Identification of Residues in Glutathione Transferase Capable of Driving Functional Diversification in Evolution

A NOVEL APPROACH TO PROTEIN REDESIGN*

Ylva IvarssonDagger §, Aaron J. Mackey§, Maryam EdalatDagger , William R. Pearson||, and Bengt MannervikDagger **

From the Dagger  Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden, the  Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, and the || Department of Biochemistry and Molecular Genetics, Charlottesville, Virginia 22908

Received for publication, November 19, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Evolution of protein function can be driven by positive selection of advantageous nonsynonymous codon mutations that arise following gene duplication. By observing the presence and degree of site-specific positive selection for change between divergent paralogs, residue positions responsible for functional changes can be identified. We applied this analysis to genes encoding Mu class glutathione transferases, which differ widely in substrate specificities. Approximately 3% of the amino acid residue positions, both near to and distant from the active site, are under statistically significant positive selection for change. Relevant human glutathione transferase (GST) M1-1 and GST M2-2 codons were mutated. A chemically conservative threonine to serine mutation in GST M2-2 elicited a 1,000-fold increase in specific activity with the GST M1-1-specific substrate trans-stilbene oxide and a 30-fold increase with the alternative epoxide substrates styrene oxide and nitrophenyl glycidol. The reverse mutation in GST M1-1 resulted in reciprocal decreases in activity. Thus, identification of hypervariable codon positions can be a powerful aid in the redesign of protein function, lessening the requirement for extensive mutagenesis or structural knowledge and sometimes suggesting mutations that would otherwise be considered functionally conservative.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The adaptive evolution of novel protein function is thought to result from a period of relaxed purifying selection immediately following gene duplication, in which mutations that provide the duplicated gene with an advantageous altered function may be positively selected (1-3). Most positive selections for change have been observed in genes involved in host-pathogen interactions (4, 5), where pathogen antigenic determinants and their complementary host recognition molecules drive continuous adaptation. Thus, the concept of hypervariable codon positions is recognized as a reasonable explanation of evolutionary change in some biological systems. In other gene families, however, the role of positive selection is unclear (6), and direct experimental support for predictions of positions driving diversification in protein evolution is weak, although positive selection for change was recently observed at amino acid positions associated with DNA binding in Pax family proteins (7).

The glutathione transferases (GSTs)1 are a family of multifunctional proteins that provide cellular defense against toxic electrophiles of both exogenous and endogenous origins. The enzymes catalyze the conjugation of electrophiles to the reactive thiol of GSH, converting the electrophile into a more water-soluble product, which can be metabolized into a mercapturic acid for urinary excretion (8). The GSTs are grouped into different classes primarily based on protein sequence similarities, and the genes of the GSTs are clustered on different chromosomes in a manner consistent with their evolutionary relationship (9).

The cytosolic GSTs are dimers (see Fig. 1), each subunit having an active site consisting of a GSH binding site (the G-site) and a hydrophobic substrate-binding site (the H-site). Whereas the structure of the G-site is well conserved among GSTs, the H-site varies widely in different classes, leading to differences in substrate selectivities. Within a class, both homodimeric and heterodimeric structures occur (10).

The human Mu class is one of the largest GST classes, with five genes clustered on chromosome 1p13.3 (11, 12). Different Mu class GSTs are expressed to varying extents in different tissues, and, despite high sequence identity (80-90%), they display major differences in their substrate selectivities. For example, GST M1-1 is expressed at the highest level in the liver and has distinctive high activity with the epoxide trans-stilbene oxide (tSO). In contrast, GST M2-2 is not detectable in the liver (13) and has negligible tSO activity (10). GST M2-2 occurs at a high level in the brain and is uniquely active with aminochrome, a toxic ortho-quinone derived from dopamine, and with other oxidation products of catecholamines (14).

The evolution of the Mu class of GSTs has involved multiple gene duplications, such that orthologous relationships between Mu class GSTs from primates and rodents are difficult to infer. However, the multiplicity of homologous sequences (see Fig. 2A) offers the possibility to identify hypervariable amino acid positions.

