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Purification and characterization of a novel glutathione transferase from Ochrobactrum anthropi1
FEMS Microbiology Letters 160 (1998) 81^86
Puri¢cation and characterization of a novel glutathione transferase from Ochrobactrum anthropi Bartolo Favaloro a; *, Sonia Melino b , Ra¡aele Petruzzelli b , Carmine Di Ilio b , Domenico Rotilio a a
Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, `Gennaro Paone' Environmental Health Center, Via Nazionale, 66030 Santa Maria Imbaro, Italy b Dipartimento di Scienze Biomediche, Facoltaé di Medicina, Universitaé di Chieti, Chieti, Italy Received 20 October 1997 ; revised 5 January 1998; accepted 6 January 1998
Abstract Glutathione transferase was purified from Ochrobactrum anthropi and its N-terminal sequence was determined to be MKLYYKVGACSLAPHIILSEAGLPY. The apparent molecular mass of the protein (24 kDa) was determined by SDSpolyacrylamide gel electrophoresis analysis. The amino acid sequence obtained showed similarities with known bacterial glutathione transferases in the range of 72^64%. Immunoblotting experiments performed with antisera raised against glutathione transferase from O. anthropi did not show cross-reactivity with two bacterial glutathione transferases belonging to Serratia marcescens and Proteus mirabilis. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Glutathione transferase ; Ochrobactrum anthropi; N-terminal sequence
1. Introduction Ochrobactrum anthropi, classi¢ed as group Vd according to the scheme of the Center for Disease Control and Prevention, is closely related to Gramnegative, aerobic, peritrichously £agellated, nonfermentative, nonfastidious bacilli that are positive for oxidase and urease production [1]. All cells are potentially exposed to toxic foreign * Corresponding author. Tel.: +39 (872) 570270; Fax: +39 (872) 578240; E-mail: [email protected] The amino acid sequence reported in this paper has been submitted to the EMBL Data Bank with accession number P81065.
chemicals (xenobiotics). The production of enzymes catalyzing detoxi¢cation reactions is among the biochemical strategies adopted by cells to cope with this type of chemical threat. This detoxi¢cation process includes two major phases; phase I is catalyzed by the cytochrome P-450 system and phase II by enzymes catalyzing the conjugation of the xenobiotic to an endogenous substrate, namely glutathione (GSH). Glutathione transferases are the enzymes usually involved in phase II. Glutathione transferases (EC 2.5.1.18) are a family of multifunctional dimeric proteins that catalyze the conjugation of the sulfur atom of glutathione to a large variety of electrophilic compounds of both endobiotic and xenobiotic origin [2,3]. Much is known about cyto-
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solic mammalian glutathione transferases which are composed of a number of isoenzymes with some overlapping substrate speci¢cities [2]. On the basis of primary structure and immunological properties, they have been grouped into four classes designated alpha, mu, pi and theta. Another class, sigma, composed mainly of cephalopod glutathione transferases [4], has also been suggested. In prokaryotes, glutathione transferase activity is very low or undetectable, but it seems to be very important in the processes involved in the biodegradation of xenobiotics, including antibiotics [5], although other roles of this enzyme have not yet been well de¢ned. In recent years, several bacterial glutathione transferases which appeared to be homodimers of about 22 kDa have been puri¢ed [6^9]. Bacterial glutathione transferases do not cross-react with antisera from mammals and plants. Moreover, they show low cross-reactivity among themselves [5]. At present it is not clear whether bacterial glutathione transferases can be classi¢ed, as is the case with mammalian enzymes, into several distinct classes. For this reason it is important to understand the structural, kinetic and immunological properties of the glutathione transferases of a wide number of bacterial strains. Here we report the puri¢cation and N-terminal sequence of a novel glutathione transferase from O. anthropi, a bacterium able to degrade atrazine [10]. We have studied the immunological properties of the enzyme of O. anthropi and compared the N-terminal sequence with known glutathione transferases puri¢ed from other bacterial strains.
