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

doi:10.1016/j.jmb.2008.10.084

J. Mol. Biol. (2009) 385, 984–1002

Available online at www.sciencedirect.com

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 Irene Axarli 1 , Prathusha Dhavala 2 , Anastassios C. Papageorgiou 2 and Nikolaos E. Labrou 1 ⁎ 1

Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855-Athens, Greece 2

Turku Centre for Biotechnology, BioCity, Turku, FIN-20521, Finland Received 29 July 2008; received in revised form 24 October 2008; accepted 29 October 2008 Available online 6 November 2008

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 kcat 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. © 2008 Elsevier Ltd. All rights reserved.

Edited by R. Huber

Keywords: diphenylether herbicides; fluorodifen; herbicide detoxification; tau class GST; X-ray structure

*Corresponding author. E-mail address: [email protected]. Abbreviations used: CDNB, 1-chloro-2,4-dinitrobenzene; Fluorodifen, 4-nitrophenyl 2-nitro-4-trifluoromethylphenyl ether; Fluazifop-butyl, butyl 2-[4-[[5-(trifluoromethyl)-2pyridinyl]oxy]phenoxy]propanoate; GSH, glutathione; GST, glutathione transferase; GST-F, glutathione transferase activity assayed with fluorodifen and GSH as substrates; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Nb-GSH, S-(p-nitrobenzyl)-glutathione; H-site, hydrophobic binding site; L-site, ligand-binding site.

Introduction Glutathione transferases (GSTs, EC 2.5.1.18, formerly known as glutathione-S-transferases) catalyse the nucleophilic addition of the sulfur atom of glutathione (γ-L-Glu-L-Cys-Gly, GSH) to the electrophilic groups of a large variety of hydrophobic molecules including organic halides, epoxides, arene oxides, α- and β-unsaturated carbonyls, organic nitrate esters, and organic thiocyanates.1

0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

985

Fluorodifen-inducible Glutathione Transferase

The conjugation of GSH to such molecules increases their solubility and facilitates further metabolic processing.2 GSTs also catalyse GSH-dependent reduction of some products of oxidative stress, such as organic hydroperoxides.3 Through these functions, GSTs have important roles in detoxification processes of xenobiotic, biosynthesis of prostaglandins and leukotriene C4, and protection against oxidative stress. Furthermore, in view of their high water solubility, their abundance in cells, and their binding properties, GSTs are implicated in the intracellular transport and storage of a broad range of structurally diverse hydrophobic ligands, such as haem, bilirubin, hormones, flavonoids, fatty acids and xenobiotics.4,5 The GST superfamily can be subdivided into a number of classes on the basis of their amino acid sequence.6 Within mammals, the following classes have been defined: alpha, mu, pi, sigma, theta, zeta, kappa and omega.7 In addition, a subfamily of chloride intracellular channel proteins has been shown to encompass members of the cytosolic GST structural family with unknown enzymatic activity.7 Several other soluble GST classes have been reported: delta and epsilon in insects;8 phi, tau, lambda, dehydroascorbate reductase in plants;9 beta10 and chi11 in bacteria. The cytosolic GSTs are related by evolution to glutaredoxin, containing a thioredoxin domain with a unique βαβαββα topology.12 In glutaredoxins, the catalytic residues are two cysteines in a CPYC motif. These cysteines form a disulfide bond when oxidized, and a mixed disulfide bond between GSH and the first cysteine during reduction. The human omega and bacterial beta GSTs also form mixed disulfide bonds with GSH.13,14 Several classes of cytosolic GSTs, including insect delta, mammalian theta and zeta, and plant tau, use a catalytic serine hydroxyl to activate GSH during catalysis.7,15 These cysteine or serine residues are located toward the N- terminal end of α- helix H1. A tyrosine hydroxyl group is used by mammalian alpha, mu, sigma and pi class enzymes.7,15 There has been a particular interest in plant GSTs with regard to herbicide selectivity and environmental safety.15,16 Different classes of herbicides such as triazines, thiocarbamates, chloroacetanilides, diphenylethers, and aryloxyphenoxypropionates can be metabolized by GSTs.17,18 The first GST reported to participate in herbicide metabolism was isolated from maize in 1970, and it was shown that this enzyme was able to conjugate atrazine.19 Herbicide tolerance is based primarily on the differential ability of plant species to detoxify a herbicide, with the formation of a herbicide-GSH conjugate in the resistant but not in the susceptible species. The plant-specific phi and tau GSTs are primarily responsible for herbicide detoxification, showing class specificity in substrate preference. For example, diphenylether herbicides are detoxified rapidly by GSTs in legumes but less efficiently in maize.20 Species-dependent GST-mediated detoxification can be explained by differences in the

expression of the distinct families of plant GST genes. For example, phi enzymes (GSTFs) are highly active toward chloroacetanilide and thiocarbamate herbicides, whereas the tau enzymes (GSTUs) are efficient in detoxifying diphenylethers and aryloxyphenoxypropionates.21,22 Here, we report the cloning, kinetic characterization and crystal structure determination of soy GSTU4-4 in complex with Nb-GSH. Analysis of its binding sites combined with differential sequence conservation among related GSTs revealed the recognition mechanism of efficient diphenylether detoxification by this enzyme. The results of the present work form the basis for a rational design of new, more selective and environmentally friendly herbicides.

Results and Discussion Cloning, expression and characterization of the recombinant enzyme Fluorodifen is a diphenylether herbicide widely used in the treatment of crops and weeds. G. max has very low, constitutively expressed glutathione transferase activity assayed with fluorodifen and GSH (GST-F) as substrates (0.03 U/mg). Treatment with fluorodifen increased the total GST-F activity in G. max seedlings. Affinity chromatography on a biomimetic Cibacron blue 3GA-GSH affinity adsorbent was used to separate the induced GST-F activity in crude extracts.23 One isoenzyme was resolved in fluorodifen-treated G. max that exhibited high GST-F activity. The GST-F eluted activity from fluorodifentreated seedlings was further purified by affinity chromatography on S-hexylglutathione, and Nterminal amino acid sequence of the purified subunit gave the sequence QDEVVLLDFWP. A BLASTp search of the NCBI database using as a query the N-terminal sequence of the fluorodifeninduced GST yielded a single 660 bp (accession number AF04897824) showing identity with the Nterminus. This sequence was used to design specific primers to the 5′ and 3′ coding region sequence. The respective 660 bp cDNA was amplified from soy leaf using RT-PCR and sequenced, showing that the fulllength sequence encoded a polypeptide of 219 amino acids, with a predicted molecular mass of 25,604.6 Da. The polypeptide showed significant identity with other tau class GSTs (GSTU, Fig. 1), around 27–30% sequence identity with the omega, zeta and delta classes, and 22% with the beta class. The sequence is similar with that reported by McGonigle et al.,24 and named GST 4 (or GmGSTU44, according to the nomenclature used by Edwards et al.16). The coding sequence was then ligated into the pQE70 plasmid for expression of the enzyme in Escherichia coli. Cell-free extract of the E. coli transformants showed high GST activity with a specific activity of 2.0 ± 0.5 U/mg protein. Recombinant GmGSTU4-4 was purified (N95% purity) in a single-

986 step procedure involving affinity chromatography on GSH-Sepharose. GSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The substrate specificity of the purified GmGSTU4-4 was investigated in order to identify catalytic activities that may be related to its biological function. To this end, a broad range of substrates was examined. Purified recombinant GmGSTU4-4 was assayed for activities as a GST and as a glutathione peroxidase, and the results are presented in Table 1. The enzyme is characterized by generally broad substrate specificity and exhibits significant differences in its individual activities for a wide variety of hydrophobic substrates. In comparison to the phi class GSTs (e.g., ZmGSTF1-125 ), recombinant GmGSTU4-4 had a much lower level of activity toward CDNB and NBC. Also unlike other phi class enzymes, GmGSTU4-4 showed a low level of activity toward triazine and acetamide herbicides atrazine and alachlor.24 However, GmGSTU4-4 was more active in detoxifying fluorodifen than any other characterized GST from maize.26 GmGSTU4-4 also conjugated ethacrynic acid and trans-4-phenyl-3-buten-2-one. These substrates are thought to form conjugates with GSH via Michael addition reaction to the α,βunsaturated ketone moiety.1,16 Also, the enzyme showed a high level of activity as a glutathione peroxidase, able to reduce cumene hydroperoxide and tert-butyl hydroperoxide, to the corresponding non-toxic alcohol. The attack of reactive oxygen species on cell components results in the production of organic hydroperoxides. GSTs participate in oxidative stress defense mechanisms by catalysing GSH-dependent reactions that inactivate such products by conjugation or reduction. Cumene hydroperoxide has been used extensively as a model substrate for the determination of such an activity. Oxidative stress also results in the production of cytotoxic alkenals. One such example is trans-2nonenal, which was generated by peroxidation of arachidonic acid in rat microsomes exposed to oxidative stress.27 The enzyme is also able to catalyse the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a blue formazan product. In addition, the enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates (R-NH-C(= S)SG).28 Isothiocyanates are abundant in a variety of

