(Received for publication, April 6, 1994; and in revised form, October 31, 1994)
From the
The common substrate for glutathione S-transferases
(GSTs), 1-chloro-2,4-dinitrobenzene (CDNB), is an inhibitor of Escherichia coli growth. This growth inhibition by CDNB is
enhanced when E. coli expresses a functional GST. Cells under
growth inhibition have reduced intracellular GSH levels and form
filaments when they resume growth. Based on this differential growth
inhibition by CDNB we have developed a simple procedure to select for
null-mutants of a human GST in E. coli. Null mutations in the
human GST gene from hydroxylamine mutagenesis or
oligonucleotide-directed mutagenesis can be selected for on agar plates
containing CDNB after transformation. The molecular nature of each
mutation can be identified by DNA sequence analysis of the mutant GST
gene. We have identified three essential amino acid residues in an
alpha class human GST at Glu, Glu
, and
Gly
. Single substitution at each of these residues, E31K,
E96K, G97D, resulted in mutant GST proteins with loss of CDNB
conjugation activity and failure in binding to the S-hexyl GSH
affinity matrix. In contrast, a mutant GST (Y8F) resulting from
substitution of the conserved tyrosine near the N terminus has much
reduced CDNB conjugation activity but was still capable of binding to
the S-hexyl GSH-agarose. Additional mutant GSTs with
substitutions at position 96 (E96F, E96Y) and 97 (G97P, G97T, G97S)
resulted in changes in both K
and k
to different extents. The in vitro CDNB conjugation activity of the purified mutant enzymes correlate
negatively with the plating efficiencies of strains encoding them in
the presence of CDNB. Based on the x-ray structure model of human GST
1-1, two of these residues are involved in salt bridges
(Arg
-Glu
,
Arg
-Glu
)) and the third
Gly
is in the middle of the helix
4. Our results
provide evidence in vivo that Tyr
,
Gly
, and the two salt bridges are important for GST
structure-function. This molecular genetic approach for the
identification of essential amino acids in GSTs should be applicable to
any GSTs with CDNB conjugation activity. It should also complement the
x-ray crystallographic approach in understanding the structure and
function of GSTs.
The glutathione S-transferases (GSTs, EC 2.5.1.18) ()are ubiquitous in nature. They are a family of dimeric
multifunctional proteins in xenobiotic biotransformation, drug
metabolism and protection against peroxidative damage (for recent
reviews, see (1, 2, 3, 4) ). To
accomplish these diverse physiological functions most organisms have
the genetic capacity to encode multiple GST
isozymes(5, 6, 7, 8) . The GST
isozymes have an absolute requirement for reduced glutathione in the
conjugation reactions with electrophiles; some of the GSTs also have
the GSH dependent peroxidase activities (for reviews, see (9) and (10) ). To form an efficient network in
transforming a variety of chemical structures in a multitude of
xenobiotic compounds, considerable diversity in the GST isozyme
structures is required in their interactions with xenobiotic compounds
(for reviews, see (9, 10, 11) ). Diversity in
the genetic repertoire of the GST gene superfamily would predict the
existence of many more new gene families than what are currently
known(5) . Recent discovery in a variety of organisms revealed
a GST isozyme complexity beyond the commonly known alpha, mu, pi, and
the microsomal GSTs(7, 8, 12, 13) .
An important area in GST research is to understand the molecular
basis of GSTs' broad substrate specificities and the mechanisms
of catalysis. Mutagenesis in vitro has been and will continue
to be a powerful tool for studying the structure/function relationship
in
GSTs(14, 15, 16, 17, 18, 19) .
The first phase of targeted mutagenesis has been directed toward the
charged amino acids conserved among the different isozyme
families(5, 14, 19) . The GST cDNA sequences
in the literature provide the data base for locating highly conserved
amino acid residues (5, 20, 21) . These
conserved residues are proposed to be important in catalysis, GSH
binding and/or structural motifs for xenobiotic substrate binding (5) because GST isozymes from different gene families have no
more than 25-35% sequence identity. Recent results from several
laboratories revealed that the conserved tyrosine residue in the
N-terminal region of different classes of GSTs is essential for
catalysis by facilitating the ionization of GSH to form the thiolate
anion(16, 17, 18, 19, 22) .
