©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Molecular Genetic Approach for the Identification of Essential Residues in Human Glutathione S-Transferase Function in Escherichia coli(*)

(Received for publication, April 6, 1994; and in revised form, October 31, 1994)

Hin-Cheung Lee Yann-Pyng S. Toung (§) Yen-Sheng L. Tu Chen-Pei D. Tu (¶)

From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 Kand 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 alpha4. Our results provide evidence in vivo that Tyr^8, 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.


INTRODUCTION

The glutathione S-transferases (GSTs, EC 2.5.1.18) (^1)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 (YY)(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.


EXPERIMENTAL PROCEDURES

Materials

Chemicals (e.g. 1-chloro-2,4-dinitrobenzene and other chloronitrobenzenes), S-hexyl GSH-agarose, S-hexyl GSH, and antibiotics (e.g. ampicillin) were purchased from Sigma-Aldrich Chemical Co. Restriction endonucleases and T4 DNA ligase were products of New England Biolabs or Boehringer Mannheim. The altered sites system for site-directed in vitro mutagenesis was purchased from Promega (Madison, WI). Exonuclease-deficient T7 DNA polymerase was kindly provided by Dr. Kenneth A. Johnson of The Pennsylvania State University. Oligonucleotides for mutagenesis (Y8F, 5`-GCTCCACTTCTTCAATG 3`; E96K, 5`-ATGTATATAAAAGGTATAGC-3`; G97D, 5`-GTATATAGAAGATATAGCAG-3`; E96F/E96Y/E96L, 5`-CCCTGATTGATATGTATATAHBBGGTATAGCAGATTTGGG-3` (oligo-96); G97P/G97T/G97S/G97L, 5`-CCCTGATTGATATGTATATAGAAHHVATAGCAGATTTGGG-3` (oligo-97)) and DNA sequencing (17 mers, 250 primer: 5`-GGGATGAAGCTGGTGCA-3`; 500 primer: 5`-GGACAAGACTACCTTGT-3`; -300 primer: 5`-AGGTTGTATTTGCTGGC-3`; -530 primer: 5`-TGTCAGCCCGGCTCAGC-3`; -780 primer: 5`-GTTGCAAAACTTTAGAA-3`) were synthesized by the Biotechnology Institute Core Facility at The Pennsylvania State University. The sequencing primer (20 mer, 5`-GGATAACAATTTCACACAGG-3`) just upstream of the EcoRI site on pKK223-3 was synthesized at Operon Technologies, Inc. (Alameda, CA). The human GST cDNA construct pGTH121 expressing GST121, a chimeric GST between human GST 1-1 and GST 2-2, was constructed by replacing the PvuII-SfaNI fragment of pGTH1-KK with that from pGTH2-KK(20, 21, 36) . The detailed construction of the chimeric GST gene and the characterization of the chimeric GST121 will be reported elsewhere. (^2)The chimeric GST121 has substitutions of R88K, V110F, C111T, and P112Q, relative to the naturally occurring GST 1-1. It has a lower Kfor GSH and a better k than its two parental GSTs, GST 1-1, and GST 2-2. This chimeric GST121 is used as the prototype or ``wild type'' GST in this study because it is a more efficient enzyme and provides a broader range of CDNB conjugation activity for manipulation than either GST 1-1 or GST 2-2. Mutagenesis by NH(2)OH was carried out with the pGTH121 DNA containing the chimeric gene construct. The E. coli hosts for mutagenesis and expression are J-M109 (endA1,recA1,gyrA96,thi,hdR17(r(K)-,m(K)+), relA1, supE44, Delta(lac-proAB), (F`,traD36,proAB,lacI^qZDeltaM15)) and BMH71-18mutS (thi, supE, Delta(lac-proAB), (mutS::Tn10), (F`, proAB, lacI^qZDeltaM15)) and DH5alpha (F, 80dlacZDeltaM15, endA1, recA1, gyrA96, thi-1, hdR17(r(K)-,m(K)+), relA1, supE44, deoR, Delta(lacZYA-argF)U169). E. coli strain 821 (thr-1, ara14, leuB6, Delta(gpt-proA)62, lacY1, tsx-33, supE44, galK2, , rac, ^s, hisG4 (Oc), rfbD1, mgl-51, gshA2, rpsL31, kdgK51, xyl-5, mtl1, argE3 (Oc), thi-1) was obtained from Dr. Barbara Bachman of the E. coli Stock Center(37) . Rabbit antisera against recombinant human GST 1-1 were prepared as described previously(36) . The GSH-400 Colorimetric Assay Kit of BIOXYTECH® S. A. (Paris, France) was purchased from Cayman Chemical Co. (Ann Arbor, MI). S-(2,4-Dinitrophenyl)-glutathione was prepared by reacting equal molar concentrations of GSH and 1-chloro-2,4-dinitrobenzene in 0.1 M potassium-P(i) buffer (pH 7.5) at 37 °C overnight. The conjugate was purified by ethyl ether extractions (38) and high performance liquid chromatography (Scientific Systems Inc., State College, PA) with a C18 reverse phase column (VYDAC, Hesperia, CA). Its concentration was determined by A with an extinction coefficient of 9600 mM.