Fluorodifen-inducible Glutathione Transferase

edible vegetables and may represent “natural substrates” for this family of enzymes.29 Although the −N = C = S function is rather reactive and therefore short-lived, a continuous supply of isothiocyanates may derive from cleavage of plant glucosinolates. Interest in isothiocyanates has recently been stimulated by the finding that these compounds offer chemoprotection against tumor formation in a variety of animal models.28,29 Steady-state kinetic analysis for selected substrates (CDNB, fluorodifen, cumene hydroperoxide and MTT) was also carried out and the kcat, and Km parameters were determined (Table 2). The Km values for GSH and CDNB were determined as 148.8 μM and 158.2 μM, respectively. With MTT as an electrophile substrate, GmGSTU4-4 exhibits poor affinity for both GSH and MTT despite an efficient catalytic rate constant. It is well-known that GSTs show a remarkable degree of catalytic diversity with single isoenzymes catalysing multiple reaction types using different substrates and employing different catalytic mechanisms. Therefore, GSTs using different substrates exhibit different kinetic constants.1,15,16,30–35 The extent and type of participation of individual residues in catalysis are highly dependent on the nature of the transition state and the rate-limiting step for the reaction in question. It is noteworthy that the large difference observed in the kcat values for GSH and fluorodifen compared to the other substrates (Table 2) may be due to the catalytic diversity of GSTs. As shown in Table 2, the Km values for GSH are highly dependent on the electrophile substrate used. GSH For example, the Km varies between 3.6 μM and 937.2 μM. Probably, this is the result of the rapid equilibrium random sequential bi-bi mechanism with intrasubunit modulation between the GSH binding site and electrophile binding site that is operated by GSTs.23,25 Conformational changes and an induced-fit mechanism upon substrate binding are well known mechanisms that have been observed for several GST isoenzymes; for example, in human GSTA1-1,33 human GST P1-135 and maize GST F1-1.30 It is of particular importance that the Km value for fluorodifen is low (115.9 ± 6.1 μM), indicating high affinity for this herbicide. In general, the enzyme is characterized by low catalytic efficiency values toward all tested substrates. This low level of catalytic efficiency has probably been integral to the evolution

Fig. 1. Sequence alignment of members of the tau family of GSTs compared with the secondary structure of GmGSTU4-4. The GmGSTU4-4 numbering is shown above the alignment and conserved areas are shown shaded. A column is framed if more than 70% of its residues are similar according to physico-chemical properties. This sequence alignment was created using the following sequences (NCBI accession numbers are in parentheses). GmGSTU4-4, Glycine max (AAC18566); GmGST10, G.max (AAG34800.1); GmGST2, G.max (CAA71784); GmGST9, G.max (AAG34799); GmGST16, G.max (AAG34806); CpGST, Carica papaya (CAA04391); NtGST, Nicotiana tabacum (CAA39707); CcGST, Capsicum chinense (CAI48072); VvGST, Vitis vinifera (ABL84692); MpGST, Malva pusilla (AAO61854); SlGST, Solanum lycopersicum (AAF22647); CmGST, Cucurbita maxima (BAC21261); Cc1GST, Castanea crenata (ABG73419); Cc2GST, Castanea crenata (ABG73420); McGST, Matricaria chamomilla (BAB32446); BnGST, Brassica napus (ABD36807); TaGST, Triticum aestivum (CAC94003); HvGST, Hordeum vulgare (ABI18247). The figure was produced using ESPript.

Fluorodifen-inducible Glutathione Transferase

Fig. 1 (legend on previous page)

987

988

Fluorodifen-inducible Glutathione Transferase

Table 1. Substrate specificity for purified recombinant GmGSTU4-4

Substrate 1-Chloro-2,4-dinitrobenzene

Structure

Specific activity (Units/mg protein) (×10− 1) 111.80

p-Nitrobenzyl chloride

2.23

Fluorodifen

0.33

Atrazine

0.02

Alachlor

0.04

Cumene hydroperoxide

4.82

tert-Butyl hydroperoxide

0.85

989

Fluorodifen-inducible Glutathione Transferase Table 1 (continued) Substrate

Structure

Specific activity (Units/mg protein) (×10− 1)

p-Nitrophenyl acetate

1.94

trans-2-Nonenal

0.93

2,3-Dichloro-4-[2-methylene-butyryl]phenoxy)acetic acid (ethacrynic acid)

0.53

trans-4-Phenyl-3-buten-2-one

0.12

Phenethyl isothiocyanate

1.84

Allyl isothiocyanate

0.63

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)

0.44

Enzyme assays were carried out under standard conditions as described in Materials and Methods. The results represent the means of triplicate determinations, with variation b5% in all cases.

990

Fluorodifen-inducible Glutathione Transferase

Table 2. Steady-state kinetic analysis of GmGSTU4-4

Substrate

Km (μM) (GSH)

CDNB 159 ± 18.9 Fluorodifen 65.6 ± 16.6 Cumene 3.60 ± 0.8 hydroperoxide MTT 937 ± 82.5

kcat (min− 1) Km (μM) kcat (min− 1) (GSH) (electrophile substrate) (electrophile substrate)

kcat/Km (×10−3) kcat/Km (×10− 3) (μΜ− 1 min− 1) (μΜ− 1 min− 1) (GSH) (electrophile substrate)

363 ± 14.4 0.36 ± 0.03 14.2 ± 0.54

158 ± 31.6 116 ± 6.1 454 ± 83.6

149 ± 18.6 2.9 ± 0.2 10.8 ± 1.2

2,283 5.5 3,994

941 25.3 24

2.30 ± 0.1

397 ± 30.6

1.03 ± 0.02

2.43

2.58

of GSTs as detoxifiers of a broad spectrum of endogenous and environmental chemicals. Structure determination of GmGSTU4-4 and quality of the final model Plant tau GSTs are a poorly characterized group among the GST superfamily and distinct in their phylogenetic position. To understand the function of tau GSTs as well as to obtain insights into the endogenous functions of this major group of plant GSTs, GmGSTU4-4 was subjected to structural determination by X-ray crystallography. The purified native recombinant GmGSTU4-4 was co-crystallized with Nb-GSH and the structure was determined to 1.75 Å resolution. The final Rcryst was 19.4% (Rfree = 24.3%) with a root-mean-square deviation (RMSD) of 0.015 Å and 1.55° in bond lengths and bond angles, respectively. Details of data collection and refinement statistics are shown in Table 3. Most of the φ,ψ pairs for non-glycine and nonproline residues fell within the energetically favorable regions of the Ramachandran plot. There were two residues (Asn82 and Glu66) in the generously allowed region. Pro12 and Pro200 are outside the allowed regions, and Pro55 is in the cis-conformation. Increased flexibility in both subunits was observed for a few residues between positions 104 and 119 in the polypeptide chain. X-ray structure of GmGSTU4-4 Overall structure GmGSTU4-4 displays the characteristic GST fold (Fig. 2) despite the low level of pairwise sequence identity with other classes. Two GmGSTU4-4 subunits form a dimer with globular shape and molecular dimensions of approximately 63 Å × 49 Å × 33 Å. The crystal structure of the monomer (Fig. 2a) consists of 219 residues. The homodimer assembly of native enzyme is shown in Fig. 2b. A prominent feature in the dimeric structure is the GST-typical very large cleft formed in the center of the dimer, which is open to the active sites and to the bulk solvent. The active site appears as a large inverted L-shape, which is different in shape and size from other plant GSTs, for example members of the phi class have a large and open cavity.30 The two independent subunits exhibit only small differences in

their secondary structure elements, and their threedimensional structures are nearly identical. Each GmGSTU4-4 subunit folds to form two spatially distinct domains; a small N-terminal α/β domain (residues 1–77) and a larger C-terminal α domain (residues 89–219). N-terminal domain The N-terminal domain comprises two large αhelices (H1, residues 14–25; H3, residues 67–77), a short α-helix (H2, residues 42–47) and a four-stranded β-sheet (Figs. 1 and 2a). The four β-strands are arranged in the order β2 (residues 31–34), β1

Table 3. Data collection and refinement statistics A. Data collection Space group Unit cell dimensions parameters a = b (Å) c (Å) α = β = γ (°) No. molecules Resolution range (Å) No. measured reflections Unique reflections Completeness (%) Mosaicity (°) bI/σ(I)N Rmerge (%) Wilson B-factor (Å2) B. Refinement Reflections (working/test) Rcryst (%)/ Rfree (%) No. protein atoms No. water molecules RMS deviation from ideal geometry Bond lengths (Å) Bond angles (°) Ramachandran plot Residues in most favourable regions (%) Residues in additionally allowed regions (%) Average B-factors (Å2) Main chain Side chain Whole chain Water molecules S-(p-Nitrobenzyl)-glutathione (4-Nitrophenyl)methanethiol

P41212 91.4 111.9 90 2 70.71–1.75 (1.78–1.75)# 2012595 48045 (2368) 99.9 (100.0) 0.57 40.1 (4.1) 5 (52.2) 26.9 45,117/2400 19.4/24.3 4243 479 0.015 1.5 93.3 5.2 24.3 (A 23.7, B 24.9) 26.6 (A 25.9, B 27.3) 25.5 (A 24.9, B 26.2) 36.5 24.7 32.3

Numbers in parentheses correspond to the highest resolution shell.

