scholarly journals Proposed Mechanism for Monomethylarsonate Reductase Activity of Human Omega-class Glutathione Transferase GSTO1

2021 ◽  
Author(s):  
Aaron J Oakley

Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

2021 ◽  
Author(s):  
Hayden Burdett ◽  
Xiahao Hu ◽  
Maxwell X Rank ◽  
Natsumi Maruta ◽  
Bostjan Kobe

TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the ″AE interface″ (mediated by helices A and E), the ″BB-loop″ (connecting β-strand B and helix B in the structure), and the ″BE interface″ (mediated by the BB-loop from one TIR and the ″DE surface″ of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact ″DE surface″ is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.


1994 ◽  
Vol 302 (2) ◽  
pp. 383-390 ◽  
Author(s):  
R J Cooke ◽  
R Björnestedt ◽  
K T Douglas ◽  
J H McKie ◽  
M D King ◽  
...  

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.


2003 ◽  
Vol 185 (14) ◽  
pp. 4074-4080 ◽  
Author(s):  
Tina Izard

ABSTRACT Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway. PPAT catalyzes the reversible transfer of an adenylyl group from Mg2+:ATP to 4′-phosphopantetheine to form 3′-dephospho-CoA (dPCoA) and pyrophosphate. The high-resolution crystal structure of PPAT complexed with CoA has been determined. Remarkably, CoA and the product dPCoA bind to the active site in distinct ways. Although the phosphate moiety within the phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites. Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme. Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion.


2001 ◽  
Vol 56 (9-10) ◽  
pp. 843-847
Author(s):  
Dessislava Nikolova Georgieva ◽  
Stanka Stoeva ◽  
Wolfgang Voelter ◽  
Nicolay Genov

Abstract The active site of Viviparus ater (mollusc) hemocyanin was investigated using the fact that the binding of dioxygen to the binuclear copper-containing sites of hemocyanins is connected with the appearance of specific dichroic bands which are very sensitive to changes in the structrure and polarity of the environment. Oxy-Viviparus ater hemocyanin exhibits near UV and visible circular dichroism spectra different from those of other molluscan and arthropo-dan hemocyanins. These differences are due probably to variations in the geometry or charge distribution in the dioxygen binding sites of the compared proteins.The thermostability of Viviparus ater hemocyanin and the significance of the copper-dioxy-gen system for the stability were also investigated. “Melting” temperatures, Tm, of 77 °C for the oxy-hemocyanin and 57 °C for the apo-protein were calculated from the denaturation curves which demonstrates the considerable role of the binuclear active site for the thermostability. Viviparus ater hemocyanin is more thermostable than other hemocyanins for which data are published.


Biochemistry ◽  
1999 ◽  
Vol 38 (49) ◽  
pp. 16268-16275 ◽  
Author(s):  
Ann Gustafsson ◽  
Maryam Etahadieh ◽  
Per Jemth ◽  
Bengt Mannervik

2009 ◽  
Vol 422 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Irene Axarli ◽  
Prathusha Dhavala ◽  
Anastassios C. Papageorgiou ◽  
Nikolaos E. Labrou

Cytosolic GSTs (glutathione transferases) are a multifunctional group of enzymes widely distributed in Nature and involved in cellular detoxification processes. The three-dimensional structure of GmGSTU4-4 (Glycine max GST Tau 4-4) complexed with GSH was determined by the molecular replacement method at 2.7 Å (1 Å=0.1 nm) resolution. The bound GSH is located in a region formed by the beginning of α-helices H1, H2 and H3 in the N-terminal domain of the enzyme. Significant differences in the G-site (GSH-binding site) as compared with the structure determined in complex with Nb-GSH [S-(p-nitrobenzyl)-glutathione] were found. These differences were identified in the hydrogen-bonding and electrostatic interaction pattern and, consequently, GSH was found bound in two different conformations. In one subunit, the enzyme forms a complex with the ionized form of GSH, whereas in the other subunit it can form a complex with the non-ionized form. However, only the ionized form of GSH may form a productive and catalytically competent complex. Furthermore, a comparison of the GSH-bound structure with the Nb-GSH-bound structure shows a significant movement of the upper part of α-helix H4 and the C-terminal. This indicates an intrasubunit modulation between the G-site and the H-site (electrophile-binding site), suggesting that the enzyme recognizes the xenobiotic substrates by an induced-fit mechanism. The reorganization of Arg111 and Tyr107 upon xenobiotic substrate binding appears to govern the intrasubunit structural communication between the G- and H-site and the binding of GSH. The structural observations were further verified by steady-state kinetic analysis and site-directed mutagenesis studies.


2000 ◽  
Vol 276 (8) ◽  
pp. 5427-5431 ◽  
Author(s):  
Anna Maria Caccuri ◽  
Giovanni Antonini ◽  
Philip G. Board ◽  
Jack Flanagan ◽  
Michael W. Parker ◽  
...  

Processes ◽  
2020 ◽  
Vol 8 (10) ◽  
pp. 1232
Author(s):  
Abeer Shokeer ◽  
Aram Ismail ◽  
Usama M. Hegazy ◽  
Rüdiger H. Kolm ◽  
Bengt Mannervik

Glutathione transferases (GSTs) are enzymes that play a critical role in cellular detoxication by catalyzing the nucleophilic attack of glutathione on the electrophilic center of a number of xenobiotic compounds, including many therapeutic drugs. Mutations of amino acid residues in the glutathione-binding site of human glutathione transferase P1–1, namely W39C, K45A, Q52A, Q52K, and Q52E, have been engineered. The recombinant mutant proteins were expressed in Escherichia coli, but only mutants K45A, Q52A, and Q52K showed measurable activity. Steady-state kinetics comparing glutathione with the alternative thiol substrate γ-glutamylcysteine demonstrated the importance of the glycine residue in glutathione for high catalytic efficiency. Inhibition experiments with a set of glutathione analogs structurally related to the therapeutic drugs Telintra and Telcyta enabled determination of binding energies that were contributed by different substituents. The effects of substituting amino acid side chains in the glutathione-binding site of the enzyme on binding the glutathione derivatives and catalysis were evaluated.


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