More recently the preferred approach for choosing targets of
mutagenesis is based on x-ray structural analysis. The structures of
porcine and human GST (23, 24) , rat GST
3-3 (Y
Y
)(25, 26) ,
human mu GST M2-2(27) , and human alpha GST 1-1 (28) have been solved by x-ray crystallography recently. For
example, recent results by site-directed mutagenesis have confirmed the
prediction of the x-ray crystallographic analysis that the N-terminal
region tyrosine is important in
catalysis(16, 17, 18, 19, 22, 29) .
A third approach based on chemical modification has been proven
informative in cases where specific affinity labels were
used(30, 31, 32) .
Targeted mutagenesis based on the x-ray structures is one of the most effective approaches in molecular enzymology for relating specific amino acid residues to enzymatic functions(18, 33, 34) . The detailed visualization of a protein's three-dimensional structure provides static views of not only the native enzyme but also enzyme complexes with substrates or inhibitors. Such structural insights allow the formulation of specific questions regarding enzyme structure and function. In general, it is easy to identify amino acid targets in close proximity to the substrate binding site(s) or are integral parts of well defined structural motifs. Important residues outside the immediate vicinity of active sites are usually difficult to be recognized with a functional perspective, however. Furthermore, the characterization of biochemical and enzymatic properties of mutant GSTs in vitro has not been matched by functional analyses in vivo(14, 15, 16, 17, 18, 19, 29, 33, 34) . Consequently, there is a pressing need to identify a convenient phenotype for GST expression and to develop a method for measuring GST activities in vivo. Satisfying such a need will also facilitate the development of a simple dominant selection method for identifying essential amino acid residues based on GST functions in vivo. In this communication we report the establishment of a procedure to measure GST activity in vivo and the identification of several essential amino acid residues in an alpha class GST based on its in vivo function. These essential amino acids were identified by selecting null mutants (i.e. loss of GST functions) of human GSTs in Escherichia coli. The molecular basis of this selection is that the most common substrate for GSTs, CDNB, has antibiotic activity (35) . This antibiotic activity is enhanced when E. coli expresses a functional GST. This method for the identification of essential amino acids for GST structure and function should have general applications for all GSTs. In addition, the severity of each mutation in GST structure/function can be tested easily in vivo before detailed biochemical and molecular analysis of mutant enzymes in vitro.
The mutants E96F/E96Y/E96L and G97P/G97T/G97S/G97L were
isolated from a pool of mutants derived from using the two 40-mer
oligonucleotides (oligo-96 and oligo-97) with degeneracy at each of the
two respective positions. The procedures are nearly identical to those
for the Y8F mutation. The DNA for mutagenesis was the single-stranded
pALTER-GTH121 DNA templates. Among products of oligo-96 mutagenesis, 8
out of 12 sequenced DNA isolates were mutants with substitutions of
either F, Y, or L. Among products of oligo-97 mutagenesis, 20 out of 36
sequenced isolates were mutants with amino acid substitutions of either
P, T, S, F (not shown), L, or a termination codon. The EcoRI
insert of each mutant plasmid DNA was purified from low melting agarose
gel, and cloned into EcoRI-digested, phosphatase-treated
pKK223-3 DNA by transformation into E. coli DH5. The
correct orientation was determined by restriction endonuclease
digestion, and the mutations were confirmed by complete DNA sequence
analysis on double-stranded DNA templates. These plasmids containing
mutant GST genes in the right orientation for expression are designated
pGTH121-E96L, pGTH121-E96F, pGTH121-E96Y, pGTH121-G97P, pGTH121-G97T,
pGTH121-G97S, pGTH121-G97L, respectively. The mutant GSTs are
designated GST121-E96L, GST121-E96F, GST121-E96Y, GST121-G97P,
GST121-G97T, GST121-G97S, GST121-G97L, respectively.
The mutant GSTs, GST121-E96F, GST121-E96Y, GST121-G97T, GST121-G97S, GST121-G97P, were purified by similar procedures from 2 liters of culture. The cultures for the E96F/E96Y mutants were grown at 30 °C.