991

Fluorodifen-inducible Glutathione Transferase

that of the phi class Arabidopsis thaliana GST and the human omega class GST.13,31 The C-terminal domain

Fig. 2. (a) A cartoon representation of GmGSTU4-4 monomer coloured from blue (N-terminus) to red (Cterminus). Secondary structure elements are labelled. The inhibitor Nb-GSH is represented as a stick and coloured according to atom type. (b) A ribbon diagram of the dimeric GmGSTU4-4 structure, the twofold axis relating the two subunits is perpendicular to the plane of the page, with subunit A coloured green and subunit B is turquoise. The proposed lock-and-key residues are shown in stick representation and labelled, except Val, which is shown in a ball representation. The three salt bridges that are formed between residues Glu66 and Lys104′ and between Glu76 and side chains of Arg92′ and Arg96′ are also shown. The inhibitor Nb-GSH is represented as a stick and coloured according to atom type. The figure was produced using PyMol [http://pymol.sourceforge.net/].

The larger C-terminal domain (residues 89–219) has an all-α structure composed of amphipathic α-helices arranged in a right-handed spiral (Fig. 2a). The first two helices (H4, resides 89–115; H5, residues 118–139) form an up-down arrangement. α-Helix H4 exhibits a sharp kink at its center and is described more accurately as two helices, termed H4a (residues 89–103) and H4b (residues 107–115). α-Helix H4a is straight and oriented nearly parallel with α-helix H3, while α-helix H4b has a bent appearance and projects over the active site located in the N-terminal domain. H5 (residues 118–139) is connected to the H6 helix (residues 152–171) by a 12 residue long linker. H6 is interrupted at position 162–163 and exhibits a sharp kink at its centre, thus it may be described as two helices, termed H6a and H6b. α-Helix H6a (residues 152–161) is oriented parallel with helix H4a. α-Helix H7 (residues 175–190) is interrupted at position 179. Helices H7 and H8 (residues 193–196) corresponded closely to similar regions in most of the other GST classes, and H9 (residues 202–211) folds back over the top of the N-terminal domain. This helix partially blocks the active site. A similar conformation is found in the human omega class enzyme.13 Likewise, the Cterminal α9-helix forms a ”lid” over the active site of the α class GST.32 The C-terminal residue Val205 makes van der Waals contacts with Trp165 and Phe170, and directs the C-terminus away from the active site. Comparison of the structure of GmGSTU4-4 with other phi class plant GST monomers30,31 reveals that, in accordance with mammalian GSTs especially, the central four-stranded β-sheet and the up and down arrangement of helices H4 and H5 are very similar. Discrepancies are observed in the linker segment, the C-terminal region and the region of the helix joining strands β2 and β3. A significant variation with respect to the A. thaliana structure31 is the region following helix 8, which differs markedly in length and sequence or conformation. Interactions between domains

(residues 5–9), β3 (residues 56–59), and β4 (residues 62–65), and exhibit a left-handed twist. The β3 strand is aligned antiparallel to the others. On one side, the β-sheet is flanked by the two α-helices (H1 and H2) with the H2 helix situated at the solvent-exposed side. Pro55, which is situated in a highly conserved region, adopts a cis-configuration. Pro55 is located at the beginning of β3 and creates the characteristic turn essential for backbone hydrogen bonding to GSH. Trp78 forms the beginning of an 11 residue linker region (residues 78–88), which forms an extended structure that connects the N- and C-terminal domains. This linker folds in a fashion similar to

The interactions between the N- and C-terminal domains are predominantly van der Waals with few polar interactions. The van der Waals interactions are developed between residues from helices H1 and H3 with those of helices H4 and H6. The side chains of Phe15 (H1), and Leu68 (H3) constitute one hydrophobic surface and interact with residues Phe152 and Phe160 (H6). There are polar side chain-to-side chain interactions formed between Arg18 and Asp103, Glu76 and Arg96, and Glu25 and Lys192. The buried surface area between the domains (937 Å2) is less than that observed in the alpha, mu and pi structures

992 (∼ 1400 Å2),33 thus being more similar in size to the theta non-mammalian and zeta class GSTs (1125 Å2).34 Interactions between subunits The dimer has an open configuration with a surface-accessible area buried at the interface of 2250 Å2, comparable with that of the omega class GST (1960 Å2), but significantly smaller than that of most other classes of GST dimer (~2799–3400 Å2). Subunit interfaces involve three types of interactions: salt bridges, hydrogen bonds and hydrophobic interactions, including a lock-and-key motif that physically anchors the two subunits together.35–37 The lock-and-key motif is a common feature of GSTs of the phi, alpha, mu and pi classes, whereas the sigma and theta classes, from which the classes

Fluorodifen-inducible Glutathione Transferase

alpha, mu, and pi have evolved, have another interface topography with fewer protruding residues and a more hydrophilic character.34–36 In the lockand-key motif observed in GmGSTU4-4 (Fig. 2b) the side chain of Val50 in a loop of the N-terminal domain (residues 48–51) functions as a ”key” that extends across the dimer interface and ”locks” into a hydrophobic pocket formed between H4 and H5 in the C-terminal domain of the opposite subunit by Phe97, Trp98, Tyr101 and Leu134. In addition to these hydrophobic contacts, three salt bridges are formed between residues Glu66 and Lys104′ and between Glu76 and side chains of Arg92′ and Arg96′ (Fig. 2b). Protein–protein interfaces appear to constitute a compromise between the stabilization contributed by the hydrophobic effect on one hand and by polar/charged interactions on the other.38 According to Argos, although aromatic residues

Fig. 3. (a) A representation of the G-site of GmGSTU4-4 with the inhibitor Nb-GSH and selected active site side chains represented as sticks. The protein is represented as a cartoon with β-strands coloured magenta and α-helices coloured turquoise. Amino acid side chains that contribute directly to G-site formation are coloured green with the exception of the catalytic serine, which is coloured yellow. Arg18 is coloured magenta and Asp103 and Lys104′ are coloured pink. Water molecules are represented as pink spheres. (b) A representation of the H-site of GmGSTU4-4 with the inhibitor Nb-GSH and selected active site side chains represented as sticks. The protein is represented as a cartoon with β-strands coloured magenta and α-helices coloured turquoise. Amino acid side chains that contribute directly to G-site formation are coloured magenta, with the exception of Trp114, which is coloured green. c, Omit Fobs–Fcalc electron density for the bound Nb-GSH. The map was calculated after a round of refinement without the ligand and is contoured at 2.8 σ. d, A molecular surface representation of GmGSTU44 dimer. The surface is coloured according to the electrostatic potential. Bound Nb-GSH is shown in balls-and-sticks.

993

Fluorodifen-inducible Glutathione Transferase

moiety of the bound molecule is located in a polar region, formed by the beginning of helices H1, H2, and H3 in the N-terminal domain (Table 4). Its γ-Glu moiety points downwards to the internal cavity and its glycine residue is oriented upwards and projects into the bulk solvent. There is no direct interaction between the glutathione portion and residues of the C-terminal domain. However, there are two indirect hydrogen bonds that are formed through a network of water molecules. These residues are Asp103 of helix H4, Lys215 of helix H9 and Lys104 of the second subunit. The γ-Glu moiety of GSH forms hydrogen bonds with Glu66, the main-chain amide and the hydroxyl group of Ser67. The complete conservation of Ser67 (Fig. 1) is consistent with its critical role in GSH binding. Glu66 is also a strictly conserved residue and shows unfavorable φ and ψ angles. The same conformation is observed for the equivalent residues Glu66 of alpha, Gln71 of mu, Gln62 of pi and Glu66 of theta. The cysteinyl moiety of the bound inhibitor forms a hydrogen bond with the peptide bond of Ile54 and the −SH group is within hydrogen bond distance from the hydroxyl group of Ser13. The glycyl moiety of the bound inhibitor forms a hydrogen bond with the side chain of Lys40. Lys53 stabilizes water molecule 370, which is within hydrogen bond distance from the glycyl N and carboxylate. Arg18 (α-helix H1) located at the bottom of the G-site is conserved among all tau GST sequences. This residue, although not involved directly in the formation of the G-site, seems to have an indirect role in GSH binding, and in stabilization of G-site architecture through a network of hydrogen bonds and electrostatic interactions. For example, the side chain of Arg18 forms a strong electrostatic interaction (2.64 Å) and stabilizes Asp103 of the H4a helix. This interaction may additionally stabilize the correct orientation of helices H1 and H4. Asp103 is Fig. 3 (legend on previous page)

have a dominant role in stabilizing protein–protein interfaces, a high proportion of charged and polar residues also contribute at the interfaces, suggesting that ion pairs contribute significantly to protein– protein binding.39 The interactions found at the GmGSTU4-4 dimer form a unique polar/hydrophobic combination of residues across the interface. This, and taking into account the fact that the residues that are involved in the dimer interface are conserved only in tau class GSTs, suggest that tau class enzymes are unable to form heterodimers with GSTs of other classes. GSH binding site (G-site) In each monomer, one molecule of Nb-GSH, a substrate product analogue and competitive inhibitor, is bound as illustrated in Fig. 3. The GSH

Table 4. S-(p-Nitrobenzyl)-glutathione-protein distances and interactions Enzyme residue (atom) Ser13(OG) Lys40(CE) Lys40(NZ) Lys53(CA) Ile54(N) Ile54(O) Ile54(O) Glu66(OE1) Ser67(N) Ser67(N) Ser67(OG) Ser67(OG) Tyr107(OH) Tyr107(OH) Tyr107(OH) Tyr107(OH) Leu212(CD2) Lys215(CB)

Distance (Å)

S-(p-Nitrobenzyl)-glutathione atom

3.2 3.3 2.7 3.3 3.0 2.8 3.1 2.8 3.3 2.9 3.4 2.5 3.3 3.0 3.2 3.4 2.9 3.3

SG2 O31 O31 O2 O2 N2 CG1 N1 O11 O12 C1 O11 C2′ C1′ C′ C6′ O41 O42

The maximum cut-off for distances was set to 3.4 Å.