Methods

Nucleic acid manipulations were carried out according to Chow et al.(36) . DNA sequence analysis was performed on double-stranded DNAs using the dideoxynucleotide-chain termination method with alpha-S-dATP(39, 40, 41) . Bacterial growth was measured from turbidity of cultures at 600 nm in a Spectronic 21 spectrophotometer.

Site-directed Mutagenesis

Construction of the Y8F mutant was carried out with the Altered Sitesin vitro mutagenesis system (Promega). The chimeric human GST gene on pGTH121 was excised by EcoRI digestion and cloned into the EcoRI-digested pALTER-1 DNA. The resulting plasmid is designated pALTER-GTH121. Single-stranded DNA templates with the chimeric gene in the sense strand was isolated after infection with helper phage R408. The beta-lactamase gene repair oligonucleotide 5`-pGTTGCCATTGCTGCAGGCATCGTGGTG- 3` (27 mer) and the phosphorylated 17-mer oligonucleotide for the Y8F mutation were annealed to the single-stranded DNA templates and processed according to manufacturer's procedure before transformation into E. coli strain BMH 71-18 mutS. The mixture of transformants were grown in the presence of 125 µg/ml ampicillin at 37 °C for 12-14 h to enrich for mutants. Plasmid DNAs from 4 ml of overnight cultures were purified from the mixture of transformants at room temperature by the rapid-alkaline lysis procedure(42) . A fraction of the isolated DNA was then transformed into E. coli strain JM109 and selected for ampicillin-resistant colonies. Twelve colonies out of several hundred transformants were selected for DNA sequence analysis on double-stranded DNA templates; five of them contained the desired Y8F mutation. The EcoRI insert from one of the mutants was purified from low melting agarose gel and cloned into EcoRI-digested, phosphatase-treated pKK223-3 DNA by transformation into E. coli DH5alpha. The correct orientation was confirmed by PstI digestion, which produced a fragment of 721 base pairs in length. The mutant plasmid was designated pGTH121-Y8F.

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 DH5alpha. 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.

General Mutagenesis of Plasmid DNA with Hydroxylamine

The plasmid DNA pGTH121 (3 µg) was incubated in a final volume of 30 µl with 0.4 M hydroxylamine-HCl, in the presence of 50 mM sodium-P(i) buffer (pH 6.7) and 0.9 mM EDTA at 4 °C for 45 min and then at 65 °C for 30 min(43, 44) . After ethanol precipitation and washing to remove hydroxylamine-HCl and sodium-P(i), the DNA was resuspended in 30 µl of TE buffer and used to transform E. coli DH5alpha. The putative GST mutants were selected for in the presence of 100 µg/ml ampicillin and 20 µg/ml CDNB as described under ``Results.''

