Catalytic loop motion in human glutathione synthetase: A molecular modeling approach

Conformational changes of three flexible loops (G, A, and S) in human glutathione synthetase (hGS) arise to accommodate the substrates inside the active site. The crystal structure of hGS, a member of the ATP-grasp superfamily, has been reported only for the product-enzyme complex. To study the function of the hGS loops, molecular dynamics simulations are performed on three different conformational models: unbound enzyme, reactant-enzyme, and product-enzyme complex of hGS. The conformational changes among the three models are analyzed and the roles of the loops during the catalytic process are described. The modeled structures of hGS show that the central portions of the G- and A-loop have a double role in the reactant complex conformation: they bind the substrates and simultaneously interact with each other through an extensive network of hydrogen bonds. The present study proposes that these favorable loop-ligand and loop-loop interactions are required for opening and closing of the active site of hGS. Additionally, this research identifies important amino acid residues and explains their function within the catalytic loops of hGS.     

The ATP-grasp enzymes

The ATP-grasp enzymes consist of a superfamily of 21 proteins that contain an atypical ATP-binding site, called the ATP-grasp fold. The ATP-grasp fold is comprised of two α+β domains that "grasp" a molecule of ATP between them and members of the family typically have an overall structural design containing three common conserved focal domains. The founding members of the family consist of biotin carboxylase, d-ala-d-ala ligase and glutathione synthetase, all of which catalyze the ATP-assisted reaction of a carboxylic acid with a nucleophile via the formation of an acylphosphate intermediate. While most members of the superfamily follow this mechanistic pathway, studies have demonstrated that two enzymes catalyze only the phosphoryl transfer step and thus are kinases instead of ligases. Members of the ATP-grasp superfamily are found in several metabolic pathways including de novo purine biosynthesis, gluconeogenesis, and fatty acid synthesis. Given the critical nature of these enzymes, researchers have actively sought the development of potent inhibitors of several members of the superfamily as antibacterial and anti-obseity agents. In this review, we will discuss the structure, function, mechanism, and inhibition of the ATP-grasp enzymes.     

The role of the glycine triad in human glutathione synthetase

Experimental kinetics and computational modeling of human glutathione synthetase (hGS) support the significant role of the G-loop glycine triad (G369, G370, G371) for activity of this ATP-grasp enzyme. Enzyme kinetic experiments indicate that G369V and G370V mutant hGS have little activity (<0.7 and 0.3%, respectively, versus wild-type hGS). However, G371V retains ∼13% of the activity of wild-type hGS. With respect to G-loop:A-loop interaction in hGS, mutations at Gly369 and Gly370 decrease ligand binding and prevent active site closure and protection. This research indicates that Gly369 and Gly370 have essential roles in hGS, while Gly371 has a lesser involvement. Implications for glycine-rich ensembles in other phosphate-binding enzymes are discussed.     

Characterization of the bifunctional gamma-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida

Glutamate-cysteine ligase (gamma-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. gamma-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal gamma-proteobacterial gamma-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an approximately 85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The gamma-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K(i) = 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.     

Structural Basis for Evolution of Product Diversity in Soybean Glutathione Biosynthesis

The redox active peptide glutathione is ubiquitous in nature, but some plants also synthesize glutathione analogs in response to environmental stresses. To understand the evolution of chemical diversity in the closely related enzymes homoglutathione synthetase (hGS) and glutathione synthetase (GS), we determined the structures of soybean (Glycine max) hGS in three states: apoenzyme, bound to γ-glutamylcysteine (γEC), and with hGSH, ADP, and a sulfate ion bound in the active site. Domain movements and rearrangement of active site loops change the structure from an open active site form (apoenzyme and γEC complex) to a closed active site form (hGSH•ADP•SO₄²⁻ complex). The structure of hGS shows that two amino acid differences in an active site loop provide extra space to accommodate the longer β-Ala moiety of hGSH in comparison to the glycinyl group of glutathione. Mutation of either Leu-487 or Pro-488 to an Ala improves catalytic efficiency using Gly, but a double mutation (L487A/P488A) is required to convert the substrate preference of hGS from β-Ala to Gly. These structures, combined with site-directed mutagenesis, reveal the molecular changes that define the substrate preference of hGS, explain the product diversity within evolutionarily related GS-like enzymes, and reinforce the critical role of active site loops in the adaptation and diversification of enzyme function.     

Interactions of GTP with the ATP-grasp domain of GTP-specific succinyl-CoA synthetase

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 A resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20beta) and with backbone atoms provide the specificity. The gamma-phosphate interacts with the side chain of an arginine residue (Arg-54beta) and with backbone amide nitrogen atoms, leading to tight interactions between the gamma-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the gamma-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the alpha- and beta-phosphates allow the gamma-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the beta-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.     

Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure

Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis–Menten constant (K_m) was determined for all three substrates (glycine, GAB and ATP): glycine K_m increased by 30–115-fold, GAB K_m decreased by 8–17-fold, and the ATP K_m was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.     