Previous attempts to rationally redesign the substrate selectivities of GSTs have relied upon structural comparisons of homologous proteins, predicting functionally important H-site residues based on stereochemical principles (15, 16). Here we use an evolutionary approach, asking whether positive selection can be observed within Mu class GST genes, by first observing the naturally occurring mutations at positions identified to be under positive selection and then directly testing whether these mutations confer altered substrate selectivity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phylogenetic Tree Construction-- An ungapped protein alignment of class Mu GSTs was assembled from 11 rodent, 7 primate, and 1 chicken amino acid sequence. The coding DNA sequences for each of the 19 GST genes were collected into a multiple alignment, using the protein sequence alignment as a guide. The PHYLIP utility dnaml was used to construct the phylogenetic tree from the DNA multiple alignment. 1,000 bootstraps on the tree. Codon-aware resamplings were used to generate 1,000 bootstrapped trees via the fastDNAml variant of dnaml.

Positive Selection Analysis-- The DNA multiple alignment and the phylogenetic tree were used as input to the codonml program of the PAML version 3.1 package (17), with CodonFreq = 3 (all 61 codon frequencies estimated) and site-specific rate variation models PAML M3 (two freely estimated categories of omega , the nonsynonymous to synonymous substitution ratio) versus PAML M3 (three categories of freely estimated omega ), PAML M1 (two categories of omega  = [0 or 1]) versus PAML M2 (three categories of omega  = [0, 1, and one freely estimated omega  category]), and PAML M7 (continuous distribution of omega  values, defined by a Beta distribution, 0 < omega  < 1) versus PAML M8 (Beta distributed omega , plus one freely estimated omega  category) (18).

Construction of GST M2-2 Mutants-- All of the GST M2-2 mutants were constructed by inverted polymerase chain reaction using Pfu DNA polymerase and custom synthesized oligonucleotides primers (Interactiva Virtual Laboratory). The GST M1-1/S210T variant was constructed as previously described (19). GST M2-2/T210S was made using the GST M2-2 clone (20) as template. The GST M2-2/T210S/A130E and GST M2-2/T210S/A130E/F104T variants were then constructed sequentially. The PCRs contained 10 ng of DNA template, 0.25 mM dNTPs, 1.5 µM concentration of each primer, 2.5 units of Pfu DNA polymerase (Stratagene, La Jolla, CA), 10 mM Tris-HCl (pH 8.8), 50 mM KCl, and 1.5 mM MgCl2. The PCR was conducted using the following temperature cycle: 1) 95 °C for 5 min, 2) 95 °C for 1 min, 3) 55 °C for 1 min, 4) 72 °C for 9 min, and 5) 72 °C for 30 min. Steps 2-4 were repeated 25 times. The PCR product was purified on agarose gel and blunt end-ligated. Escherichia coli XL1 Blue cells (Stratagene, La Jolla, CA) were transformed with the ligation mixture by electroporation. The mutations were confirmed by DNA sequence analysis.

Expression and Purification-- E. coli XL1 Blue carrying the expression vector pKK-D with wild-type GST DNA (M1-1, M2-2) or mutant GST DNA (GST M2-2/T210S, GST M2-2/T210S/A130E, GST M2-2/T210S/A130E/F104T, or GST M1-1/S210T) was grown in 2TY (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl) at 37 °C. Overnight cultures were diluted 250-fold in 2TY and were allowed to grow at 37 °C to A600 approx  0.3. Protein expression was induced via the addition of isopropyl-1-thio-beta -D-galactopyranoside to a final concentration of 0.2 mM, and the cells were grown overnight. The cells were harvested by centrifugation and lysed by ultrasonication.

GST M2-2, GST M1-1, and the mutant GST proteins, respectively, were purified from lysates by affinity chromatography on glutathione-Sepharose affinity gel (Amersham Biosciences) (21). The purity of the enzyme samples was confirmed using SDS-PAGE with Coomassie Brilliant Blue staining. Protein concentrations were determined by absorbance measurements at 280 nm, using, for GST M2-2 and the mutant GST M2-2 proteins, an extinction coefficient of 81,680 M-1 cm-1 and a molecular mass of 51 kDa and, for GST M1-1 and the M1-1 mutant, an extinction coefficient of 78,000 M-1 cm-1 and a molecular mass of 51 kDa.

