Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism [Biochimica et Biophysica Acta 1597 (2002) 157-163]

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (α9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the α9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k_cat and k_cat/K_m for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the α9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Δ⁵-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k_cat/K_m. The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Crystallographic and Functional Characterization of the Fluorodifen-inducible Glutathione Transferase from Glycine max Reveals an Active Site Topography Suited for Diphenylether Herbicides and a Novel L-site

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrphobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k_cat regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.     

Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase

The effect of hexahistidine-tag (His-tag) location at either the C or N-terminus on the catalytic activity of 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis was studied. The kinetic parameters of 3HBDHs with C and N-terminal His-tags were investigated, and the enzyme with an N-terminal His-tag was found to have approximately 1,200-fold higher catalytic efficiency than its C-terminal counterpart. Furthermore, the effect of His-tag location on the catalytic activity of 3 engineered variants of 3HBDH that were previously developed for the conversion of levulinic acid to 4-hydroxyvaleric acid was also investigated. All of the N-terminal variants exhibited higher catalytic efficiency for levulinic acid than did the C-terminal counterparts. The structural basis of the His-tag effect was studied by investigating the structure of 3HBDH obtained from in silico His-tag modification, and the results revealed that the modification of the C-terminal structure could deform the hinge region of the active site entry loop, disrupting the catalytic motion of the enzyme. In contrast, due to the location of the N-terminus far from the active site of the enzyme, the catalytic activity of the enzyme was not severely affected by the N-terminal His-tag.     

Divergence in structure and function of tau class glutathione transferase from Pinus tabulaeformis,P. yunnanensis and P. densata

Glutathione transferases (GSTs) are a family of enzymes that play important roles in stress tolerance and detoxification in plants. The plant GSTs are divided into four classes (phi, tau, zeta and theta), among which tau is the most numerously represented. To date, studies on GSTs in plants have focused largely on crop species. There is extremely little information on the molecular characteristics of GSTs in gymnosperms. Generalization on GST characteristics unique to gymnosperms and the patterns of GST evolution in plants cannot be made before more members of the gene family in conifers are described. In this study we report three new GSTs from Pinus tabulaeformis, Pinus densata and Pinus yunnanensis. Structural and phylogenetic analyses placed these three GSTs in tau class. The tau GST class is subdivided into three clades and this subdivision seems an ancient event that may have pre-dated the gymnosperm and angiosperm split. Sequence analysis revealed a highly conserved N-terminal domain in contrast to a highly variable C-terminal domain. Mutations even outside the critical glutathione-binding site in N-terminal domain can have pronounced effect on GST catalytic property. Thus, sequence similarity does not parallel functional specificity. The high diversity in C-terminal domain determines a wide range of substrate selectivity and specificity among tau GSTs. Thus the a few conserved residues in C-terminal domain seem essential to maintain the structure of the domain and the protein dimer. More extensive data on GST family organization and a thorough gene-by-gene analysis in conifers are needed to advance our understanding of the true diversity and evolution of GST in structure and function in plants.     

An Extended Structure of the APOBEC3G Catalytic Domain Suggests a Unique Holoenzyme Model

Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of human immunodeficiency virus-1, other retroviruses, and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an α1-helix (residues 201-206) that was not apparent in the shorter protein, and it contributes to catalytic activity through interactions with hydrophobic core structures (β1, β3, α5, and α6). Both A3G catalytic domain solution structures have a discontinuous β2 region that is clearly different from the continuous β2 strand of another family member, APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain, including the interdomain linker and some of the last α-helix. These structured residues (residues 191-196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal β2 regions are too distant from each other to participate in this interaction.     

Characterization of porcine alpha-class glutathione transferase A1-1

An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissues and animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence of our clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to our cloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1^∗. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactions was at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.     

A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily

In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme''s catalytic efficiency and specificity.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha 9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha 9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters K(cat) and K(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha 9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on K(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD⁺-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD⁺-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD⁺-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.     

Novel enzymatic activity derived from the Semliki Forest virus capsid protein

The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and k_cat/K_M values up to 5 × 10⁵ s⁻¹ M⁻¹. The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pK_a1 = 6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k₂ = 4.4±0.6 s⁻¹ and k₃ = 1.6±0.5 s⁻¹, respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M_w of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M_w of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K_m value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer.     

Probing the two-domain structure of homodimeric prokaryotic and eukaryotic catalase–peroxidases

Catalase–peroxidases (KatGs) are ancestral bifunctional heme peroxidases found in archaeons, bacteria and lower eukaryotes. In contrast to homologous cytochrome c peroxidase (CcP) and ascorbate peroxidase (APx) homodimeric KatGs have a two-domain monomeric structure with a catalytic N-terminal heme domain and a C-terminal domain of high sequence and structural similarity but without obvious function. Nevertheless, without its C-terminal counterpart the N-terminal domain exhibits neither catalase nor peroxidase activity. Except some hybrid-type proteins all other members of the peroxidase–catalase superfamily lack this C-terminal domain. In order to probe the role of the two-domain monomeric structure for conformational and thermal stability urea and temperature-dependent unfolding experiments were performed by using UV–Vis-, electronic circular dichroism- and fluorescence spectroscopy, as well as differential scanning calorimetry. Recombinant prokaryotic (cyanobacterial KatG from Synechocystis sp. PCC6803) and eukaryotic (fungal KatG from Magnaporthe grisea) were investigated. The obtained data demonstrate that the conformational and thermal stability of bifunctional KatGs is significantly lower compared to homologous monofunctional peroxidases. The N- and C-terminal domains do not unfold independently. Differences between the cyanobacterial and the fungal enzyme are relatively small. Data will be discussed with respect to known structure and function of KatG, CcP and APx.     

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-l-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-l-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding.     

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 Å resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional ¹H,¹⁵N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.     

A conserved hydrogen-bond network stabilizes the structure of Beta class glutathione S-transferases

We identified a network of hydrogen bonds that is conserved in the structures of bacterial Beta class glutathione S-transferases (GSTs). It is formed by three residues: a serine, a histidine and a glutamate, together with a water molecule that links the serine with the histidine. This network connects the first helix of the N-terminal glutaredoxin-like domain with the last helix of the C-terminal GST-specific all helical domain. Here we show that substitution of Ochrobactrum anthropi GST His15 and Glu198 with alanine greatly compromises the catalytic efficiency of the enzyme, even though none of these residues takes part to the enzyme active site. Thermal and chemical denaturation experiments point to a role for this network in global structure stabilization. Furthermore, we show that OaGST structure looses compactness at alkanine pHs and that this behavior may be ascribed to partial disruption of the H-bond network, pointing to an important role in zippering the N-terminal and C-terminal domains of the protein.     

Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro¹⁶ and Pro³⁰ of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro³⁸. This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.