994 involved in hydrogen bonds with W64, W280 and W53, all interacting with the γ-Glu moiety of GSH and contributing to its correct orientation. Furthermore, Asp103 forms a water-mediated (W280) hydrogen bond with the side chain of Lys104′ of the neighbor subunit, contributing to the subunit interface and therefore to the spatial organization of the dimer. Electrophilic binding site (H-site) The H-site of GmGSTU4-4 is situated next to the Gsite and is formed by residues from the C-terminal domain. In general, the H-site of GSTs exhibits a low degree of sequence identity and hence a unique structure that reflects its different substrate specificity compared to other tau and phi class plant enzymes. The H-site of GmGSTU4-4 is typically hydrophobic, and is built predominantly by hydrophobic residues from the C-terminal domain (Fig. 3b): helix H4a, (Tyr107, Arg111), helix H6 (Trp163) helix H9 (Phe208, Leu212, Lys215 and Leu216), and Phe10 and Leu37 from the N-terminal domain. All these residues are oriented towards the centre of the active site. These residues are not conserved among other classes and probably modulate substrate recognition by affecting the size, the shape and the binding characteristics of the H-site. The 4-nitrobenzyl moiety of the bound ligand is pointing towards the bulk solvent. It is bound in a hydrophobic cleft formed on one side by Tyr107 and Trp163, and on the other side by Phe10, Phe208 and Leu212 (Fig. 3). This hydrophobic pocket might explain its high level of activity toward hydrophobic substrates (Table 1). The nitro group of the inhibitor is closer than 4 Å to the side chain of Lys215 and Leu212. Orientation and conformation of the bound inhibitor resemble that of Nb-GSH of human GST.33 Lys215, which is not a conserved residue among tau class GSTs, is located at the solvent-exposed end of the cleft and its side-chain acts as a lid over the entrance to the site. The presence of this positive charged group capping the H-site may have a significant effect on the substrate specificity. Trp114 is strictly conserved in all tau class enzymes. The side chain of this residue is about 10 Å away from the 4-nitrobenzyl moiety in the complex and, therefore, it may not participate directly in ligand binding. However, it may participate indirectly in the formation of the H-site architecture. It is well established that glutathione reacts predominantly as a thiolate anion and that its formation is facilitated by an active site tyrosine or serine residue by donation of a hydrogen bond.1,15,16 In the beta and omega structures, a cysteine residue has been suggested to be important in catalysis.13,14 Analysis of the GmGSTU4-4 crystal structure shows that there is one serine residues, Ser13, that is strongly conserved and could be catalytically important. In the structure, the hydroxyl group of Ser13 is 3.24 Å from the S atom of the bound inhibitor and forms a hydrogen bond with the sulphur group of GSH.

Fluorodifen-inducible Glutathione Transferase

A striking characteristic of the active site in GmGSTU4-4 is the central position of Tyr107, which is not a conserved residue and has been replaced by other polar or non-polar residues in other tau class GSTs (Fig. 1). The hydroxyl group of Tyr107 points towards the aromatic ring of the bound inhibitor, and is able to make an on-face hydrogen bond between the hydroxyl group of its side chain and the π-electron cloud of the benzyl group of 4-nitrobenzyl. This interaction may stabilize aromatic substrates at their productive orientation. Tyr108 in human and GSTP1-1 has been found at equivalent position and has been implicated in the catalytic mechanism.40 The topmost region of α-helix H4 contains one arginine residue (Arg111) that is not conserved among tau class GSTs. The guanidino group of Arg111 makes a hydrogen bond (3.10 Å) with the hydroxyl group of Tyr107, which may provide the correct orientation for the hydroxyl group. In addition, the positive charge of Arg111 may stabilize the high-energy Meisenheimer intermediate complex that is formed during nucleophile substitution reactions. Arg111, together with Lys215 from H9 and Arg18, Lys40 and Lys53 from the N-terminal domain, make the approach to the H-site significantly basic (Fig. 4d). These basic residues form a positively charged region at the active site, which should enable the enzyme to bind negatively charged substrates (e.g. ethacrynic acid, Table 1). In addition, this positive electrostatic field may contribute to −SH ionisation of GSH. The involvement of positively charged residues in the electrostatic field regulation has also been observed in other GSTs.41,42 Another characteristic of the structure is the high flexibility of the loop between α-helices H4 and H5,

Fig. 4. A representation of the putative L-site of GmGSTU4-4 with the ligand (4-nitrophenyl)methanethiol. Selected binding site residues are shown as sticks. The ligand is coloured according to atom type. The protein is represented as a cartoon with β-strands coloured magenta and α-helices coloured turquoise. Amino acid side chains that contribute to the L-site formation are coloured blue with the exception of Arg18, which is coloured green. Nb-GSH is shown in a stick representation and coloured yellow.

Fluorodifen-inducible Glutathione Transferase

995

and of α-helix H5 itself. In GmGSTU4-4 the B-factors of the loop and helix are very high, clearly demonstrating increased flexibility. The high level of flexibility can be explained by the presence of Gly118, which is located at the end of the loop. Ligand binding site (L-site) In each monomer, one molecule of (4-nitrophenyl) methanethiol is bound, as illustrated in Fig. 4. The source of this compound in the crystallisation mixture is unknown. However, it is more conceivable that this compound is a degradation product of NbGSH. The compound is located in a hydrophobic surface pocket formed by Trp11, Arg20, Tyr30, Tyr32, Leu199 and Pro200. Important interactions involved in the binding are given in Table 5. In particular, the nitro-group of (4-nitrophenyl)methanethiol makes four hydrogen bonds (two with Arg20, two with the main-chain peptide bond of Trp11 and Pro200) and several van der Waals contacts (e.g. Tyr32, Lys197, Ser198 and Leu199). In total, the ligand makes 37 van der Waals contacts (b4 Å). There is only one water-mediated interaction with the inhibitor identified in the crystal structure. In both monomers, water molecule 457 is within hydrogen bonding distance from one of the − NO2 oxygen atoms. In addition, an edge-to-face aromatic interaction may be formed between the benzyl ring of the bound ligand and the benzyl ring of Tyr32. The possibility that (4-nitrophenyl)methanethiol is actually a glutathione conjugate in which the glutathionyl moiety is invisible due to too high flexibility and lack of a defined position in the structure was excluded for a number of reasons. For example, the B-factors for the surrounding residues are about 20 Å 2 , indicating that there is low flexibility in the region. Superposition of the NbGSH onto (4-nitrophenyl)methanediol shows that the glycyl moiety of the inhibitor Nb-GSH will make clashes with Tyr30. In addition, contouring of the electron density map to low σ levels revealed no density that would fit glutathione. The main binding residues (Trp11, Arg20, Tyr30 and Tyr32) are, in general, conserved within the tau GST family (Fig. 1). This probably indicates that this newly identified binding site may be of biological interest. It has been reported that GSTs, in addition Table 5. (4-nitrophenyl)methanethiol-protein distances and interactions in the putative L-site Enzyme residue (atom) Trp11 (CB) Arg20 (NH1) Arg20 (NH1) Tyr30 (OH) Tyr32 (CB) Lys197 (O) Lys197 (O) Leu199 (C) Pro200 (N)

Distance (Å)

4-Nitrophenyl-methanethiol atom

3.4 3.1 3.4 3.4 3.5 3.2 3.2 3.2 3.1

O41 O41 N41 C′ SG2 C2′ C3′ O42 O42

Fig. 5. Difference spectra titrations of p-nitrophenol in the presence of GmGSTU4-4•Nb-GSH complex. The figure depicts the dependence of difference absorbance at 410 nm as a function of the total concentration of p-nitrophenol (0–20 μM).