Oligonucleotide-directed Mutagenesis on Double-stranded DNA Template

Approximately 2 µg of CsCl gradient-purified pGTH121 DNA was denatured in 0.2 N NaOH or heated in boiling water for 3 min to generate single-stranded DNA templates. A fraction of the denatured template DNA (0.18 µg or 0.05 pmol) was annealed with 5 pmol of the appropriate mutant oligonucleotide in 1 times annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl(2), 50 mM NaCl). The mixture was incubated at 65 °C for 10 min and slowly cooled to 10 °C over a period of 6 h. The 10 times synthesis buffer (100 mM Tris-HCl, pH 7.5, 5 mM dNTPs, 10 mM ATP, 20 mM dithiothreitol) was added to the mixture, together with 10 units of T4 DNA polymerase and 2 units of T4 DNA ligase to a final volume of 30 µl and 1 times synthesis buffer. After incubation at 0 °C for 5 min, followed by room temperature for 5 min, and 37 °C for 2 h, the reactions were stopped by the addition of 3 µl of stop solution (0.25% SDS, 5 mM EDTA, pH 8.0) and incubated at 65 °C for 5 min. The mixture was used to transform E. coli BMH71-18 mutS. Plasmid DNAs were isolated from the transformation mixture after overnight growth at 37 °C. An aliquot of the DNA was used to transform E. coli DH5alpha. The mutants were selected for by plating the transformation mixture on LB agar plates containing 100 µg/ml ampicillin and 20 µg/ml CDNB. Only mutants with a loss of GST function appeared as colonies on the agar plates.

Purification of GST121 and Mutant GSTs

A single colony of E. coli DH5alpha containing various GST gene constructs was inoculated into 3 ml of LB medium in a 16 times 100-mm test tube containing 100 µg/ml ampicillin at 37 °C overnight. The culture was diluted 100-fold into 50 ml of LB in 100 µg/ml ampicillin in a 37 °C waterbath shaker until late log phase. This culture was then diluted 100-fold into 8 liters of prewarmed LB containing 100 µg/ml ampicillin in 1- and 2-liter flasks and grown overnight at 37 °C or 30 °C in a New Brunswick G26 shaker (250 rpm). GST121 and mutant GSTs (e.g. GST121-Y8F) were expressed in E. coli DH5alpha at high levels without induction by isopropyl-1-thio-beta-galactopyraonside. Consequently, no isopropyl-1-thio-beta-galactopyraonside was used in any of the GST preparations. The cells were harvested by centrifugation (6000 rpm for 5 min in a GS3 rotor, DuPont-Sorvall RC5B centrifuge). The cell pellets were resuspended by a vortex mixer in 15 ml of 25 mM Tris-HCl, pH 8.0, per liter of original culture. All of the following steps were carried out at 4 °C. The cell suspension was immersed in ice and sonicated with a regular probe of a Branson Sonifier (model 450) at 75% of maximum power. Twelve of 15-s sonications were applied with 2 min cooling in between for a total of 3 min sonication. The sonicated cells were centrifuged in an HB-4 rotor at 11,000 rpm for 20 min. The supernatant fraction (140 ml from 8 liters of culture) was dialyzed overnight against 2 liters of 25 mM Tris-HCl, pH 8.0, and followed by centrifugation to remove traces of denatured proteins. The supernatant fraction (143 ml from 8 liter of original culture) was loaded on an S-hexyl GSH column (1-ml bed volume/liter of original culture) previously equilibrated with 25 mM Tris-HCl, pH 8.0, at 60 ml/h flow rate. The column (8-ml bed volume) was washed with 300-400 ml of 25 mM Tris-HCl, pH 8, and 0.2 M KCl overnight. The washings were reduced for some of the mutant GSTs. The GST protein was eluted with 25 mM Tris-HCl, pH 8, containing 0.2 M KCl, 2.5 mM GSH, and 5 mMS-hexyl GSH. A total of 12 fractions (40 drop or 1.5 ml) were collected by a Gilson microfractionator. Main fractions determined by the CDNB conjugation assay and detected by SDS-PAGE were combined and dialyzed overnight with two changes against 1 liter of 25 mM Tris-HCl, pH 8. Protein concentration was determined by the Bradford method (45) and the BCA protein assay kit (46) from Pierce Chemical Co. in duplicate sets. CDNB conjugation and GSH peroxidase assays for GSTs were carried out as described previously (36, 47, 48) using a Gilford Response Spectrophotometer. The yield for GST121 and GST121-Y8F was 11 mg of pure protein/liter of culture.