Figure 1: Sequencing strategy and DNA sequence patterns of GST 121 mutations. Panel A, Sequencing strategy. Six oligonucleotides were used as primers in the dideoxy chain termination sequencing protocol with direction and extent of DNA sequence determination shown by the arrows. Oligonucleotide 1 (20-mer, 5`-GGATAACAATTTCA-CACAGG-3`) is located just upstream of the EcoRI site near the tac promoter. Oligonucleotide 2 (17-mer, 5`-GGGATGAAGCTGGTGCA) is at position 250 reading in the sense direction. Oligonucleotide 3 (17- mer, 5`-GGACAAGACTACCTTGT-3`) is at position 500 reading in the sense direction. Oligonucleotide 4 (17-mer, 5`-GTTGCAAAACTTTAGAA-3`) is at position 780 reading in the antisense direction (-780). Oligonucleotide 5 (17-mer, 5`-TGTCAGCCCGGCTCAGC-3`) is located at position 530 reading in the antisense direction (-530). Oligonucleotide 6 (17-mer, 5`-AGGTTGTATTTGCTGGC-3`) is located at position 300 reading in the antisense direction (-300). The nucleotide position 1 is the beginning of the cDNA sequence in pGTH1 as described previously by Tu and Qian(20) . Amino acid residues are assigned from the N-terminal Ser in the GST121 as isolated from E. coli. The Met does not appear in the GST121 and is not counted. Panel B, identification of the Y8F mutation demonstrates the change of TAC codon in the wild type GST121 to TT*C in the mutant. Panel C, identification of the E31K mutation demonstrating the change of the GAG codon in the wild type to A*AG in the mutant. Panel D, identification of the E96K and G97D mutations demonstrating the changes of GAA to A*AA (E96K) and of GGT to GA*T (G97D).
Figure 2:
Western blot of E. coli crude
extracts from GST121 mutants derived from hydroxylamine mutagenesis.
The crude extracts were prepared from 5-ml overnight cultures by
sonication in 1-ml suspension in 25 mM Tris-HCl, pH 8.0.
One-twentieth of each one of the crude extracts (50 µl each) was
used for SDS-PAGE and transferred to nitrocellulose membranes according
to Towbin et al.(65) . The membrane was reacted with
antiserum against recombinant human GST 1-1 followed by alkaline
phosphatase-conjugated goat anti-rabbit IgG. The bands were developed
with the chromophore BCIP as described
previously(36, 65) . The lanes are: 1, S-hexyl GSH affinity purified GST121 (10 µg); 2-10, crude extracts from E. coli DH5
containing pGTH121 (2), pGTH121-Y8F (3), mutant E96K (4), mutant Gln
* (5), mutant M93I/E96K (6), mutant G97D (7), mutant G26E/V27I/M93I/G97D (8), pGTH121 (9) and mutant E31K (10). The arrow points at the position of the GST121 polypeptide. In lane 5, the truncated peptide is revealed in a faster
migrating species.
To determine the
nature of these mutations the 12 mutant genes were sequenced in their
entirety by a set of six oligonucleotide primers according to the
sequencing strategy in Fig. 1. Sequences surrounding each
mutation and the corresponding wild type sequence are shown in Fig. 1, Panels B, C, and D. Two of
the 12 mutants have multiple mutations (M93I/E96K and
G26E/V27I/M93I/G97D) (not shown), three of them are single mutations
(E31K, E96K, or G97D), one has a premature termination
(Gln*) (Fig. 2, lane 5), and the rest are
identical to the ones just described. Unlike the Y8F mutation, none of
these mutant GSTs binds to the S-hexyl GSH-agarose affinity
matrix. To confirm the sequencing results and to validate the selection
procedure, the mutations E96K and G97D were reproduced by site-directed
mutagenesis.