2. Materials and methods 2.1. Microorganism isolation and identi¢cation The microorganism was isolated from activated sludge contaminated with atrazine. Culture morphology and biochemical reaction in the API ID 32 E and ID 32 GN (Biomeèrieux) system were consistent with the identi¢cation of O. anthropi [10]. 2.2. Assay of glutathione transferase activity The activity was assayed by the spectrophotomet-
ric method [11] using 1 mM GSH and 1 mM 1chloro-2,4-dinitrobenzene (CDNB) as substrates. Protein concentration was determined by the bicinchoninic acid assay (BCA, Pierce) with bovine serum albumin as standard. 2.3. Puri¢cation of glutathione transferase from O. anthropi Cells were grown overnight at 37³C in 2 l of LB broth. All operations described below were performed at 4³C. The bacteria were harvested by centrifugation at 13 000Ug for 20 min suspended in 30 ml of 10 mM potassium phosphate bu¡er (pH 7), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl £uoride (PMSF) (bu¡er A), and disrupted by sonication. The particulate material was removed by centrifugation at 100 000Ug for 1 h and the supernatant applied to a GSH-agarose a¤nity column (1U10 cm) (Pharmacia). The column was exhaustively washed with buffer A and supplemented with 200 mM KCl. The enzyme was eluted with 50 mM Tris-HCl (pH 9.6) containing 10 mM GSH. The GST fractions were dialyzed and concentrated using Centriprep-10 (Amicon), with 10 mM potassium phosphate bu¡er (pH 7) and stored at 380³C. SDS-PAGE was carried out and proteins were detected by silver staining. 2.4. N-terminal amino acid sequencing SDS-PAGE of the puri¢ed enzyme was performed. The protein band was then transferred from the unstained gel onto a polyvinylidenedi£uoride membrane (PVDF, ProBlott Applied Biosystem), and analyzed by a protein sequencer (470A Applied Biosystem model). 2.5. Preparation of anti-glutathione transferase antibodies A speci¢c antiserum against glutathione transferase antigen was prepared in our laboratory using the puri¢ed enzyme. Three di¡erent lanes were loaded with 10 Wg of protein and run on 12.5% polyacrylamide gels. After electrophoresis, the protein bands were located by staining with copper chloride. The three gel slices were excised and fragmented by repeated passages through a syringe, and then injected
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subcutaneously into a New Zealand white rabbit. After primary immunization, the animal was given additional injections on days 21 and 42, it was then bled 14 days later and the antiserum was used for immunoblotting. 2.6. Immunoblot analysis Puri¢ed enzymes from di¡erent sources (O. anthropi, Serratia marcescens and Proteus mirabilis) were analyzed by SDS-PAGE on 12.5% polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose (for 3 h at 1 A) using an immunoblot transfer apparatus (Hoefer). After transfer, the nitrocellulose was incubated for 2 h at 40³C in 5% dry milk powder (Merck) in Tris 20 mM, 500 mM NaCl, pH 7.5 (milk-TBS) to block nonspeci¢c binding. The blot was incubated overnight at 4³C with milk-TBS containing antiserum at a dilution of 1:500. After three 20-min washes with TBS, containing 0.25% dry milk powder and 0.1% Tween20 (milk-T-TBS), the blot was incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit immunoglobulin (Sigma) diluted 1:1000 in milk-TBS. The blot was again washed three times with milk-T-TBS and twice with TBS. Antibodies were visualized using the chemiluminescence detection system (ECL, Amersham).
3. Results 3.1. Puri¢cation Bacterial lysates, obtained as described in Section 2, showed a very low GSH-conjugating activity toward CDNB; a similar value was detected when bacteria were cultivated in succinate and atrazine (data not shown). After one-step chromatography on a GSH-agarose a¤nity column, the speci¢c activity, evaluated as described in Section 2, was 1.54 U/mg, corresponding to a 1400-fold puri¢cation, as reported in Table 1. Eluates from this GSH-agarose a¤nity column produced a single band of about 24 kDa on SDS-PAGE (Fig. 1). This molecular mass re£ects the range of those reported in the literature and is compatible with that of bacterial glutathione transferases.
Fig. 1. SDS-PAGE of GST puri¢ed from O. anthropi. Lane 1, crude extract. Lane 2, puri¢ed GST through a GSH-agarose column. Lane 3, molecular mass standards: myosin (200 kDa), phosphorylase L (97.4 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.3 kDa).
3.2. Immunological properties The speci¢city of the antiserum, raised against puri¢ed enzyme, was assayed by immunoblotting experiments. These antibodies recognized only the glutathione transferase puri¢ed from O. anthropi when in the presence of two other glutathione transferases puri¢ed from S. marcescens and P. mirabilis, as shown in Fig. 2. The antiserum raised against glutathione transferase from P. mirabilis did not recognize puri¢ed enzyme from O. anthropi (data not shown). 3.3. N-terminal amino acid sequencing Fig. 3 shows the N-terminal amino acid sequence
Fig. 2. Western blot analysis using an antiserum raised against glutathione transferase from O. anthropi. Lane 1, 1 Wg S. marcescens glutathione transferase ; lane 2, 1 Wg P. mirabilis glutathione transferase; lane 3, 1 Wg O. anthropi glutathione transferase.