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.

Determination of Intracellular GSH Levels during Bacterial Growth

A single colony of E. coli DH5alpha (pGTH121) or E. coli DH5alpha (pGTH121-G97D) was inoculated into 5 ml of LB medium containing 100 µg/ml ampicillin and grown overnight at 37 °C in a waterbath shaker. Each overnight culture was transferred into a separate 1-liter flask containing 500 ml of LB (ampicillin) and cultured in a 37 °C waterbath shaker at 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 diluted 100-fold to each culture to a final concentration of 1 mM. Cultures continued to grow at 37 °C in the waterbath at 200 rpm. Bacterial growth was monitored by A measurement and by colony plate counts on LB plates containing 100 µg/ml ampicillin. At various time points, 50 ml from each culture were collected for assays of intracellular GSH and CDNB conjugation activity of GST(47) . Cells were collected by centrifugation at 5000 rpm in a Dupont-Sorvall GSA rotor at 4 °C. The cell pellet was washed by resuspension in 25 ml of ice-cold 50 mM potassium-P(i) (pH 7.0), concentrated in 0.5 ml of 50 mM potassium-P(i) (pH 7.0), and sonicated with the microprobe of a Branson Sonifier (model 450) at 50% duty cycle and output control setting 3 for 45 s. The sonicated cells were centrifuged in a microcentrifuge at 4 °C for 20 min. The supernatant fluid was collected as the ``crude extract'' fraction. 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. An aliquot (300 µl) of each crude extract was mixed with an equal volume of 10% (w/v) metaphosphoric acid on a vortex mixer. Precipitated proteins were removed by centrifugation in a microcentrifuge at 4 °C for 20 min. The supernatant fluid was collected for GSH assay according to the manufacturer's procedure. Briefly, a fraction of the supernatant fluid was diluted with buffer to 900 µl and thoroughly mixed with 50 µl of solution R1 first, and then with 50 µl of solution R2. The mixtures were incubated for 10 min at 25 °C in the dark followed by measuring absorbance at 400 nm within the hour. The GSH concentration in each original crude extract was deduced from the standard curve. Comparisons of intracellular GSH levels of the original cultures were made in terms of nanomoles of GSH/mg of protein. The specific activities of CDNB conjugation reactions were also determined for these crude extracts(47) .


RESULTS

Characterization of GST121-Y8F

The nucleotide sequence patterns in Fig. 1(Panel B) demonstrated the sequence change from the wild type TAC (Y) to the mutant TTC (F). The mutant enzyme has a much reduced but detectable activity (0.3%) toward CDNB conjugation relative to GST121. The analysis of kinetic parameters revealed a 2-3-fold decrease in the affinity for substrates (K(m) (GSH), 0.54 mM (mutant) versus 0.26 mM (wild type); K(m) (CDNB), 0.68 mM (mutant) versus 0.22 mM (wild type)) and a much reduced turnover number (k (GSH), 0.31 s (mutant) versus 132 s; k (CDNB), 0.36 s (mutant) versus 93 s). The overall catalytic efficiency is over 800-fold in favor of the wild type GST121 (Table 1). These results are consistent with previous reports on other GSTs(16, 17, 18, 19, 22, 29) .


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).





Growth Inhibition of E. coli by CDNB

The compound CDNB inhibits E. coli (DH5alpha) growth at concentrations greater than 0.8 mM in liquid cultures at 37 °C. At appropriate CDNB concentrations the cells expressing a functional GST (e.g. E. coli DH5alpha containing pGTH121) are more sensitive to CDNB than a E. coli without it (e.g. E. coli DH5alpha with or without pGTH121-Y8F). Other chloronitrobenzenes such as 1,2-dichloro-4-nitrobenzene (DCNB), 1-chloro-3,4-dinitrobenzene, 1,2,3-trichloro-4-nitrobenzene, and 1,2,4-trichloro-5-nitrobenzene also affect E. coli growth to different extent at various concentrations (see below and Table 2). Some of them (e.g. 1,2-dichloro-4-nitrobenzene, 1,2,3-trichloro-5-nitrobenzene) are potentially useful in the mutant selection of different GSTs. In this communication we concentrated on the ``universal'' GST substrate, CDNB.