Figure 4:
Western blot analysis of E96L and G97P
grown at different temperature. DH5 carrying pGTH121 (lanes 1 and
2), pGTH121-E96L (lanes 3 and 4) and pGTH121-G97P (lanes 5 and 6) were grown overnight in 5 ml of LB
media containing 100 µg/ml ampicillin at either 30 °C (lanes 1, 3, 5) or 42 °C (lanes
2, 4, 6). Cells were harvested and resuspended
in 0.5 ml of 25 mM Tris-HCl, pH 8.0. Crude extracts of enzymes
were obtained by sonicating cell suspensions with a microprobe of a
Branson Sonifier (model 450; duty cycle at 50%; output control at 2.5)
for 20 s. Twenty-five µg of each crude extract were loaded into
each lane of a 12% gel for SDS-PAGE. Electrophoresis was carried out at
constant current of 30 mA for 4 h, and proteins were then
electro-transferred onto an (PVDF) Immobilon-P membrane (Millipore) by
a Bio-Rad Trans-blot cell at a constant current of 90 mA for 2 h. The
blot was then probed with anti-serum (1:1000) against GST 1-1, and
protein bands were detected by the alkaline phosphatase method using
BCIP/NBT as substrates. Both E96L and G97P showed the
temperature-sensitive phenomenon.
The first in vivo test is a
simple streaking experiment where a loopful of bacterial colony was
evenly streaked across a fixed area on the agar plates. Three identical
plates containing 20 µg/ml CDNB were streaked for growth at 30, 37,
and 42 °C. There was a control without CDNB for each temperature.
The results shown in Fig. 3indicated that the mutants E96L and
G97P may be temperature sensitive because they showed more growth at 42
°C than at 30 °C or 37 °C. On the other hand, G97S behaved
like the wild type GST121 and was not temperature-sensitive. The
temperature sensitive nature of the E96L and G97P mutants were
investigated further by Western blot analyses of crude extracts.
Results shown in Fig. 4revealed that E96L protein levels were
much lower than those of the wild type GST121, and that the amount of
mutant GST protein was even lower when cultures were grown at 42
°C. This was reproducible whether the crude extracts were obtained
by sonication or by lysis with lysozyme. For the G97P protein mutant,
the level at 30 °C was similar to that of the wild type, but was
reduced relative to GST121 for cultures grown at 42 °C. The
specific activities in CDNB conjugation for the G97P crude extracts
were the same for cells grown at 30 °C and at 37 °C. However,
the purified G97P protein lost 14% CDNB conjugation activity after
5 min at 50 °C or 50% of activity after 3 h. Under the same
condition, GST121's activity did not change over the course of
the incubation. These results indicated that GST121-E96L was probably
much less stable than GST121-G97P and that such instability was
enhanced when cultures were grown at elevated temperature. Since the
promoter and ribosome binding site are the same for expressing GST121
and these mutant GSTs, the major difference in the amount of proteins
detected by antibody was most likely due to proteolytic degradation of
unstable mutant proteins, relative instability of mutant mRNAs or both.
Figure 3:
Temperature effect on the growth of
mutants E96L and G97P on agar plate containing CDNB. One loopful of
overnight culture (37 °C) was evenly streaked onto LB agar plate
containing 100 µg/ml ampicillin and 20 µg/ml CDNB. Another
loopful of the same culture was streaked onto LB agar plate containing
100 µg/ml ampicillin to serve as control for normal growth. Plates
were then incubated at 30 °C, 37 °C or 42 °C for 24 h.
Plates on the left were LB agar plate containing 100 µg/ml
ampicillin, and the corresponding LB agar plates containing 100
µg/ml ampicillin and 20 µg/ml CDNB were on the right. Bacteria (E. coli DH5) expressing wild type or mutant enzymes were
indicated by different numbers (1, pGTH121; 2,
pGTH121-Y8F; 3, pKK223-3; 4, pGTH121-E96L; 5, pGTH121-G97P; 6, pGTH121-G97S). Growth of E96L and
G97P on plates containing CDNB at 42 °C increased significantly,
suggesting that both mutants are temperature-sensitive. Growth of
non-temperature-sensitive mutant G97S was similar to that of the
chimeric GST121.
The mutant proteins E96F and E96Y also appear to be temperature sensitive by these simple streaking tests (data not shown). Subsequently plating efficiency, which is a more quantitative measure of in vivo activity, was determined in the presence of CDNB for each of the strain expressing mutant GSTs. The results in Table 3confirmed the negative correlation between mutant GST activity in vitro and plating efficiency in vivo at 30 °C. Those mutants with very low (e.g. Y8F) or no CDNB (e.g. E96K, G97D) activities showed a 3 to 4 orders of magnitude growth advantage over the wild type in the presence of CDNB. The temperature sensitivity of the mutations E96F and E96Y was confirmed with the purified mutant GST proteins. The thermal inactivation time courses of GST121-E96F and GST121-E96Y were compared with that of GST121 at 50 °C. Results in Fig. 5clearly indicated that both of them were thermolabile at 50 °C, losing 98% (E96F) and 62% (E96Y), respectively, of the CDNB conjugation activities in 5 min. For 30 min of incubation at various temperatures (4-70 °C), GST121-E96F lost 50% of the CDNB conjugation activity at 43.6 °C, whereas GST121-E96Y lost 50% of the conjugation activity at 44.7 °C by interpolation of thermal inactivation curves (data not shown). The wild type GST121 lost 50% of the activity at 60 °C under the same experimental condition.