to their catalytic function, may act as ligand-binding proteins and bind hydrophobic molecules in a nonsubstrate manner into a distinct site (termed L-site) sequestering them away from critical intracellular targets.43–48 To analyse whether this site exhibits the characteristics of the L-site (saturation binding, high affinity, stoichiometry close to unity) the binding of p-nitrophenol to the GmGSTU4-4•Nb-GSH complex was investigated by difference spectroscopy (Fig. 5). The results showed a hyperbolic dependence of binding on p-nitrophenol concentration and the formation of one type of complex with stoichiometry (defined after Scatchard analysis) between enzyme subunit and p-nitrophenol close to unity (0.9 ± 0.1). The KD was 11.6 ± 0.1 μM, which indicates high affinity with the enzyme. All this kinetic evidence, together with the crystallographic data, point to the conclusion that this binding site represents a novel L-site. Although many compounds have been identified as ligands and their binding parameters have been characterized, there is little information about the exact location and nature of the L-site in GSTs. For example, a similar hydrophobic surface pocket was found in human P1-1 enzyme that binds part of the compound sulfasalazine. In this case, the sulfasalazine sits in a hydrophobic pocket lined with the side chains of Pro9, Val10, Pro202 and Gly205.43 On the other hand, the L-site of GST from Schistosoma japonica44 is located at the dimer interface. In the case of the Arabidopsis enzyme,31 the L-site is located next to the G-site between the side chains of helices α3″/ α3‴ and α 5″, whereas the L-site of the human pi class GST is located in the H-site.45 Several GST isoenzymes are able to interact with plant hormones and flavonoids with distinct binding specificities 46–48. For example, the recombinant GSTF2 from A. thaliana binds indole-3-acetic acid and the artificial auxin 1-N-naphthylphthalamic acid.47 The GST isoenzymes AN9 from petunia and GST III from maize are inhibited by several

996 flavonoids.46 The GST isoenzymes GST1-1 and GST2-1 from Nicotiana tabacum could be inhibited by 2,4-dichlorophenoxyacetic acid, but only the GST1-1 isoenzyme is inhibited by the structurally related compound 2,4-dichlorobenzoic acid.48 The precise functions of ligandin GmGSTU4-4 binding to non-substrate ligands is unknown. One possibility is that binding of non-substrate ligands to GmGSTU44 prevents oxidation of the molecules in vivo. Another possibility is that GmGSTU4-4 prevents cellular damage by cytotoxic and genotoxic compounds that can oxidize protein and intercalate into DNA. The third possibility is that GSTs may facilitate delivery of the bound ligands to specific receptors (proteins) or cellular compartments. Considering that the proposed binding site in GmGSTU4-4 is located at the surface of the protein and is accessible to the bulk solvent, it is unlikely to have a protective role for bound ligands. It is therefore more conceivable that this site may be involved in transfer and delivery of bound ligands to specific protein receptors. When GSTs perform their ligand-binding or ligandin function, their catalytic activity is usually inhibited in a non-competitive manner. The structural basis whereby catalytic function is modulated by the occupation of these sites is unclear. However, analysis of the present crystal structure showed that a plausible mode of structural communication between the L-site and G-site exists. For example, this may be achieved through Arg20 (Fig. 4), which may influence the conformation of its neighbour Arg18 for which an important structural role has been assigned in the stabilization of G-site architecture (see above). It is well established that protein–protein interactions take place physically between interface residues of two complementary proteins. Studies focusing on protein interfaces have revealed that binding energies are not distributed uniformly along the protein interfaces. Instead, there are certain critical residues called hot spots. These residues comprise only a small fraction of interfaces, yet account for the majority of the binding energy.49 Systematic analysis has found the hot spots to be particularly enriched in Trp, Tyr and Arg. In addition, these are largely surrounded by hydrophobic rings, probably to occlude bulk solvent.50 It is important to point out that the proposed L-site in GmGSTU4-4 exhibits the characteristics of a putative protein–protein interaction interface. This observation further support the significance of the new (4-nitrophenyl)methanethiol site identified in GmGSTU4-4 and points to the conclusion that it may be involved in transfer and delivery of bound ligands to specific protein receptors.

Fluorodifen-inducible Glutathione Transferase

Molecular docking of the fluorodifen to the H-site was done to analyse the high level of catalytic activity of GmGSTU4-4 towards this important herbicide. The model for the binding of fluorodifen to GmGSTU4-4 (Fig. 6) was constructed on the basis of: (i) the proposed role of Ser13, Tyr107 and Arg111 in catalysis; (ii) the orientation of Nb-GSH conjugate in our structure; (iii) the location of the electrophile carbon atom of fluorodifen; and (iv) manual fitting to obtain optimum interactions between the hydrophobic chain of fluorodifen and the hydrophobic residues of the H-site. The results of the docking showed that the 4-nitrobenzyl ring of fluorodifen is located towards the solvent, at the same position found in the Nb-GSH conjugate. The ether group and the electrophile carbon of 2-nitrobenzyl ring is positioned close to the −SH group of GSH, making it susceptible to nucleophilic attack. The important Tyr107 and Arg111 are well positioned close to the electrophile reaction centre. The 2-nitrobenzyl ring is likely to interact with Phe15 from the N-terminal domain. Site-directed mutagenesis The mechanism of fluorodifen reaction with GSH proceeds via an SnAr nucleophilic attack of GSH at the site of substitution to give a negatively charged Meisenheimer complex. This S nAr reaction is favoured by the electron withdrawing substituent on the ring (− NO2) since mesomerically withdrawing substituents at positions ortho (e.g. − NO2) to the site of substitution are particularly effective in promoting SnAr reaction by decreasing π-electron density and facilitate approach of a nucleophile. The hydroxyl group of Tyr107 is located close to the ether oxygen atom. The side chain of Arg111 makes a hydrogen bond with the phenolic oxygen of Tyr107. The hydroxyl group of Ser13 makes a hydrogen bond with the sulfur atom of GSH and is located in a position that would allow it to stabi-

Fluorodifen binding site The H-site is significantly larger and more hydrophobic than that observed for other classes of GSTs. The active site cleft is large, and is able to accommodate with high affinity long hydrophobic substrates such as fluorodifen (see the structure in Table 1).

Fig. 6. The predicted mode of interaction between fluorodifen and GmGSTU4-4. The ligand is represented as a stick and coloured according to atom type. Mutated residues are represented as sticks.

997

Fluorodifen-inducible Glutathione Transferase Table 6. Kinetic parameters of mutant enzymes for the fluorodifen-GSH reaction GSH Enzyme

Km (μM)

kcat (min− 1)

Wild type Ser13Ala Tyr107Ala Arg111Ala

65.6 ± 16.6 159 ± 7.2 23.9 ± 6.4 35.6 ± 2.8

0.36 ± 0.03 0.05 ± 0.02 0.11 ± 0.007 0.15 ± 0.004

Fluorodifen kcat/Km (× 10− 3) (μΜ− 1 min− 1)

Km (μM) ()

kcat (min− 1)

5.5 0.31 4.6 4.2

116 ± 6.1 186 ± 8.1 188 ± 77.3 54.7 ± 5.1

2.9 ± 0.2 0.04 ± .0.01 0.39 ± 0.13 0.19 ± 0.013

lize the thiolate anion of glutathione and enhance its nucleophilicity. Site-directed mutagenesis was used to evaluate the role of Ser13, Tyr107 and Arg111 in catalysis; Ser13, Tyr107 and Arg111 were mutated to Ala. The mutants were expressed, purified and their kinetic parameters kcat and Km toward fluorodifen were determined by steady-state kinetic analysis (Table 6). The results showed that the mutated enzymes Ser13Ala, Tyr107Ala and Arg111Ala exhibited 115-, 12.7- and 7.2-fold decreases in kcat/Km values, respectively, compared to the wild type enzyme. The mutations do not change appreciably the affinity of the H-site for the electrophile substrate. It is noteworthy that mutant Ser13Ala showed an increased value of Km for GSH, indicating that Ser13 contributes significantly to GSH binding and stabilization in the active site. On the other hand, the mutants Tyr107Ala and Arg111Ala showed decreased values of Km for GSH, although their catatalytic efficiency (kcat/Km) towards GSH remained essentially unaltered. The effect of viscosity on the kinetic parameters was measured in order to analyse the rate-limiting step of the catalytic reaction. A decrease of kcat induced by increasing the medium viscosity should indicate that the rate-limiting step of the reaction is related to the product release or to diffusioncontrolled structural transitions of the protein.51 A o o plot of the inverse relative rate constant kcat /kcat (kcat o is determined at viscosity η ) versus the relative viscosity, η/ηo should be linear, with a slope equals to unity when the product release is limited by a strictly diffusional barrier or a slope approaching zero if catalytic reaction chemistry is rate-limiting.52 The o inverse relative rate constant kcat /kcat for GmGSTU4-4 shows linear dependence on the relative viscosity with a slope of 0.87± 0.1 (Fig. 7). The intermediate value of the slope (0 b slope b 1) observed indicates that the rate-limiting step in the enzyme is not dependent on a diffusional barrier (e.g. product release) and other viscosity-dependent motions or conformational changes of the protein contribute to the rate-limiting step of the catalytic reaction.51 The effect of viscosity was also evaluated using two additional substrates: p-nitrobenzyl chloride and MTT. The slopes obtained for p-nitrobenzyl chloride and MTT were 0.70 ± 0.1 and 0.79 ± 0.1, respectively, supporting the results obtained using fluorodifen as substrate. In contrast, the mutants Ser13Ala, Tyr107Ala and Arg111Ala exhibit kcat values with different degrees of viscosity-dependence, compared to the

kcat/Km (× 10− 3) (μΜ− 1 min− 1) 25.3 0.22 2.0 3.5

wild type enzyme (Fig. 7). It is important to note that glycerol does not induce changes in the enzyme secondary structure as detected by far-UV difference spectroscopy (spectra not shown). Furthermore, glycerol does not have a non-specific inhibitory effect on catalysis. The significantly lower values of the slopes (slopes approaching zero) observed in the mutants Ser13Ala (slope 0.084), Tyr107Ala (slope 0.095) and Arg111Ala (slope 0.203) indicate that probably the catalytic reaction chemistry contributes more significantly to the rate-limiting step of the catalytic reaction in the case of mutant enzymes. Taking this conclusion into account and considering that the mutated amino acid residues are not located at mobile or flexible regions (as observed by analysing the crystallographic B-factors), it is more conceivable to conclude that Ser13, Tyr107 and Arg111 do not seem to contribute to catalysis through modulating specific conformational changes in the enzyme. Therefore, the effect of mutations in catalytic efficiencies may be plausibly explained assuming their direct involvement in the reaction chemistry. The double mutant may be informative with respective to the catalytic mechanism. However, the presence of two mutations at a catalytically important and conserved region (e.g. α-helix H4 in the present study) may cause secondary effects and structural perturbations.