Selection of GST-null Mutants in E. coli

Since the effects of CDNB's antibiotic activity are enhanced by the presence of a functional GST gene, a strategy for selecting GST-null mutations in E. coli based on the differential sensitivity to CDNB was developed. We expected that E. coli containing a null mutation in GST will outgrow the wild type in the presence of CDNB. To quantitate the differences in CDNB growth inhibitions we compared the plating efficiency of E. coli DH5alpha containing pGTH121 with that of DH5alpha containing pGTH121-Y8F in the presence of various concentrations of CDNB. The former strain expresses a fully functional GST, whereas the latter strain expresses a mutant GST with 0.3% of the wild type specific activity. The plating efficiency at 20 µg/ml CDNB was over 2000-fold in favor of the E. coli strain with the mutant GST121-Y8F. At higher or lower concentrations of CDNB the difference in plating efficiency became smaller (Table 2). A similar preference was observed with E. coli DH5alpha containing the expression vector (pKK223-3) alone (data not shown). It is conceivable that many other GST-null mutants in E. coli demonstrate a similar growth advantage in the presence of CDNB relative to E. coli expressing the wild type GST121. Therefore, null mutations originated from hydroxylamine mutagenesis or site-directed mutagenesis at essential amino acid residues of the human GST gene on pGTH121 can be selected for on plates in the presence of appropriate concentration of CDNB. This procedure should be generally applicable to any GSTs with CDNB conjugation activities as demonstrated below.

Hydroxylamine Mutagenesis

Hydroxylamine-treated pGTH121 DNA (3 µg) was used to transform E. coli DH5alpha, and colonies were selected on LB agar plates containing 100 µg/ml ampicillin and 20 µg/ml CDNB. Ninety-five transformants were obtained, of which 66 grew in the presence of 30 µg/ml CDNB. A dozen of them were randomly chosen for further characterization. The CDNB conjugation activity in crude extracts was determined for each mutant candidate. This was then followed by Western blot analysis of each crude extract with antiserum against human GST 1-1. This antiserum reacts with the known human alpha class GSTs and their mutants(36) . Eleven of the 12 mutant crude extracts have a drastic decrease in specific activity (CDNB conjugation) without a loss of signal on Western blots (Fig. 2), whereas the 12th mutant did not show a band at the 25.6 kDa position. In general, the decrease of CDNB conjugation activity for each of these mutants relative to GST121 was more pronounced than the mutant GST121-Y8F. These results are consistent with the principle of CDNB selection.


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 DH5alpha 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.

Site-directed Mutagenesis on Double-stranded DNA

To confirm the effects of the E96K and G97D mutations we performed separately oligonucleotide-directed mutagenesis on the pGTH121 DNA template. To facilitate mutant isolation and to test the utility of the CDNB selection procedure for GST-null mutants, we relied on CDNB to select directly for GST-null mutations in colonies after transformation. The plasmid DNA from the BMH71-18 mutS strain after initial mutagenesis was used to transform E. coli DH5alpha and plated on LB agar plates containing 100 µg/ml ampicillin and 30 µg/ml CDNB at a control transformation frequency of 1.1 times 10^6 transformants/µg of pKK223-3 DNA. The presence of 30 µg/ml CDNB reduced the transformation frequency to a level of 3.1 times 10 in the E96K experiment and to a level of 8.5 times 10 in the G97D experiment. Among six transformants from each of the two mutagenesis experiments all but one of them has the mutation expected from the mutant oligonucleotides. When these mutant crude extracts were analyzed for CDNB conjugation activity, the results were the same as those mutants originally derived from hydroxylamine mutagenesis. Therefore, the two amino acid residues at positions 96 (E) and 97 (G) appear to serve important functions in the structure integrity and/or catalytic activity of GST121.