Figure 5: Thermostability of GST121-E96F and GST121-E96Y at 50 °C. The GST 121 and mutant proteins (200 µl each) at 50 µg/ml were incubated in a 50 °C water bath. Aliquots of ten µl were taken every 5 min and assayed for conjugation activity against CDNB. The thermo-inactivation curves shown above were the average of two measurements. Deviations ranged from 0.5 to 7%.
Figure 6:
Effects of CDNB and DNPG on bacterial
growth. Effects of CDNB (Panel A) and DNPG (Panel B)
on growth were tested on E. coli strains DH5
(GSH-sufficient), 821 (GSH-deficient) and their derivatives expressing
wild type and mutant GST121 proteins. Forty µl of overnight
cultures of DH5
(1 in Panels A and B)
or 821 (6 in Panel A and 5 in Panel
B) were used to inoculate 4 ml of LB medium (control), 4 ml of LB
containing 0.4 mM CDNB (Panel A) and 4 ml of LB
containing 0.4 mM DNPG (Panel B). Forty µl of
overnight cultures of derivative strains of DH5
or 821 (DH5
carrying pKK223-3 (2 in Panels A and B),
DH5
carrying pGTH121 (3 in Panels A and B), DH5
carrying pGTH121-Y8F (4 in Panels A and B), DH5
carrying pGTH121-E96K (5 in Panel
A), 821 carrying pKK223-3 (7 in Panel A),
821 carrying pGTH121 (8 in Panel A and 6 in Panel B), 821 carrying pGTH121-Y8F (9 in Panel A and 7 in Panel B), 821 carrying pGTH121-E96K (10
in Panel A)) were used to inoculate 4 ml of LB containing 100
µg/ml ampicillin (control), 4 ml of LB containing 100 µg/ml
ampicillin and 0.4 mM CDNB (Panel A) or 4 ml of LB
containing 100 µg/ml ampicillin and 0.4 mM DNPG (Panel
B). All cultures were allowed to grow in a shaking water bath at
37 °C (200 rpm). Growth was monitored by absorbance measurements at
600 nm (A
). Ratio of A
of
CDNB or DNPG-treated cells/control cells were plotted against
time.
Figure 7:
Effect
of CDNB on intracellular GSH level. E. coli DH5 (pGTH121)
and DH5
(pGTH121-G97D) were grown at 37 °C in a waterbath
shaker (200 rpm) until A
= 0.3. An
aliquot of 50 ml of culture was collected as the 0-h sample. CDNB stock
solution in ethanol was then added to each culture to a final
concentration of 1 mM. Cultures continued to grow at 37 °C
in a waterbath shaker at 200 rpm. Bacterial growth was monitored by A
and colony plate counts. At various time
points, 50 ml from each culture were collected for assays of
intracellular GSH and CDNB conjugation activity of GST (47) after sonication. The levels of GSH were determined by a
GSH-400 Colorimetric Assay Kit (BIOXYTECH® S.A., Paris, France)
using a GSH standard curve of 10-100 µM GSH. Circles represented readings (A
, GSH
level, colony plate count, or CDNB conjugation activity) for DH5
(pGTH121), whereas triangles represented readings (A
, GSH level, colony plate count, or CDNB
conjugation activity) for DH5
(pGTH121-G97D). Broken lines represented A
measurements in all three
panels, whereas solid lines represented other variables. Panel A shows the effect of CDNB on intracellular GSH level in
nanomoles/mg of protein in the sonicated crude extracts. Panel B shows the effect of CDNB on colony plate counts in the presence of
100 µg/ml ampicillin, and Panel C shows a decrease of CDNB
conjugation activity in the crude extract of DH5
(pGTH121). The
crude extract of DH5
(pGTH121-G97D) did not have detectable CDNB
conjugation activity because of the mutation G97D in GST121. The
GST121-G97D was made at the same level as GST121 in the respective E. coli cultures (data not shown).
