Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys10 and His106

2008 ◽  
Vol 417 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Xin-Yu Wang ◽  
Zai-Rong Zhang ◽  
Sarah Perrett
Keyword(s):  
Escherichia Coli ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Thiol Group ◽  
Imidazole Ring ◽  
Oxidoreductase Activity ◽  
Cellular Detoxification ◽  
Free Thiol Group ◽  
The Stability ◽  
Gst Activity

GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys10 and His106 as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys10 and His106 to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys10 and His106 leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His106 plays an important role in the catalytic mechanism of the enzyme, whereas Cys10 is involved in binding of the substrate, glutathione. Engineering of the Cys10 site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys10 plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys10 and the substrate, glutathione.

Crystal structures and kinetic studies of human Kappa class glutathione transferase provide insights into the catalytic mechanism

2011 ◽  
Vol 439 (2) ◽  
pp. 215-225 ◽  
Author(s):  
Bing Wang ◽  
Yingjie Peng ◽  
Tianlong Zhang ◽  
Jianping Ding
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Cellular Localization ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Substrate Binding ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Kinetic Mechanism ◽  
Cellular Detoxification

GSTs (glutathione transferases) are a family of enzymes that primarily catalyse nucleophilic addition of the thiol of GSH (reduced glutathione) to a variety of hydrophobic electrophiles in the cellular detoxification of cytotoxic and genotoxic compounds. GSTks (Kappa class GSTs) are a distinct class because of their unique cellular localization, function and structure. In the present paper we report the crystal structures of hGSTk (human GSTk) in apo-form and in complex with GTX (S-hexylglutathione) and steady-state kinetic studies, revealing insights into the catalytic mechanism of hGSTk and other GSTks. Substrate binding induces a conformational change of the active site from an ‘open’ conformation in the apo-form to a ‘closed’ conformation in the GTX-bound complex, facilitating formations of the G site (GSH-binding site) and the H site (hydrophobic substrate-binding site). The conserved Ser16 at the G site functions as the catalytic residue in the deprotonation of the thiol group and the conserved Asp69, Ser200, Asp201 and Arg202 form a network of interactions with γ-glutamyl carboxylate to stabilize the thiolate anion. The H site is a large hydrophobic pocket with conformational flexibility to allow the binding of different hydrophobic substrates. The kinetic mechanism of hGSTk conforms to a rapid equilibrium random sequential Bi Bi model.

scholarly journals The Stability of Acyl Carrier Protein in Escherichia coli

1973 ◽  
Vol 248 (12) ◽  
pp. 4461-4466
Author(s):  
Gary L. Powell ◽  
Michael Bauza ◽  
Allan R. Larrabee
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
The Stability

scholarly journals Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

2021 ◽  
Vol 22 (9) ◽  
pp. 4769
Author(s):  
Pablo Maturana ◽  
María S. Orellana ◽  
Sixto M. Herrera ◽  
Ignacio Martínez ◽  
Maximiliano Figueroa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Conformational Dynamics ◽  
High Specificity ◽  
Arginine Decarboxylase ◽  
Space Groups ◽  
X Ray Crystallography ◽  
High Dynamics ◽  
Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

scholarly journals 1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S643-S643
Author(s):  
Maria F Mojica ◽  
Christopher Bethel ◽  
Emilia Caselli ◽  
Magdalena A Taracila ◽  
Fabio Prati ◽  
...  
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Covalent Bond ◽  
Thiol Group ◽  
Viable Cell ◽  
Free Thiol ◽  
Transition State Analogs ◽  
Cell Counts ◽  
Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Imidazole Ring-Opened Purines: Occurence and Repair in Escherichia Coli and Mammalian Cells

1989 ◽  
pp. 25-36 ◽  
Author(s):  
Jacques Laval ◽  
Timothy O’Connor ◽  
Claudine d’Hérin-Lagravere ◽  
Patricia Auffret Van der Kemp ◽  
Serge Boiteux
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Imidazole Ring
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