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Fig. 3. Comparison of the N-terminal amino acid sequence of O. anthropi glutathione transferase with other bacterial, plant, insect and mammalian glutathione transferases. N-terminal sequencing was performed by Edman degradation. Dots indicate deletions. The black squares indicate invariant or conservatively replaced residues among glutathione transferases from di¡erent species. The gray boxes indicate the invariant serine in bacteria and plant/theta glutatione transferases. Pseudomonas sp. LB400 GST [14]; S. marcescens GST [7]; P. mirabilis GST [16]; X. campestris GST [8]; E. coli K-12 GST [9]; maize GST III [23]; A. thaliana GST [22]; theta, Lucilia cuprina GST [17]; Pi, human GST P1 [15]; mu, rat M1 [18]; alpha, human A1 [19]; theta, rat Yrs [24].
determined for the glutathione transferase from O. anthropi. The corresponding structures of the other glutathione transferases are also provided. Comparison with known N-terminal amino acid sequences of bacteria, plant, insect and mammalian glutathione transferases demonstrates that plant, insect and mammalian enzymes are distantly related (less than 24% identity) to O. anthropi, whereas those of bacteria are more similar (68% identity). This analysis
indicates that some amino acid residues, such as Tyr5 , Leu18 and Gly22 , are highly conserved among the glutathione transferases of prokaryotes and eukaryotes (plants, insects and mammals).
4. Discussion Glutathione transferases have been found in in-
Table 1 Puri¢cation of GST from O. anthropi
Crude extract GSH-agarose
Protein (mg)
Total activity (units)
Speci¢c activity (units mg31 )
Puri¢cation (fold)
924 0.022
1.02 0.034
0.0011 1.54
1 1400
One unit of enzyme produces 1 Wmol of S-(2,4-dinitrophenyl)glutathione per min at 30³C.
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sects, plants, parasites and fungi, and they are involved in the mechanism of resistance against agricultural medicines and insecticides [12]. In prokaryotes, glutathione transferase activity is often very low or completely undetectable, as described for Bacillus subtilis and Pseudomonas putida [13]. Although glutathione transferases have been puri¢ed from several bacterial species [6^9], little is known about their biological functions, structures and regulation. Di Ilio et al. have demonstrated that glutathione transferases from several bacterial species have distinct antigenic determinants. In fact, an antiserum against glutathione transferase from O. anthropi did not cross-react with bacterial enzymes, such as P. mirabilis and S. marcescens. These results reinforce the hypothesis of immunological speci¢city among bacterial glutathione transferases. The sequence of our enzyme compared to other known bacterial glutathione transferases showed a homology of more than 64%. The highest level of similarities was found in the enzyme from Pseudomonas LB 400. In this strain, a glutathione transferase-encoding gene was located within the bph locus that contains genes encoding polypeptides of polychlorinated biphenyl-degrading enzymes [14]. O. anthropi was isolated from activated sludge contaminated with atrazine, and was able to grow on plates containing atrazine as the only source of carbon [10]. Therefore, we hypothesize that the glutathione transferase may be involved in the metabolism of this type of herbicide. The N-terminal sequence of glutathione transferase from O. anthropi, as compared to mammalian enzymes, showed similarities of less than 24%; although identities between glutathione transferases from prokaryotes and eukaryotes were low, several amino acid residues were highly conserved. One of the conserved residues was Tyr5 , which aligned with Tyr7 of the human glutathione transferase P1 [15] and with corresponding Tyr residues in other species [7,9,14,16^19]. In mammalian enzymes of the classes alpha, mu and pi, this tyrosyl residue was shown to be spatially located adjacent to the thiol group of GSH by X-ray crystallography [20]. This tyrosyl residue is considered to be a catalytic residue in mammalian glutathione transferases for the activation of the thiol group of GSH [21]. Although several bacterial N-terminal glutathione transferase sequences, including ours, have conserved this residue, it is un-
85
likely that this amino acid has the same function in bacteria. In fact, site-speci¢c mutagenesis studies in glutathione transferase from Escherichia coli showed that the conserved tyrosyl residue was not essential for catalysis [9]. Moreover, the three-dimensional structure of glutathione transferase from the herbicide-conjugating plant Arabidopsis thaliana indicates the lack of a tyrosine residue in the active site [22], while a Ser9 residue is reported to be essential to the catalytic activity of class theta glutathione transferase from Lucilia cuprina pupae [17]. Although the Nterminal sequence of class theta glutathione transferases is not similar to those from bacteria, the sequence of O. anthropi contains a serine residue that is highly conserved among bacterial glutathione transferases. Since only the Ser9 residue of the Nterminal sequence from P. mirabilis aligns to the conserved Ser9 from plant/theta enzymes [17,22^24], the highly conserved Ser11 residue in bacterial glutathione transferases could be the counterpart of the `catalytic' serine residue of class theta. The knowledge of the three-dimensional structure of bacterial glutathione transferases will be a useful tool to classify bacterial enzymes as either theta or belonging to a speci¢c class.