Fig. 7. The effect of viscosity on kcat for the fluorodifenGSH reaction catalysed by GmGSTU4-4 and its mutants. A plot of the reciprocal of the relative turnover number (kocat/ kcat) as a function of relative viscosity (η/ηo) with glycerol as cosolvent for the wild type (●) and for the mutants Ser13Ala (○), Tyr107Ala (□) andArg111Ala, ( ). Lines were calculated by least-squares regression analysis.

▪

998 In conclusion, we have addressed questions regarding the structure and function of a tau class GST from soy, a relatively poorly characterized group among the GST superfamily. Detailed studies of tau GSTs are justified because of the considerable agronomic potential of these enzymes. Important residues that contribute to fluorodifen binding and catalysis have been determined. Since the function of a given GST cannot be predicted from the primary structure, studies based on protein crystallography in combination with expression and activity assays, such as those presented here, provide a powerful approach for investigating GST-mediated herbicide tolerance. The results of the present work may form the basis for a rational design of new, more selective and environmentally friendly diphenylether herbicides.

Materials and Methods Materials Poly(A) mRNA purification kits, first-strand cDNA synthesis kits, dNTPs and restriction enzymes were obtained from Promega (UK). Reduced glutathione, 1-chloro-2,4dinitrobenzene (CDNB), Nb-GSH and all other enzyme substrates were obtained from Sigma-Aldrich, USA. XL1Blue E. coli cells and Pfu DNA polymerase were purchased from Stratagene, USA. Methods Plant growth G. max seeds were pre-germinated on plates, on filter paper (Whatman 3MM, soaked in distilled water). The plates were kept for 72 h at 30 °C. After germination, they were transferred into soil in plastic pots. The plants were grown in a controlled environment (12 h light at 25 °C/ 12 h dark at21 °C, 65 % relative humidity, watered with deionized water every two days. The soy seedlings were treated with or without fluorodifen (0.2 mM) for 2 days. Purification of fluorodifen-induced GST Protein extraction from soy was typically carried out by mixing whole plants (5 g) with 15 mL of 50 mM sodium phosphate buffer pH 5.0. The mixture was disintegrated in a blender (15 s bursts for a total of 10 min) and placed on a rotary mixer for 60 min at 4 °C. The mixture was subsequently centrifuged at 10,000g for 30 min at 4 °C. The supernatant was collected and passed through a 0.45 μm pore size filter. The amount of total protein extracted was determined by the Bradford assay and GSTs were measured by a fluorodifen-GSH activity assay. Soy seedlings were treated with or without fluorodifen for two days and the GST present was isolated by affinity chromatography on Cibacron blue 3GA-GSH biomimetic adsorbent23 as follows: a solution of plant extract (2 mg of total protein) previously dialysed against 20 mM Tris–HCl, pH 7.2 was applied to the affinity adsorbent (1 mL). The adsorbent was washed with 10 mL of equilibration buffer. Bound proteins were eluted with equilibration buffer containing different concentrations of GSH (1–13 mM). Fractions (2 mL) were collected, assayed for GST-F activity and total protein was determined by the

Fluorodifen-inducible Glutathione Transferase

Bradford assay. The fractions containing GST activity were combined and dialysed against equilibration buffer (20 mM Tris–HCl pH 7.5) and loaded onto an affinity column of S-hexylglutathione coupled to epoxy-activated agarose at a flow rate of 1 mL min− 1. The column was washed with equilibration buffer, followed by 20 mM Tris–HCl pH 7.5 containing 50 mM NaCl. Material with GST activity was eluted with 20 mM Tris–HCl pH 7.5 containing 10 mM GSH. The eluted fractions were assayed for GSH conjugating activity toward fluorodifen, and those with high activity were pooled, concentrated and desalted using Centricon-10 centrifugal concentrators (Millipore, Watford, UK). The desalted enzyme solution was analysed by SDS-PAGE and transferred electrophoretically onto PVDF membranes. The band of interest was excised from the membrane and sequenced with an Applied Biosystems protein sequencer, (model 477A) equipped with an on-line phenylthiohydantoin analyser (model 120 A). Molecular cloning Poly(A) mRNA was purified from crude plant seedlings using an RNA Isolation Kit. First-strand cDNA synthesis was done using oligo-p(dT)15 primer and AMV reverse transcriptase. The PCR-primers 5′-TTT TGC ATG CAG GAT GAG GTA GTG TTA TTA G-3′ (forward primer) and 5′-TTA AGC TTC TAC TCA ATG CCT AAC TTC TTT C-3′ (reverse primer) were designed according to the GmGSTU4-4 gene sequence.24 SphI and HindIII restriction sites were introduced at the forward and reverse primers, respectively. The GmGSTU4-4 gene was amplified using the polymerase chain reaction (PCR). The reaction contained 1 μg of template cDNA, 0.2 mM each of the four deoxyribonucleotide triphosphates (dNTPs), 10 × Pfu buffer, 1.5 U of Pfu DNA polymerase and 4 pmol of each primer. A total of 30 cycles of the PCR reaction were carried out with denaturation at 94 °C for 1.5 min, annealing at 55 °C for 2 min and polymerization at 72 °C for 2 min, followed by 10 min at 72 °C. This program gave, on a 1.2% (w/v) agarose gel, a single band at the correct size (670 bp) that was extracted from gel with QIAquick. For the subcloning of PCR product into the pQE-70 vector, both the purified PCR product and vector were digested with the restriction enzymes SphI and HindIII. The digested products were submitted to agarose gel electrophoresis and extracted from the gel before being used in a ligation reaction. This ligation mixture was used to transform competent E. coli XL1-Blue cells. Positive clones were selected after digestion with the restriction enzymes SphI and HindIII. All sequences were verified by DNA sequencing on Applied Biosystems Sequencer 373A with the DyeDeoxy Terminator Cycle sequencing kit. Expression and purification of recombinant GmGSTU4-4 E. coli M15[pREP4] cells harbouring recombinant plasmid were grown at 37 °C in 1 L of LB medium containing ampicillin (100 μg/mL) and kanamycin (50 μg/mL). The synthesis of GST was induced by the addition of 1 mM isopropyl 1-thio-β-galactopyranoside when the absorbance at 600 nm was 0.6. At 4 h after induction, cells were harvested (∼3 g) by centrifugation at 5000g for 15 min, resuspended in 9 mL of 0.1 M potassium phosphate buffer pH 6.5, sonicated, and centrifuged at 13,000 rpm for 5 min. The supernatant was loaded onto a column of glutathione coupled to epoxy-activated Sepharose (1,4-butanediol diglycidyl ether-GSH-SepharoseCL6B, 1 mL of affinity adsorbent), which was previously

999

Fluorodifen-inducible Glutathione Transferase

equilibrated with 20 mM potassium phosphate buffer pH 7. Non-adsorbed protein was washed off with 10 mL of equilibration buffer. Bound GST was eluted with equilibration buffer containing 10 mM glutathione. The protein purity was judged by SDS-PAGE.

Deoxy Terminator Cycle sequencing kit. The mutant enzymes were expressed and purified as described for the wild type enzyme.