Assay of Enzyme Activity-- Six alternative electrophiles were used to monitor the effect of the substitutions in GST M1-1 and GST M2-2 (see Fig. 3). Specific GST activities with tSO were determined spectrophotometrically at 235 nm (Delta epsilon 235 = -20,300 M-1 cm-1) in 250 mM Tris-HCl containing 5% EtOH (v/v) (pH 7.2), using 4 mM GSH and 150 µM tSO. The specific activities with SO were measured in the same buffer at 234 nm (Delta epsilon 235 = 760 M-1 cm-1) using 5 mM GSH and 1.6 mM SO. Specific activities with CDNB, cyanoDMNG, NPG (10), and aminochrome (22) were determined by spectrophotometric assays at 30 °C under standard conditions.

Steady-state Kinetic Measurements-- Kinetic constants were determined using steady-state kinetic analysis at a saturating concentration of glutathione (5 mM); tSO was used in the concentration range 7.5-150 µM, and CDNB was used in a concentration range of 0.05-1.5 mM. All kinetic data were obtained at 30 °C. Steady-state kinetic parameters were determined by fitting the Michaelis-Menten equation to the data points using Prism 2.0 (GraphPad Software Inc., San Diego, CA). The values of kcat were expressed per subunit (25,600 Da).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Positive Selection Analysis-- To test for the presence of positively selected residues among the Mu class GST, both maximum likelihood-based (17, 18, 23, 24) (see Fig. 2B and Table I) and Bayesian (25) (data not shown) analyses of mutational rate variability among codons were applied. Both types of analysis suggested that about 3% of the codon positions exhibit a higher rate of nonsynonymous mutation than synonymous mutation, consistent with positive selection for change. Under different models of positive selection (18) (see "Experimental Procedures"), residues 130, 210, and 214 (numbered with respect to Mu class glutathione transferase 1, denoting the initiator methionine as number 1) were consistently identified as likely to be under positive selection (see Fig. 2 and Table I), whereas residues 104, 205, and 206 were less strongly indicated. Comparison of human GST subunits M2 and M3 shows that their primary structures differ in all of these six hypervariable positions (Figs. 1 and 2). Between GST M1-1 and GST M2-2, residues 205, 206, and 214 are identical and can consequently not be responsible for the distinct substrate selectivities of the two enzymes.

                              
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Table I
Maximum log likelihood scores, parameter estimates, and likelihood ratio test (LRT) statistics of models for positive selection within GST genes


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Fig. 1.   Heterodimeric human GST M2-3 consisting of subunit M2 (left, Calpha wire) and subunit M3 (right, flat ribbon). The model is based on crystal structure 3GTU in the Protein Data Bank. The focus of catalytic function in GST subunit M2 is indicated by the red hydroxyl groups of active site residues (green) Tyr7 and Tyr116. Residues Phe104, Ala130, and Thr210 (blue) were judged from evolutionary analysis as being under positive selection for change. Corresponding hypervariable residues in GST subunit M3 (with a four-residue N-terminal extension) are Val108, Glu134, and Asn214 (blue).


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Fig. 2.   Evolutionary tree, polymorphic residues at sites identified to be under positive selection, and relative substrate specificities of Mu class glutathione transferases. A, Mu class glutathione transferase genes are from human (h), macaque (q), mouse (m), rat (r), and chicken (c). Shown are residues with posterior probability of being under positive selection greater than 0.5. Residue positions are numbered with reference to human Mu class glutathione transferase M1-1. Relative specific activities are represented by a plus sign for each order of magnitude. B, the probability of each tested model (18) for positive selection for change is calculated as p(iLRT >=  chi 2, df = 2), in comparison with an equivalent base model with no positive selection for change. The LRT statistic, when measured between models with nested parameters, is conservatively distributed as chi 2, with degrees of freedom equal to the number of additional parameters estimated by the larger model (24).

Expression and Purification-- Single, double, and triple mutations were made to human GST M2-2 at positions 210, 130, and 104 (T210S, A130E, F104T), sequentially replacing the original GST M2-2 residues with the corresponding human GST M1-1 residues. The effect of the reverse mutation of GST M1-1 at residue 210 (S210T) was also studied. The recombinant enzymes were successfully purified from E. coli XL1 Blue. A single band on SDS-PAGE confirmed the purity of the samples. The GST M2-2 wild-type and M2-2 mutants were obtained in yields ranging between 40 and 90 mg/liter. The yield of GST M1-1 protein was 11 mg/liter, and the yield of the mutant GST M1-1 S209T was 1.6 mg/liter. All enzymes could be stored on ice for several months without loss of activity.