Gamma-glutamylcysteine synthetase-glutathione synthetase: domain structure and identification of residues important in substrate and glutathione binding

In most organisms, glutathione (GSH) is synthesized by the sequential action of distinct enzymes, gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase (GS). In Streptococcus agalactiae, GSH synthesis is catalyzed by a single enzyme, gamma-glutamylcysteine synthetase-glutathione synthetase (gamma-GCS-GS). The N-terminal sequence of gamma-GCS-GS is similar to Escherichia coli gamma-GCS, but the C-terminal sequence is an ATP-grasp domain more similar to d-Ala, d-Ala ligase than to any known GS. In the present studies, C-terminally and N-terminally truncated constructs were characterized in order to define the limits of the gamma-GCS and GS domains, respectively. Although WT gamma-GCS-GS is nearly uninhibited by GSH (K(i) approximately 140 mM), shorter gamma-GCS domain constructs were unexpectedly found to be strongly inhibited (K(i) approximately 15 mM), reproducing a physiologically important regulation seen in monofunctional gamma-GCS enzymes. Because studies with E. coli gamma-GCS implicate a flexible loop region in GSH binding, chimeras of S. agalactiae gamma-GCS-GS were made containing gamma-GCS domain flexible loop sequences from Enterococcus faecalis and Pasteurella multocida gamma-GCS-GS, isoforms that are inhibited by GSH. Inhibition remained S. agalactiae-like (i.e., very weak). C-Terminal constructs of gamma-GCS-GS have GS activity (0.01-0.04% of WT), but proper folding and significant GS activity required a covalently linked gamma-GCS domain. In addition, site-directed mutants in the middle region of the gamma-GCS-GS sequence established that GS activity depends on residues in a region that is also part of the gamma-GCS domain. Our results provide new insights into the structure of gamma-GCS-GS and suggest gamma-GCS-GS evolved from a monomeric gamma-GCS that became C-terminally fused to a multimeric ATP-grasp protein.     

Phosphatidylinositol phosphate kinase: a link between protein kinase and glutathione synthase folds.

Comparisons of serine/threonine protein kinase (PK) and type IIbeta phosphatidylinositol phosphate kinase (PIPK) structures with each other and also with other proteins reveal structural and functional similarity between the two kinases and proteins of the glutathione synthase fold (ATP-grasp). This suggests that these enzymes are evolutionarily related. The structure of PIPK, which clearly resembles both PK and ATP-grasp, provides a link between the two proteins and establishes that the C-terminal domains of PK, PIPK and ATP-grasp share the same fold. The functional implications of the proposed homology are discussed.     

The Role of Strong Electrostatic Interactions at the Dimer Interface of Human Glutathione Synthetase

The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein–protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.     

Valine 44 and valine 45 of human glutathione synthetase are key for subunit stability and negative cooperativity

It was hypothesized that residues Val44 and Val45 serve as important residues for human glutathione synthetase (hGS) function and stability given their location at the dimer interface of this enzyme. Computational studies suggest that mutation at Val45 has more impact on the structure and stability of hGS than does mutation at Val44. Experimentally, enzymes with mutations at the 44 and or 45 positions of hGS were prepared, purified and assayed for initial activity. Val45 position mutations (either to alanine or tryptophan) have a greater impact on enzyme activity than do mutations at Val44. Differential scanning calorimetry experiments reveal a loss of stability in all mutant enzymes, with V45 mutations being less stable than the corresponding Val44 mutations. The γ-GluABA substrate affinity remains unaltered in V44A and V45A mutant enzymes, but increases when tryptophan is introduced at either of these positions. Hill coefficients trend towards less negative cooperativity with the exception of V45W mutant hGS. These results imply that residues V44 and V45 are located along the allosteric pathway of this negatively cooperative dimeric enzyme, that their mutation impacts the allosteric pathway more than it does the active site of hGS, and that these residues (and by extension the dimer interface in which they are located) are integral to the stability of human glutathione synthetase.     

Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase 4 (AtITPK4) is an outlier to a family of ATP-grasp fold proteins from Arabidopsis

The Arabidopsis genome encodes a family of inositol 1,3,4-trisphosphate 5/6-kinases which form a subgroup of a larger group of ATP-grasp fold proteins. An analysis of the inositol 1,3,4-trisphosphate 5/6-kinase family might, ultimately, be best rewarded by detailed comparison of related enzymes in a single genome. The enzyme encoded by At2G43980, AtITPK4; is an outlier to its family. At2G43980 is expressed in male and female organs of young and mature flowers. AtITPK4 differs from other family members in that it does not display inositol 3,4,5,6-tetrakisphosphate 1-kinase activity; rather, it displays inositol 1,4,5,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate isomerase activity.     

Crystal structure of a lysine biosynthesis enzyme,LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.     

Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC

The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.     

Enhanced multispecificity of arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2

Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E(2)17betaG uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E(2)17betaG uptake, neither competes with DNP-GS for uptake nor is subject to E(2)17betaG-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.     

A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity.

The recently developed PSI-BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide-binding fold, referred to as palmate, or ATP-grasp fold. In addition to D-alanine-D-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three-dimensional structures, the ATP-grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin-tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP-dependent carboxylate-amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP-grasp superfamily also includes succinate-CoA ligase (both ADP-forming and GDP-forming variants), malate-CoA ligase, and ATP-citrate lyase, enzymes with a carboxylate-thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP-grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis.     

Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.     

Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase

In Escherichia coli, the PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of glycinamide ribonucleotide (GAR) in the de novo pathway for purine biosynthesis. On the basis of amino acid sequence analyses, it is known that the PurT transformylase belongs to the ATP-grasp superfamily of proteins. The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 A from one of the gamma-phosphoryl oxygens of the AMPPNP.     

A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.