Acknowledgments The authors wish to thank A. Tamburro for his valuable suggestions, R. Bertazzi for her expert assistance in the preparation of the ¢gures, D. Spadano, M.G. Mencuccini, P. Di Nardo and M.P. De Simone for helping prepare the manuscript.
References [1] Kern, W.V., Oethinger, M., Kaufhold, A., Rozdzinski, E. and Marre, R. (1993) Ochrobactrum anthropi bacteremia: report of four cases and short review. Infection 21, 306^310. [2] Jakobi, W.B., Habig, W.H., Keen, J.H., Ketley, J.N. and Pabst, M.J. (1976) Glutathione S-transferases: catalytic aspects. In: Glutathione : Metabolism and Function, pp. 189^ 211. Raven Press, New York. [3] Wilce, M.C.J. and Parker, M.W. (1994) Structure and function of glutathione S-transferase. Biochim. Biophys. Acta 1205, 1^18. [4] Meyer, D.J., Muimo, R., Thomas, M., Coates, D. and Isaac, R.E. (1996) Puri¢cation and characterization of prostaglan-
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[5]
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[14]
[15]
B. Favaloro et al. / FEMS Microbiology Letters 160 (1998) 81^86 din-H E-isomerase, a sigma-class glutathione S-transferase, from Ascaridia galli. Biochem. J. 313, 223^227. Piccolomini, R., Di Ilio, C., Aceto, A., Allocati, N., Faraone, A., Cellini, L., Ravagnan, G. and Federici, G. (1989) Glutathione transferase in bacteria: subunit composition and antigenic characterization. J. Gen. Microbiol. 135, 3119^ 3125. Di Ilio, C., Aceto, A., Piccolomini, R., Allocati, N., Faraone, A., Cellini, L., Ravagnan, G. and Federici, G. (1988) Puri¢cation and characterization of three forms of glutathione transferase from Proteus mirabilis. Biochem. J. 255, 971^975. Di Ilio, C., Aceto, A., Piccolomini, R., Allocati, N., Faraone, A., Bucciarelli, T., Barra, D. and Federici, G. (1991) Puri¢cation and characterization of a novel glutathione transferase from Serratia marcescens. Biochim. Biophys. Acta 1077, 141^146. Di Ilio, C., Aceto, A., Allocati, N., Piccolomini, R., Bucciarelli, T., Dragani, B., Faraone, A., Sacchetta, P., Petruzzelli, R. and Federici, G. (1993) Characterization of glutathione transferase from Xanthomonas campestris. Arch. Biochem. Biophys. 305, 110^114. Nishida, M., Kong, K.H., Inoue, H. and Takahashi, K. (1994) Molecular cloning and site-directed mutagenesis of glutathione S-transferase from Escherichia coli. The conserved tyrosyl residue near the N terminus is not essential for catalysis. J. Biol. Chem. 269, 32536^32541. Laura, D., De Socio, G., Frassanito, R. and Rotilio, D. (1996) E¡ects of atrazine on Ochrobactrum anthropi membrane fatty acids. Appl. Environ. Microbiol. 62, 2644^2646. Habig, W.H. and Jakoby, W.B. (1981) Assay for di¡erentiation of glutathione S-transferases. Methods Enzymol. 77, 398^ 405. Fahey, R.C. and Sundquist, A.R. (1991) Evolution of glutathione metabolism. Adv. Enzymol. Relat. Areas Mol. Biol. 64, 1^53. Sheehan, D. and Casey, J.P. (1993) Evidence for alpha and Mu class glutathione S-transferase in a number of fungal species. Comp. Biochem. Physiol. B 104, 7^13. Hofer, B., Backhaus, S. and Timmis, K.N. (1994) The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB 400 encodes four additional metabolic enzymes. Gene 144, 9^16. Kano, T., Sakai, M. and Muramatsu, M. (1987) Structure and expression of a human class P glutathione S-transferase messenger RNA. Cancer Res. 47, 5626^5630.