The reason that such selection scheme has been effective in E. coli is the absence of any GST(s) as effective toward CDNB as the mammalian GSTs. One of the candidates for E. coli GST is the stringent starvation protein (SSP), which has sequence homology to the Drosophila GST D27(49) . The SSP is a dimeric protein inducible in E. coli under nutrient starvation. CDNB conjugation activity for purified SSP has not been tested.
The
antibiotic activity of CDNB can be used for selection of mutants
produced by generalized or regional mutagenesis, and for providing a
measurement of mutant GST activities in vivo. Many chemical
mutagens (e.g. hydroxylamine, bisulfite) can be used for
random or regional mutagenesis in mutant isolation. Mutations can also
be introduced by oligonucleotide-directed mutagenesis. Other regional
or random mutagenesis schemes based on PCR should be satisfactory as
well(51) . The organization of the GST expression plasmid(s)
has three major components: plasmid replication and segregation, the
-lactamase gene and the GST gene. In principle, mutations could
occur anywhere on the plasmid DNA in hydroxylamine mutagenesis.
Mutations in the replication regions and in the
-lactamase gene
will not emerge under CDNB selection because of their failure to
replicate or confer ampicillin resistance. Only mutations negatively
affecting the GST structural gene and the regulatory sequence for its
expression will appear as colonies in the presence of CDNB. As far as
mutations affecting levels of GST expression (e.g. promoter
mutations and mutations affecting translation initiation) are
concerned, the GST band intensity on Western blots from crude extracts
should provide an effective screening.
This CDNB selection procedure allows identification of amino acid residues essential in the structure/function of GST based on its in vivo conjugation activity. In principle, such a selection should be applicable to any GSTs with activities toward CDNB. Results in Table 2suggested that such in vivo activity based selection can be expanded to include other substrates for GSTs such as DCNB. This selection procedure based on function of GST in vivo (or a lack of it) for identification of essential residues in GST function should complement x-ray crystallography in the analysis of GST structure and function relationship. In some instances this molecular genetic approach should identify essential amino acid(s) not immediately obvious from the three-dimensional structure (e.g. G97 in GST121). Another important application of this CDNB selection procedure is in the isolation of temperature sensitive GST mutants. Those E. coli defective in GST activities at elevated temperatures should form colonies in the presence of CDNB at 42 °C. These colonies can be tested for temperature sensitivity of GST activity in the presence of CDNB at 30 °C. For example, E. coli expressing a temperature-sensitive GST should grow well at 42 °C but not at 30 °C in the presence of CDNB because of the expression of conjugation activity. The temperature sensitive plasmid replication mutants can be eliminated by the selection of ampicillin resistance during growth at 42 °C. In the absence of such a function-based procedure a systematic selection for conditional defective mutant in mammalian GSTs would have been nearly impossible. Such mutants should be very valuable in elucidating the biological functions of GSTs and in the study of protein structures and functions in general. Thus, two general strategies have be established in the structure/function analysis of GSTs: 1) identification of null mutations and replacement by ``weaker alleles'' and 2) isolation of temperature sensitive mutants.
The CDNB selection procedure could have other applications. It could be used to search among xenobiotic compounds and natural products for antibiotics acting though GSH conjugation; these compounds should also be good substrates for GSTs. The action of these compounds should be selective according to the substrate specificity of the target (bacterial or mammalian) GSTs. The fact that bacterial GSTs are much less active toward CDNB than mammalian and other eukaryotic GSTs makes it possible to search for new antibiotics targeted specifically against bacterial GSTs. Results in Table 2indicated that this approach may be feasible because other chloronitrobenzenes showed growth inhibition in E. coli expressing a functional GST.