Assay of enzyme activity and protein

Difference spectral titrations were done with a PerkinElmer Lamda16 double beam double monochromator UVVIS spectrophotometer at 30 °C. Enzyme solution (0.5 mg of wild-type GmGSTU4-4 in 1 mL of 20 mM potassium phosphate, pH 7, 10 mM Nb-GSH) and 1 mL of enzyme solvent (20 mM potassium phosphate, pH 7, 10 mM NbGSH) were placed in the sample and reference black-wall silica cuvettes (10 mm pathlength), respectively, and the baseline difference spectrum was recorded in the range 700–300 nm. Identical volumes (2–5 μL) of 0.5 mM pnitrophenol solution were added to both cuvettes and the difference spectra were recorded after each addition. The difference of absorption at 410 nm was measured relative to a zero-absorbance reference area at 700 nm. The data were analysed using Scatchard analysis and according to Axarli et al.,4 using the equation:

Enzyme assays for the CDNB and fluorodifen conjugation reactions were done as described.25,53 Assays were carried out at 37 °C (CDNB assay) or at 30 °C (fluorodifen assay) in order to increase the sensitivity. The reaction velocities were corrected for spontaneous reaction rates when necessary. All initial velocities were determined in triplicate in buffers equilibrated at constant temperature. Turnover numbers were calculated on the basis of one active site/subunit. Glutathione peroxidase activity was determined as described.54 The reactions were done at 37 °C in 600 μL of 0.1 M potassium phosphate buffer, pH 7.5, 1 mM EDTA, 1 mM GSH, 1.5 mM cumene hydroperoxide, 0.2 mM NADPH, one unit of glutathione reductase, and GmGSTU4-4. All other conjugation reactions were done as described.11,24,55 Protein concentration was determined by the Bradford assay using bovine serum albumin (fraction V) as the standard. Kinetic analysis Steady-state kinetic measurements for the wild type and mutant enzymes were done at 37 °C (CDNB assay) or at 30 °C (fluorodifen assay).53 Initial velocities were determined in the presence of 5 mM GSH, and fluorodifen was used in the concentration range 3–60 μM. Alternatively, fluorodifen was used at a fixed concentration (0.05 mM), and the concentration of glutathione was varied in the range 25–1000 μM. Solutions of glutathione or analogues were prepared fresh each day. Steady-state data were fit to the Michaelis-Menten equation by non-linear regression analysis using GraFit (Erithacus Software Ltd.). Viscosity dependence of kinetic parameters The effect of viscosity on kinetic parameters was assayed at 30 °C with various concentrations of glycerol as described.25 Site-directed mutagenesis Site-directed mutagenesis was done as described.56 The pairs of oligonucleotide primers used in the PCR reactions were as follows. For the Ser13Ala mutation: 5′-TTCTGGCCAGCTCCATTTGGGATG-3′ 5′-CATCCCAAATGGAGCTGGCCAGAA-3′

For the Tyr107Ala mutation: 5′-AAGAAGATAGCTGATCTTGGAAGG-3′ 5′-CCTTCCAAGATCAGCTATCTTCTT-3′

For the Arg111Ala mutation: 5′-GATCTTGGAGCGAAGATTTGGACA-3′ and 5′-TGTCCAAATCTTCGCTCCAAGATC-3’

All mutations were verified by DNA sequencing with an Applied Biosystems Sequencer 373A with the Dye-

Difference spectroscopy

DA =

DAmax ½pQnitrophenol KD + ½pQnitrophenol

where ΔA is the difference of absorption at 410 nm after each addition of p-nitrophenol, and ΔA max is the maximum difference of absorption at 410 nm at saturated concentration of p-nitrophenol. Bioinformatics analysis and molecular modelling Sequences homologous to GmGSTU4-4 were sought in the NCBI using BLAST.57 The resulting sequence set was aligned with ClustalW.58 ESPript† was used for alignment, visualization and manipulation. Electrostatic calculations and graphics were produced using the CCP4 molecular graphics program.59 The Discovery Studio suite of programs was employed for predicting the geometry of the fluorodifen bound to the enzyme‡. Crystallisation Purified GmGST4-4 was concentrated to 12.5 mg/mL in 10 mM Hepes buffer pH 7.0 and mixed with an S-(pnitrobenzyl)-glutathione solution to a final concentration of 10 mM. Crystallization trials were set-up using the INDEX crystal screen (Hampton). Crystals appeared after four to five days in condition 70 (0.2 M NaCl, 25% (w/v) PEG 3350, 0.1 M Bis-Tris, pH 5.5). Data collection and processing Data were collected from a single crystal on station X11 in EMBL-Hamburg. Before data collection, the crystal was soaked briefly in a mother liqueur solution containing 20% (v/v) glycerol as a cryoprotectant and was subsequently flash-cooled in a stream of liquid nitrogen. A total of 250 diffraction images were collected at 0.8088 Å wavelength using a MAR CCD detector with a crystal-to-detector distance of 142 mm and Δφ 0.5°. The raw diffraction data † http://prodes.toulouse.inra.fr/ESPript/cgi-bin/ ESPript.cgi_exe.cgi ‡ http://accelrys.com

1000 were indexed, processed and scaled using the HKL suite.60 The data collection statistics are given in Table 3. Structure determination and refinement The structure of GmGST4-4 was determined by molecular replacement using PHASER61 and the rice GST1 structure (PDB code 1OYJ) as a search model. All residues in the search model were truncated to alanine. The best solution was obtained in space group P41212. Building of the side chains was done with ARP/wARP62 and manual intervention for corrections. Refinement was carried out using REFMAC63 through the CCP4i.64 Medium NCS restraints were applied in the early stages of the refinement and were released at the final stages. The progress of refinement was monitored with Rfree and inspection of 2|Fo|–|Fc| and |Fo|–|Fc| maps in COOT.65 When Rfree reached 27.3%, TLS refinement was applied and the Rfree dropped to 25.3%.. Stereochemical analysis was done with PROCHECK66 in CCP4i. Protein Data Bank accession code The refined coordinates of the model and the structure factors have been deposited with the Protein Data Bank under the accession code 2vo4.

Acknowledgements This work was supported by a grant from AUA (code no. 020083). I.A. gratefully acknowledges the A.S. Onassis Public Benefit Foundation for financial support. A.C.P. thanks the Sigrid Jusélius Foundation, the Centre for International Mobility (CIMO), and the Academy of Finland (grant no. 121278) for research funding. Access to EMBL Hamburg (c/o DESY) was provided by the European Community (Access to Research Infrastructure Action of the Improving Human Potential Programme to the EMBL Hamburg Outstation, contract number: HPRI-CT-1999-00017).

References 1. Armstrong, R. N. (1997). Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem. Res. Toxicol. 10, 2–18. 2. Rea, P. A. (1999). MRP subfamily ABC transporters from plants and yeast. J. Exp. Bot. 50, 895–913. 3. Hayes, J. D., Flanagan, J. U. & Jowsey, I. R. (2005). Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45, 51–88. 4. Axarli, I. A., Rigden, D. J. & Labrou, N. E. (2004). Characterization of the ligandin site of maize glutathione S-transferase I. Biochem. J. 382, 885–893. 5. Dixon, D. P., Lapthorn, A., Madesis, P., Mudd, E. A., Day, A. & Edwards, R. (2008). Binding and glutathione conjugation of porphyrinogens by plant glutathione transferases. J. Biol. Chem. 283, 20268–20276. 6. Mannervik, B. & Danielson, U. H. (1988). Glutathione transferases-structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283–337.

Fluorodifen-inducible Glutathione Transferase

7. Oakley, A. J. (2005). Glutathione transferases: new functions. Curr. Opin. Struct. Biol. 15, 716–723. 8. Alias, Z. & Clark, A. G. (2007). Studies on the glutathione S-transferase proteome of adult Drosophila melanogaster: responsiveness to chemical challenge. Proteomics, 7, 3618–3628. 9. Dixon, D. P., Davis, B. G. & Edwards, R. (2002). Functional divergence in the glutathione transferase superfamily in plants. Identification of two classes with putative functions in redox homeostasis in Arabidopsis thaliana. J. Biol. Chem. 277, 30859–30869. 10. Allocati, N., Favaloro, B., Masulli, M., Alexeyev, M. F. & Di Ilio, C. (2002). Proteus mirabilis glutathione S-transferase B1-1 is involved in protective mechanisms against oxidative and chemical stresses. Biochem. J. 373, 305–311. 11. Wiktelius, E. & Stenberg, G. (2007). Novel class of glutathione transferases from cyanobacteria exhibit high catalytic activities towards naturally occurring isothiocyanates. Biochem. J. 406, 115–123. 12. Xia, B., Vlamis-Gardikas, A., Holmgren, A., Wright, P. E. & Dyson, H. J. (2001). Solution structure of Escherichia coli glutaredoxin-2 shows similarity to mammalian glutathione-S-transferases. J. Mol. Biol. 310, 907–918. 13. Board, P. G., Coggan, M., Chelvanayagam, G., Easteal, S., Jermiin, L. S., Schulte, G. K. et al. (2000). Identification, characterization, and crystal structure of the Omega class glutathione transferases. J. Biol. Chem. 275, 24798–24806. 14. Rossjohn, J., Polekhina, G., Feil, S. C., Allocati, N., Masulli, M., De Illio, C. & Parker, M. W. (1998). A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications. Structure, 6, 721–734. 15. Frova, C. (2006). Glutathione transferases in the genomics era: new insights and perspectives. Biomol. Eng. 23, 149–169. 16. Edwards, R., Dixon, D. P. & Walbot, V. (2000). Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health. Trends Plant Sci. 5, 193–198. 17. Edwards, R. & Dixon, D. P. (2005). Plant glutathione transferases. Methods Enzymol. 401, 169–186. 18. Edwards, R. & Dixon, D. P. (2000). The role of glutathione transferases in herbicide metabolism. In Herbicides and Their Mechanisms of Action (Cobb, A. H. & Kirkwood, R. C., eds), pp. 38–71, Sheffield Academic Press, Sheffield, UK. 19. Lamoureux, G. L., Shimabukuro, R. H., Swanson, H. R. & Frear, D. S. (1970). Metabolism of 2-chloro4-ethylamino-6-isopropylamino-s-triazine (atrazine) in excised sorghum leaf sections. J. Agric. Food Chem. 18, 81–86. 20. Shimabukuro, R. H., Frear, D. S., Swanson, H. R. & Walsh, W. C. (1971). Glutathione conjugation. An enzymatic basis for atrazine resistance in corn. Plant Physiol. 47, 10–14. 21. Jepson, I., Lay, V. J., Holt, D. C., Bright, S. W. J. & Greenland, A. J. (1994). Cloning and characterization of maize herbicide safener-induced cDNAs encoding subunits of glutathione S-transferase isoforms I, II and IV. Plant Mol. Biol. 26, 1855–1866. 22. Thom, R., Cummins, I., Dixon, D. P., Edwards, R., Cole, D. J. & Lapthorn, A. J. (2002). Structure of a tau class glutathione S-transferase from wheat active in herbicide detoxification. Biochemistry, 41, 7008–7020. 23. Melissis, S. C., Rigden, D. J. & Clonis, Y. D. (2001). New family of glutathionyl-biomimetic ligands for

1001

Fluorodifen-inducible Glutathione Transferase

24.