The Structure and Function Relationship of Glu and Gly Mutants

The mutations E96K and G97D resulted in mutant GSTs with total loss of CDNB conjugation activity and the inability to bind to the S-hexyl GSH affinity matrix. To understand the biochemical basis of these two mutations in the function of GST121, additional amino acid substitutions were introduced into these two positions to generate ``weaker alleles.'' A leucine substitution at position 96 (E96L) produced a mutant GST unable to bind to the S-hexyl GSH affinity column but can be detected on Western blot, although at a much reduced level relative to GST121 and GST121-Y8F (see below, Fig. 4). This mutant GST121-E96L in the crude extract has a level of CDNB conjugation activity between those for GST121-Y8F and the background (e.g. E. coli DH5alpha with or without GST121-E96K). The mutant GSTs, GST121-E96F, and GST121-E96Y, can be purified to electrophoretic homogeneity in good yield when cultures were grown at 30 °C. The catalytic efficiency (k/K(m)) for both mutant GSTs was only reduced by a factor of about 3-6 relative to that for GST121. Such a decrease is due to a combination of a slight increase in K(m) and a slight decrease in k (Table 1). For substitutions at Gly, (L, P, S, T), they resulted in mutant GSTs with a broad range of reduced CDNB conjugation activities relative to GST121. The proline substitution produced a mutant GST121-G97P which is slightly more active than the GST121-Y8F with a better k but a weaker affinityfor the substrates (Table 1). On the other hand, the serine and threonine substitutions each produced a mutant GST with nearly 50% of the wild type activities. Again, the 5-11-fold reduction in catalytic efficiencies was due to changes in both K(m) and k values. The leucine substitution at 97 in GST121-G97L turned out to be just as inactive as GST121-E96L in the crude extract despite the fact that it was made at a level comparable to GST121 on Western blots. It has weak but detectable binding to the S-hexyl GSH affinity matrix (data not shown).


Figure 4: Western blot analysis of E96L and G97P grown at different temperature. DH5alpha 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.



Analyses of Mutant GST Activity in Vivo

Comparison of plating efficiency of mutant GST121-Y8F and GST121 in E. coli indicated that it is negatively correlated with CDNB conjugation activities of the purified GST proteins. Such correlation provided an independent confirmation in vivo of mutant GST activities determined from purified proteins, especially for the ``weaker alleles'' isolated by site-directed mutagenesis. Since the properties of these mutant GSTs were usually unpredictable and protein purification might sometimes result in damages to mutant GSTs to various extents, such an in vivo activity-based procedure should be essential to ascertain the mutant GSTs' behavior in vitro. This combination of in vitro and in vivo analyses for mutant GSTs is necessary for reaching an accurate conclusion for the impact of a particular amino acid substitution on GST structure and function.

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 DH5alpha) 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%.



Application to Other GSTs

The CDNB selection procedure should be applicable in mutant isolation for any other GSTs with CDNB conjugation activities. To demonstrate this point we made an expression construct for the Drosophila GST D27 (49, 50) in pKK223-3, designated pGTDm27-KK. The antisense construct was designated pGTDm27-KK`. The GST D27 protein has been purified from E. coli cultures by S-hexyl GSH affinity chromatography to electrophoretic homogeneity. Its specific activity for CDNB conjugation is 2.4 units/mg, much lower than the mammalian GSTs. (^3)When the plating efficiency was compared between E. coli DH5alpha carrying the two plasmids, the strain with the antisense construct is clearly favored by a factor of over 200 in the presence of 20 µg/ml CDNB (Table 4). Such a difference should be sufficient to allow selection for null mutations in Drosophila gstD27 and the subsequent identification of essential amino acids in GST D27 function.