The principle of this CDNB selection procedure could be expanded
into screening for chemicals (drugs) capable of inhibiting the activity
of a particular GST from mammalian, plant or insect species. The action
of such a compound could alleviate E. coli DH5 with a
functional GST from growth inhibition by CDNB, for example. Since alpha
class GSTs have been proposed to play a clinically significant role in
the acquired resistance to cancer chemotherapy involving alkylating
agents, the successful selection of antagonistic compounds to alpha
class GST should be valuable in efforts to enhance the efficacy of
alkylating agents in cancer
chemotherapy(1, 4, 52, 53) .
The
biochemical bases of mutations at Glu, Glu
,
and Gly
can be rationalized in light of the recently
published x-ray structure of the native human GST 1-1 (26) ,
which differs from GST121 by only four amino acids at R88K, V110F,
C111T, P112Q. The first two residues are involved in salt bridges,
Glu
with Arg
and Glu
with
Arg
. The substitutions by lysine at both positions
obviously disrupted the important electrostatic interactions, which in
turn perturbed the structural stability and/or active conformations for
catalysis. The in vivo activity based plating efficiency in
the presence of CDNB provided an independent confirmation on the
structural and/or functional importance of these salt bridges. The
importance of Gly
in GST 1-1 structure/function was not
highlighted from the structural discussions(26) . However,
residues 85-110 form a long helix (
4) which is part of the
dimer interface. Furthermore, these two amino acids (Glu
,
Gly
) are conserved in all of the human and rat alpha class
GSTs(21) . It is possible that E96K and G97D mutations have
each interfered with dimer formation. The substitutions of L, F, or Y
at 96 and of D, L, T, S at 97 should not affect the helix stability per se because all of these amino acids have a higher
propensity in helix formation than the wild type residues(54) .
Only the proline substitution at 97 may have an unfavorable structural
effect according to this criterion. Therefore, additional interactions
may be affected because of consequences of these other substitutions at
positions 96 and 97. For example, all three substitutions in the 96
position resulted in temperature-sensitive phenotypes with different
severity in enzyme activities. The leucine substitution was somehow
much more severe in mutant GST stability than the Phe and Tyr
substitutions. The latter two mutants retain considerable enzyme
activities and can be purified by the S-hexyl GSH affinity
chromatography. Although Phe
and Tyr
are as
hydrophobic as leucine, they are different in that the aromatic side
chains can pack against the backbone of a neighboring helix at medium
separation. Also the size and the orientation of the side chains of
Phe
and Tyr
are different from that of
leucine. It is possible that the aromatic rings of Phe
or
Tyr
might interact with helix
3 (residues
67-78) of the neighboring monomer. These interactions are likely
to stabilize the dimer as well as the overall structure for a
functional enzyme to a certain extent. Although R68 are still unable to
form a salt-bridge with either Phe
or Tyr
,
there might be some interactions between the side chain of R68 and the
electron-rich aromatic side chain. On the other hand, the aromatic side
chains might have introduced some steric effects on the surrounding
structure, which might have caused their thermal instability. E96F is
less thermostable than E96Y. E96F retains only 2.4% of the activity
after incubation at 50 °C for 5 min whereas E96Y retains 37.6% of
the activity after the same treatment (Fig. 5). It will be
interesting to know how the extra OH group on Tyr
contribute to a better thermostability than Phe
.
According to the structure of GST 1-1(26) , some of the
important residues responsible for GSH binding are located in this area (e.g. Gln
, Thr
, Asp
,
Glu
, and Arg
). Therefore, mutations of E96F
and E96Y might affect binding of GSH indirectly by alterations in the
local structure. The overall active site conformation for catalysis
might also be slightly affected resulting in minor decreases in the k
values.
The important role of glycine at
position 97 is somewhat more difficult to understand. Both a negatively
charged residue (D) and a hydrophobic residue (L) resulted in loss of
enzyme activities. However, the substitutions of polar amino acids with
hydroxyl groups (T, S) retained considerable activity. It is possible
that a precise configuration at Gly may be essential for
optimal conformation and activity. It is fortunate that most of these
mutant GSTs have been purified to electrophoretic homogeneity. The
x-ray structural analyses should aid in a better understanding of their
contributions to structure and function of alpha class GSTs.