Enzymatic Specific Activities-- The specific activities of the purified enzymes were determined with six alternative substrates (Fig. 3). Two of these compounds, aminochrome and cyanoDMNG, are distinctive GST M2-2 substrates, whereas the epoxides used (tSO, SO, and NPG) are preferred GST M1-1 substrates, and CDNB is a general GST substrate (10). A 1,000-fold increase in the specific activity of GST M2-2 by the mutation T210S was obtained with tSO, the epoxide substrate characteristic for GST M1-1 (Table II). The reverse mutation S210T in GST M1-1 caused a 100-fold loss in specific activity with the same substrate. The specific activities with the alternative epoxide substrates (SO oxide and NPG) increased 30-fold in the GST M2-2 mutant. In contrast, no major effects of the mutations were seen with the GST M2-2-specific substrates aminochrome and cyanoDMNG or with CDNB, which give activities of the same magnitude with both GST M1-1 and GST M2-2 (Table II). Mutation of residues 104 and 130 in GST M2-2 did not result in marked alterations of the catalytic properties of the T210S mutant.


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Fig. 3.   Electrophilic substrates used to monitor catalytic activities and effects of the mutations in GST M2-2 and GST M1-1.

                              
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Table II
Specific activities of wild-type and mutant human Mu class GSTs with alternative electrophilic substrates

Catalytic Efficiencies-- Steady-state kinetic parameters were determined with the two alternative substrates CDNB and tSO at a saturating concentration of GSH (5 mM). Due to the low solubility of tSO, only kcat/Km values could be determined accurately (Table III). The catalytic efficiencies of the mutant enzymes with CDNB were in the same range as those of the wild-type GSTs. Wild-type GST M1-1 has a uniquely high catalytic efficiency (kcat/Km value) with tSO; wild-type GST M2-2 has ~3,000-fold lower efficiency. The catalytic efficiencies of the GST M2-2 mutants had increased more than 200-fold to 8-13% of the value characterizing GST M1-1 with tSO as the electrophilic substrate.

                              
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Table III
Catalytic efficiencies (kcat/Km) of wild-type and mutant human Mu class GSTs with CDNB and tSO


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this investigation, nonsynonymous/synonymous evolutionary rate analysis was used to identify positions within Mu class GSTs likely to be under positive selection for change. Six residues (104, 130, 205, 206, 210, and 214; numbered with respect to Mu class glutathione transferase M1-1) were identified as hypervariable, three of which (205, 206, and 214) are identical between GST M1-1 and GST M2-2. These results complement those of a recent study of Pax gene family members, where two of three hypervariable positions were found to drive changes of differences in site-specific DNA binding (7).

The three hypervariable residues distinguishing GST M1-1 and GST M2-2 were mutated in order to evaluate their significance for the differential substrate selectivities of the two enzymes. Of the three positions targeted for mutation in GST M2-2, only residue 210 markedly influenced the measured catalytic activity (Table II). This is also the one of the variable residues that appears to be capable of contacting the substrate in the H-site, based on crystal structures (26). The observed difference between GST M1-1 and GST M2-2 at residue 210 is a serine/threonine interconversion (Fig. 2), which normally would be considered a conservative, function-conserving exchange.

The T210S mutation in GST M2-2 elicited a 1,000-fold increase in specific activity with tSO and a parallel, somewhat smaller, increase with the alternative epoxides substrates SO and NPG. The CDNB activity, which is high for both GST M2-2 and GST M1-1, was also largely unchanged. In contrast, mutation of residue 210 did not markedly alter the activity of GST M1-1 or GST M2-2 with aminochrome, an ortho-quinone substrate distinguishing GST M2-2 from other GSTs (19), or with cyanoDMNG, another GST M2-2-specific substrate (Table II). The six alternative electrophilic substrates monitor the effect of the mutations on four different types of reaction: Michael addition to aminochrome, denitrosation of cyanoDMNG, aromatic substitution of CDNB, and conjugation of the three epoxide substrates (Fig. 3). The functional consequences of the mutations are highly selective and depend on the chemical mechanisms of the catalyzed reaction. Apparently, the different reaction chemistries require different structural complements in the active site.

