Class Sigma Glutathione Transferase Unfolds via a Dimeric and a Monomeric Intermediate:  Impact of Subunit Interface on Conformational Stability in the Superfamily†

Biochemistry ◽  
1998 ◽  
Vol 37 (44) ◽  
pp. 15534-15541 ◽  
Author(s):  
Julie M. Stevens ◽  
Judith A. T. Hornby ◽  
Richard N. Armstrong ◽  
Heini W. Dirr
Keyword(s):  
Glutathione Transferase ◽  
Conformational Stability ◽  
Subunit Interface ◽  
Monomeric Intermediate

scholarly journals The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

2003 ◽  
Vol 372 (1) ◽  
pp. 241-246 ◽  
Author(s):  
Chris NATHANIEL ◽  
Louise A. WALLACE ◽  
Jonathan BURKE ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Conformational Stability ◽  
Wild Type ◽  
Catalytic Function ◽  
Evolutionarily Conserved ◽  
Glutathione S Transferases ◽  
Equilibrium Unfolding ◽  
The Stability ◽  
Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

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

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

Class Pi Glutathione Transferase Unfolds via a Dimeric and Not Monomeric Intermediate: Functional Implications for an Unstable Monomer

Biochemistry ◽  
2010 ◽  
Vol 49 (24) ◽  
pp. 5074-5081 ◽  
Author(s):  
Samantha Gildenhuys ◽  
Louise A. Wallace ◽  
Jonathan P. Burke ◽  
David Balchin ◽  
Yasien Sayed ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Functional Implications ◽  
Monomeric Intermediate

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.

Structural contributions of Delta class glutathione transferase active-site residues to catalysis

2010 ◽  
Vol 428 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Jantana Wongsantichon ◽  
Robert C. Robinson ◽  
 Albert J. Ketterman
Keyword(s):  
Crystal Structures ◽  
Glutathione Transferase ◽  
Substrate Binding ◽  
Conformational Stability ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Molecular Rearrangement ◽  
Hydrophobic Substrate ◽  
Substrate Processing ◽  
First Time

GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr111 indirectly stabilizes GSH binding, Tyr119 modulates hydrophobic substrate binding and Phe123 indirectly modulates catalysis. Mutations at Tyr111 and Phe123 also showed evidence for positive co-operativity for GSH and 1-chloro-2,4-dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit–subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic ‘zipper’ in the H-site contributing a network of aromatic π–π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the ‘zipper’ that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe123 mutations. The three crystal structures provide comprehensive evidence of the aromatic ‘zipper’ residues having an impact upon protein stability, catalysis and specificity. Consequently, ‘zipper’ residues appear to modulate and co-ordinate substrate processing through permissive flexing.

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