The
mechanism by which CDNB causes growth inhibition is intriguing. We have
shown that the GSH conjugate of CDNB, S-(2,4-dinitrophenyl)glutathione, when it is added
extracellularly, is not an inhibitor for bacterial growth (Fig. 6). We have also shown that the GSH deficient strain E. coli 821 (60) is more sensitive to CDNB than E. coli DH5, which is normal in GSH synthesis. In the
absence of any heterologous GSTs, the sensitivity to CDNB growth
inhibition in E. coli 821 became more pronounced as the CDNB
concentration in the media increased from 0.4 mM up to 1.5
mM (data not shown). This is consistent with the notion that
GSH is depleted faster by CDNB in E. coli 821 where the GSH
level was much below the normal level(37) . In the E. coli DH5
, the growth inhibition by CDNB is enhanced in the
presence of an active GST (e.g. GST121 from pGTH121) over that
seen in the presence of a mutant GST (e.g. GST121-G97D). As
shown in Fig. 7, the reduction or depeletion of intracellular
GSH persisted throughout most of the
20-h period of growth
inhibition. The GSH level in DH5
(pGTH121) in the presence of 1
mM CDNB began to recover just before the turbidity of the
culture began to increase (Fig. 7A). It subsequently
continued its increase in parallel to the increase in culture turbidity (A
). On the other hand, the intracellular GSH
level in DH5
expressing the mutant GST121-G97D essentially
paralleled its relatively normal growth curve except for an early drop
soon after the addition of 1 mM CDNB (Fig. 7A). During the
20-h period of growth
inhibition in the DH5
(pGTH121) culture, the GSH level remained
low and GST121's CDNB conjugation activity was relatively high.
This is consistent with the notion that GST121 in the cells continued
to consume GSH in the conjugation with CDNB. It is interesting to note
that the GST121 specific activity continued to decrease upon addition
of 1 mM CDNB to the culture and eventually to a level of
5% of cultures without CDNB. During the growth phase (i.e. increase in A
) after the period of growth
inhibition, the increase of intracellular GSH level correlated with the
continuous decrease in GST121's specific activity (Fig. 7C). The decrease in GST121's specific
activity in the crude extracts was due to a decrease in the GST121
protein level as analyzed by SDS-PAGE (data not shown). When E.
coli DH5
(pGTH121) resumed growth after the period of growth
inhibition by CDNB, the growth corresponded to an increase in cell
volume (i.e. increase A
) but not in
viable cell counts. The viable cell counts continued to decrease over
the period of growth inhibition and leveled off during the 35-h
recovery period (Fig. 7B). When cells during the growth
period were examined under a microscope, they were found to be in the
form of elongated filaments (data not shown), suggesting that cell
division was still impaired during the recovery phase (30-65 h).
CDNB has been used to deplete GSH in
eucaryotes(55, 56) . In erythocytes, depletion of GSH
by CDNB caused rapid oxidative damage of hemoglobin even though GSH
peroxidase and GSH reductase were not affected by CDNB(57) . In
bacteria, depletion of GSH by diamide caused growth
inhibition(58) , and oxidative stress response was manifested
in alarmone synthesis(35, 59) . It was also reported
that addition of CDNB (0.6 mM) or N-ethylmaleimide
(0.5 mM) to E. coli cells elicited rapid K
efflux probably due to formations of S-(2,4-dinitrophenyl)glutathione or Nethyl-succinimido-S-glutathione. Neither chemical
reaction of GSH with iodoacetate nor depletion of GSH in biosynthetic
mutants resulted in major K
loss from cells (60) . The K
and glutamate levels are known to
play a role during osmotic adaptation of E. coli(61) .
However, it is not clear how CDNB-mediated K
efflux
leads to growth inhibition. The molecular mechanism of the signal
transduction from GSH depletion to inhibition of bacterial growth is
intriguing and worthy of further investigation. The enhancement of this
growth inhibition by the presence of an active heterologous human GST
suggests that GSH metabolism is clearly involved.
Since the original submission of this manuscript, several reports on expressing mammalian GSTs in Salmonella typhimurium tester strains to study the activation/detoxification of several mutagenic compounds have appeared(62, 63, 64) . Their emphasis was on the xenobiotic compounds or their conjugation products with GSH. None of the compounds tested has turnover numbers nearly as high as those for CDNB. It was less likely that the increased reversion frequency was due to depletion of intracellular GSH. Nevertheless, it should be interesting to monitor the GSH levels of those engineered tester strains in the presence of various mutagenic xenobiotics.