):  
Lawrence C. Thompson ◽  
John Walters ◽  
Jonathan Burke ◽  
James F. Parsons ◽  
Richard N. Armstrong ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Double Mutation ◽  
Subunit Interface

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 Functional Role of the Lock and Key Motif at the Subunit Interface of Glutathione Transferase P1-1

2003 ◽  
Vol 279 (10) ◽  
pp. 9586-9596 ◽  
Author(s):  
Usama M. Hegazy ◽  
Bengt Mannervik ◽  
Gun Stenberg
Keyword(s):  
Functional Role ◽  
Glutathione Transferase ◽  
Subunit Interface

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 Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

2015 ◽  
Vol 35 (6) ◽  
Author(s):  
Jantana Wongsantichon ◽  
Robert C. Robinson ◽  
Albert J. Ketterman
Keyword(s):  
Drosophila Melanogaster ◽  
Active Site ◽  
Active Sites ◽  
Glutathione Transferase ◽  
Glutathione Transferases ◽  
Conserved Motif ◽  
Subunit Interface ◽  
Dimeric Subunit

Analysis of a new structure of an Epsilon class glutathione transferase from Drosophila melanogaster reveals a highly conserved motif that spans the dimeric subunit interface and connects the two active sites.

scholarly journals The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kenichi Kamata ◽  
Kenji Mizutani ◽  
Katsuya Takahashi ◽  
Roberta Marchetti ◽  
Alba Silipo ◽  
...  
Keyword(s):  
Immune System ◽  
Sialic Acid ◽  
Ligand Binding ◽  
Steric Hindrance ◽  
Sequence Similarity ◽  
Binding Assays ◽  
Subunit Interface ◽  
Nmr Techniques ◽  
And Control ◽  
Asialo Gm1

AbstractSeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac$$\alpha$$ α (2-3)Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc) and its precursor, asialo-GM1 (Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the $$\beta$$ β -trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

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