Possible Mechanism of CDNB-mediated Growth Inhibition of E. coli

The fact that functional expression of GST enhances the antibiotic action of CDNB suggests that the initial conjugation product S-(2,4-dinitrophenyl)glutathione (DNPG) and/or their metabolites might be the active ingredient of the antibiotic activity. However, the S-(2,4-dinitrophenyl)glutathione added to any bacterial culture at 0.4 mM did not inhibit bacterial growth (Fig. 6). Alternatively, accelerated reduction or depletion of reduced GSH inside E. coli due to GST action or DNPG itself or its metabolites might be a signal for the growth inhibition. In the presence of 0.4 mM CDNB, E. coli DH5alpha expressing GST121 clearly suffered from growth inhibition (Fig. 6A, curve 3), whereas DH5alpha without GST121 or with inactive mutant GSTs grew much more normally (Fig. 6A, curves 1, 2, 4, and 5). Furthermore, the CDNB growth inhibition was much more enhanced in the GSH-deficient strain 821 whether it expressed any GST or not (Fig. 6A, dashed lines). To document the reduction of intracellular GSH level in the presence of CDNB growth of E. coli containing a functional GST (pGTH121) or an inactive GST (pGTH121-G97D) in the presence of 1 mM CDNB was compared by turbidity measurements of A and by colony plate counts at various time points. The intracellular GSH level and specific activity of CDNB conjugation were also determined at various time points of cell growth. As shown in Fig. 7growth inhibition by 1 mM CDNB occurred over a period of 20 h for E. coli DH5alpha (pGTH121) containing the active GST. During this period of growth inhibition the intracellular GSH level remained very low at 60% below normal. Also, the colony plate counts (i.e. number of viable bacteria) continued to drop, so was the specific activity for CDNB conjugation. In contrast, the intracellular GSH level paralleled the A curve for E. coli DH5alpha containing the inactive mutant GST121-G97D except for an early drop. The colony counts also paralleled the increase in A except for the decrease in the first 6 h after the addition of 1 mM CDNB.


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 DH5alpha (GSH-sufficient), 821 (GSH-deficient) and their derivatives expressing wild type and mutant GST121 proteins. Forty µl of overnight cultures of DH5alpha (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 DH5alpha or 821 (DH5alpha carrying pKK223-3 (2 in Panels A and B), DH5alpha carrying pGTH121 (3 in Panels A and B), DH5alpha carrying pGTH121-Y8F (4 in Panels A and B), DH5alpha 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 DH5alpha (pGTH121) and DH5alpha (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 DH5alpha (pGTH121), whereas triangles represented readings (A, GSH level, colony plate count, or CDNB conjugation activity) for DH5alpha (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 DH5alpha (pGTH121). The crude extract of DH5alpha (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).




DISCUSSION

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 beta-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 beta-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 DH5alpha 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 (alpha4) 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 alpha3 (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 DH5alpha, 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 DH5alpha, 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 DH5alpha (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 DH5alpha 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 DH5alpha (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 leq5% 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 DH5alpha (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.


FOOTNOTES

*
The project described was supported by National Institute of Environmental Health Science, National Institutes of Health Grant ES 05661. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biological Sciences, Stanford University, Stanford, CA 94305.

To whom correspondence should be addressed: 401 Paul M. Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802. Tel.: 814-863-2096; Fax: 814-863-7024.

(^1)
The abbreviations used are: GST, glutathione S-transferase; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; DNPG, S-(2,4-dinitrophenyl)glutathione; PAGE, polyacrylamide gel electrophoresis. The amino acid residue numbers are according to the actual sequences of human GST 1-1 as purified from the E. coli expression system. No methionine was detected at the N terminus in the mature protein isolated from E. coli.

(^3)
H.-C. Lee and C.-P. D. Tu, unpublished results.

(^2)
C. L. Strickland, H.-C. Chen, K. Zeng, Y.-S. L. Tu, J. P. Rose, B.-C. Wang, and C.-P. D. Tu, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Professor Bi-Cheng Wang and Ke Zeng for providing structure coordinates of GST121 before publication and helpful discussions. We also thank Chian-Yu Peng for technical assistance in acquiring data for Table 3, and Eileen McConnell for expert secretarial assistance. The efforts and results by P.-C. Benjamin Tu in the early phase of this project is also acknowledged.


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