The structure of the homologous rat GST M1-1 (27) has revealed a role of Tyr116 in the protonation of the oxygen of epoxide substrates and an auxiliary function of Ser210, which forms a hydrogen bond to the phenolic oxygen of Tyr116 (Fig. 4B). Quantum mechanical/molecular mechanical simulations suggest that Ser210 may also interact directly with the oxirane oxygen of the substrate (28). The primary hydrogen bond donor Tyr116, as well as another active site tyrosine residue (Tyr7), is conserved in the Mu class GSTs. A structure of human GST M2-2 in complex with an appropriate active site ligand is not available. However, it is likely that the side chain methyl group of Thr210 interferes with the formation of a hydrogen bond from Thr210 that would facilitate protonation of the oxirane ring of epoxide substrates, thus explaining the effects of the T210S exchange. In addition, the simulations indicate that the number of hydrogen bonds from active site water molecules decreases to the glutathione thiolate and increases to the oxirane oxygen along the reaction coordinate (28). This dynamic process contributes to catalysis by desolvation of the nucleophilic sulfur and stabilization of the nascent oxyanion. The proximity of the Thr210 methyl substituent in GST M2-2 may sterically hinder rate-contributing interactions of water molecules with the reactants that are possible with Ser210.


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Fig. 4.   Structures of the active sites of human GST M2-2 and the homologous rat GST M1-1. A, active site of human GST M2-2 (Protein Data Bank code 1HNA), the conserved active site residues Tyr7 and Tyr116, the hypervariable Thr210, and the bound ligand GSH are shown. B, rat GST M1-1 enzyme in complex with the conjugate of GSH and phenantrene 9,10-oxide (Protein Data Bank code 2GST).

In enzyme-catalyzed reactions, the specificity constant kcat/Km reflects catalytic efficiency. Further, the ratio of specificity constants for alternative substrates is a measure of the substrate selectivity of the enzyme (29). The value of kcat/Km for the substrate tSO increases by almost 3 orders of magnitude by the mutations in GST M2-2 (Table III), and the discrimination against alternative substrates increases by a similar factor (cf. Table II). Thus, the substrate selectivity is drastically altered in parallel with the increased catalytic efficiency with epoxide substrates. The enhanced catalytic efficiency is clearly not a general increase of catalytic activity in the mutated GST M2-2 but a reflection of preferential transition state stabilization of reactions between epoxides and the nucleophilic sulfur of glutathione. The T210S mutation in GST M2-2 affords a decrease in the transition state energy (Delta Delta GDagger ) of ~14.7 kJ/mol for the tSO reaction.

There are several approaches to the redesign of protein function: rational protein redesign, stochastic methods, and combinations of these methods. In rational design, the residues targeted for mutagenesis are selected on the basis of detailed knowledge of protein structure, function, and mechanism (15, 16, 30). Thus, rational design requires an understanding of the function of residues in the active site, information that partly can be obtained from crystal or solution structures. The stochastic approach uses random mutagenesis and DNA shuffling, followed by screening or selection (31, 32).

In the present study, evolutionary rate analysis served as the basis for redesigning the substrate selectivity of human Mu class GSTs. The remarkable change in activity with epoxide substrates caused by the interchange of serine and threonine in position 210 provides experimental support for the notion that hypervariable codons can drive divergent protein evolution. Furthermore, this is the first example of protein redesign based on mutations of hypervariable codons. This approach is not dependent on any structural information about the active site or other functional regions. However, it requires knowledge of DNA sequences for a number of closely related proteins, and it relies on a phylogenetic relationship among the analyzed sequences. The evolutionary approach to the engineered diversification of protein function will become more practical as larger numbers of closely related (diverged in the past 100 million years) genomes are sequenced and dozens of orthologous and paralogous sequences become available for additional gene families.

    ACKNOWLEDGEMENTS

We thank Dr. Ziheng Yang for advice on PAML and Dr. Lars O. Hansson for making the GST M1-1/S210T mutant available.

    FOOTNOTES

* This work was supported by the Swedish Research Council and a grant from the United States National Library of Medicine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: Dept. of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden. Tel.: 46-18-471-45-39; Fax: 46-18-55-84-31; E-mail: Bengt.Mannervik@biokem.uu.se.

Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M211776200

    ABBREVIATIONS

The abbreviations used are: GST, glutathione transferase; CDNB, 1-chloro-2,4-dinitrobenzene; cyanoDMNG, 2-cyano-1,3-dimethyl-1-nitrosoguanidine; NPG, (2R,3R)-(+)-3-(4-nitrophenyl)glycidol; SO, styrene-7,8-oxide; tSO, trans-stilbene oxide; LRT, likelihood ratio test.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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