From the 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
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ABSTRACT |
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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.
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.
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 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
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 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 ( 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).
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.
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.
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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, the nonsynonymous to
synonymous substitution ratio) versus PAML M3 (three
categories of freely estimated
), PAML M1 (two categories of
= [0 or 1]) versus PAML M2 (three categories of
= [0, 1, and one freely estimated
category]), and PAML
M7 (continuous distribution of
values, defined by a Beta
distribution, 0 <
< 1) versus PAML M8 (Beta
distributed
, plus one freely estimated
category) (18).
0.3. Protein expression was induced via the addition of
isopropyl-1-thio-
-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.
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.
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
(
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, C
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
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
2, with degrees of freedom equal to the number of
additional parameters estimated by the larger model (24).
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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.
Specific activities of wild-type and mutant human Mu class GSTs with
alternative electrophilic substrates
Catalytic efficiencies (kcat/Km) of
wild-type and mutant human Mu class GSTs with CDNB and tSO
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
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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 (G
) 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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ziheng Yang for advice on PAML and Dr. Lars O. Hansson for making the GST M1-1/S210T mutant available.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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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.
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