25.

26. 27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

affinity chromatography of glutathione-recognising enzymes. J. Chromatogr. A, 917, 29–42. McGonigle, B., Keeler, S. J., Lau, S. M., Koeppe, M. K. & O'Keefe, D. P. (2000). A genomics approach to the comprehensive analysis of the glutathione Stransferase gene family in soybean and maize. Plant Physiol. 124, 1105–1120. Labrou, N. E., Mello, L. V. & Clonis, Y. D. (2001). Functional and structural roles of the glutathionebinding residues in maize (Zea mays) glutathione S-transferase I. Biochem. J. 358, 101–110. Sommer, A. & Buger, P. (1999). Characterization of recombinant corn glutathione S-transferase isoforms I, II, III and IV. Pestic. Biochem. Physiol. 63, 127–138. Esterbauer, H., Benedetti, A., Lang, J., Fulceri, R., Fauler, G. & Comporti, M. (1986). Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation. Biochim. Biophys. Acta, 876, 154–166. Kolm, R. H., Danielson, U. H., Zhang, Y., Talalay, P. & Mannervik, B. (1995). Isothiocyanates as substrates for human glutathione transferases: structure-activity studies. Biochem. J. 311, 453–459. Fenwick, G. R., Heaney, R. K. & Mullin, W. J. (1983). Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr. 18, 123–201. Prade, L., Huber, R. & Bieseler, B. (1998). Structures of herbicides in complex with their detoxifying enzyme glutathione S-transferase-explanations for the selectivity of the enzyme in plants. Structure, 6, 1445–1452. Reinemer, P., Prade, L., Hof, P., Neuefeind, T., Huber, R., Zettl, R. et al. (1996). Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 Å resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J. Mol. Biol. 255, 289–309. Kuhnert, D. C., Sayed, Y., Mosebi, S., Sayed, M., Sewell, T. & Dirr, H. W. (2005). Tertiary interactions stabilise the C-terminal region of human glutathione transferase A1-1: a crystallographic and calorimetric study. J. Mol. Biol. 349, 825–838. Sinning, I., Kleywegt, G. J., Cowan, S. W., Reinemer, P., Dirr, H. W., Huber, R. et al. (1993). Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. J. Mol. Biol. 232, 192–212. Thom, R., Dixon, D. P., Edwards, R., Cole, D. J. & Lapthorn, A. J. (2001). The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism. J. Mol. Biol. 308, 949–962. Hegazy, U. M., Mannervik, B. & Stenberg, G. J. (2004). Functional role of the lock and key motif at the subunit interface of glutathione transferase p1-1. J. Biol. Chem. 279, 9586–9596. Sayed, Y., Wallance, L. A. & Dirr, H. W. (2000). The hydrophobic lock-and-key intersubunit motif of glutathione transferase A1-1: implications for catalysis, ligandin function and stability. FEBS Lett. 465, 169–172. Vargo, M. A., Nguyen, L. & Colman, R. F. (2004). Subunit interface residues of glutathione S-transferase A1-1 that are important in the monomer-dimer equilibrium. Biochemistry, 43, 3327–3335. Veselovsky, A. V., Ivanov, Y. D., Ivanov, A. S., Archakov, A. I., Lewi, P. & Janssen, P. (2002).

39. 40.

41.

42.

43.

44.

45.

46.

47.

48.

49. 50.

51.

52.

53.

Protein-protein interactions: mechanisms and modification by drugs. J. Mol. Recognit. 15, 405–422. Hubbard, S. J. & Argos, P. (1995). Evidence on close packing and cavities in proteins. Curr. Opin. Biotechnol. 6, 375–381. Lo Bello, M., Oakley, A. J., Battistoni, A., Mazzetti, A. P., Nuccetelli, M., Mazzarese, G. et al. (1997). Multifunctional role of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1. Crystallographic and kinetic studies on the Y108F mutant enzyme. Biochemistry, 36, 6207–6217. Patskovsky, Y. V., Patskovska, L. N. & Listowsky, I. (1999). Functions of His107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a. Biochemistry, 38, 1193–1202. Patskovsky, Y. V., Patskovska, L. N. & Listowsky, I. (2000). The enhanced affinity for thiolate anion and activation of enzyme-bound glutathione is governed by an arginine residue of human Mu class glutathione S-transferases. J. Biol. Chem. 275, 3296–3304. Hayeshi, R., Chinyanga, F., Chengedza, S. & Mukanganyama, S. (2006). Inhibition of human glutathione transferases by multidrug resistance chemomodulators in vitro. J. Enzyme Inhib. Med. Chem. 21, 581–587. McTigue, M. A., Williams, D. R. & Tainer, J. A. (1995). Crystal structures of a schistosomal drug and vaccine target: glutathione S-transferase from Schistosoma japonica and its complex with the leading antischistosomal drug praziquantel. J. Mol. Biol. 246, 21–27. Oakley, A. J., Lo Bello, M., Nuccetelli, M., Mazzetti, A. P. & Parker, M. W. (1999). The ligandin (nonsubstrate) binding site of human Pi class glutathione transferase is located in the electrophile binding site (H-site). J. Mol. Biol. 291, 913–926. Mueller, L., Goodman, C. D., Silady, R. A. & Walbot, V. (2000). AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein. Plant Physiol. 123, 1561–1570. Smith, A. P., Nourizadeh, S. D., Peer, W. A., Xu, J., Bandyopadhyay, A., Murphy, A. S. & Goldsbrough, P. B. (2003). Arabidopsis AtGSTF2 is regulated by ethylene and auxin, and encodes a glutathione Stransferase that interacts with flavonoids. Plant J. 36, 433–442. Droog, F., Hooykaas, P. & Van Der Zaal, B. J. (1995). 2,4-Dichlorophenoxyacetic acid and related chlorinated compounds inhibit two auxin-regulated type-III tobacco glutathione S-transferases. Plant Physiol. 107, 1139–1146. Bogan, A. A. & Thorn, K. S. (1998). Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280, 1–9. Ma, B., Elkayam, T., Wolfson, H. & Nussinov, R. (2003). Protein-protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces. Proc. Natl Acad. Sci. USA, 100, 5772–5777. Johnson, W. W., Liu, S., Ji, X., Gilliland, G. L. & Armstrong, R. N. (1993). Tyrosine 115 participates both in chemical and physical steps of the catalytic mechanism of a glutathione S-transferase. J. Biol. Chem. 268, 11500–11511. Sampson, N. S. & Knowles, J. R. (1992). Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase. Biochemistry, 31, 8488–8494. Dixon, D. P., McEwen, A. G., Lapthorn, A. J. & Edwards, R. (2003). Forced evolution of a herbicide

1002

54.

55. 56.

57. 58.

59.

detoxifying glutathione transferase. J. Biol. Chem. 278, 23930–23935. Wilson, S. R., Zucker, P. A., Huang, R. R. C. & Spector, A. (1989). Development of synthetic compounds with glutathione peroxidase activity. J. Am. Chem. Soc. 111, 5936–5939. Habig, W. H., Pabst, M. J. & Jakoby, W. B. (1974). Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77, 51–59. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Thompson, J. D., Higgins, D. J. & Gibson, T. J. (1994). CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K. & Noble, M. (2002). The CCP4 molecular-graphics project. Acta Crystallogr. D, 58, 1955–1957.

Fluorodifen-inducible Glutathione Transferase

60. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collection in oscillation mode. Methods Enzymol. 276, 307–326. 61. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. (2005). Likelihood-enhanced fast translation functions. Acta Crystallogr. D, 61, 458–464. 62. Morris, R. J., Perrakis, A. & Lamzin, V. S. (2003). ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 374, 229244. 63. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D, 53, 240–255. 64. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. (2003). A graphical user interface to the CCP4 program suite. Acta Crystallogr. D, 59, 1131–1137. 65. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr. D, 60, 2126–2132. 66. Laskowski, R., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291.