[16] Mignogna, G., Allocati, N., Aceto, A., Piccolomini, R., Di Ilio, C., Barra, D. and Martini, F. (1993) The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes. Eur. J. Biochem. 211, 421^425. [17] Wilce, M.C.J., Board, P.J., Feil, S.C. and Parker, W. (1995) Crystal structure of a theta-class glutathione transferase. EMBO J. 14, 2133^2143. [18] Ding, G.J.-F., Lu, A.Y.H. and Pickett, C.B. (1985) Rat liver glutathione S-transferase. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit. J. Biol. Chem. 260, 13268^ 13271. [19] Board, P.G. and Webb, G.C. (1987) Isolation of a cDNA clone and localization of human glutathione S-transferase 2 genes to chromosome band 6p12. Proc. Natl. Acad. Sci. USA 84, 2377^2381. [20] Sinning, I., Kleywegt, G.J., Cowan, S.W., Reinemer, P., Dirr, H.W., Huber, R., Gilliland, G.L., Armstrong, R.N., Ji, X., Board, P.G., Olin, B., Mannervik, B. and Jones, T.A. (1993) Structure determination and re¢nement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. J. Mol. Biol. 232, 192^212. [21] Kolm, R.H., Sroga, G.E. and Mannervik, B. (1992). Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1. Biochem. J. 285, 537^540. [22] Reinemer, P., Prade, L., Hof, P., Neuefenind, T., Huber, R., Zettl, R., Palme, K., Schell, J., Koelln, I., Bartunik, H.D. and Bieseler, B. (1996). Three-dimensional structure of glutathione î resolution: S-transferase from Arabidopsis thaliana at 2.2 A structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J. Mol. Biol. 255, 289^309. [23] Grove, G., Zarlengo, R.P., Timmermann, K.P., Li, N.-Q., Tam, M.F. and Tu, C.-P. (1988) Characterization and heterospeci¢c expression of cDNA clones of genes in the maize GSH S-transferase multigene family. Nucleic Acid Res. 16, 425^ 438. [24] Ogura, K., Nishiyama, T., Okada, T., Kajita, J., Hiroyuki, N., Watabe, T., Hiratsuka, A. and Watabe, T. (1991) Molecular cloning and amino acid sequencing of rat liver class theta glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters of carcinogenic arylmethanols. Biochem. Biophys. Res. Commun. 181, 1294^1300.
FEMSLE 8028 17-2-98
Puri¢cation and characterization of a novel glutathione transferase from Ochrobactrum anthropi Bartolo Favaloro a; *, Sonia Melino b , Ra¡aele Petruzzelli b , Carmine Di Ilio b , Domenico Rotilio a a
Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, `Gennaro Paone' Environmental Health Center, Via Nazionale, 66030 Santa Maria Imbaro, Italy b Dipartimento di Scienze Biomediche, Facoltaé di Medicina, Universitaé di Chieti, Chieti, Italy Received 20 October 1997 ; revised 5 January 1998; accepted 6 January 1998
Abstract Glutathione transferase was purified from Ochrobactrum anthropi and its N-terminal sequence was determined to be MKLYYKVGACSLAPHIILSEAGLPY. The apparent molecular mass of the protein (24 kDa) was determined by SDSpolyacrylamide gel electrophoresis analysis. The amino acid sequence obtained showed similarities with known bacterial glutathione transferases in the range of 72^64%. Immunoblotting experiments performed with antisera raised against glutathione transferase from O. anthropi did not show cross-reactivity with two bacterial glutathione transferases belonging to Serratia marcescens and Proteus mirabilis. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Glutathione transferase ; Ochrobactrum anthropi; N-terminal sequence
1. Introduction Ochrobactrum anthropi, classi¢ed as group Vd according to the scheme of the Center for Disease Control and Prevention, is closely related to Gramnegative, aerobic, peritrichously £agellated, nonfermentative, nonfastidious bacilli that are positive for oxidase and urease production [1]. All cells are potentially exposed to toxic foreign * Corresponding author. Tel.: +39 (872) 570270; Fax: +39 (872) 578240; E-mail: [email protected] The amino acid sequence reported in this paper has been submitted to the EMBL Data Bank with accession number P81065.
chemicals (xenobiotics). The production of enzymes catalyzing detoxi¢cation reactions is among the biochemical strategies adopted by cells to cope with this type of chemical threat. This detoxi¢cation process includes two major phases; phase I is catalyzed by the cytochrome P-450 system and phase II by enzymes catalyzing the conjugation of the xenobiotic to an endogenous substrate, namely glutathione (GSH). Glutathione transferases are the enzymes usually involved in phase II. Glutathione transferases (EC 2.5.1.18) are a family of multifunctional dimeric proteins that catalyze the conjugation of the sulfur atom of glutathione to a large variety of electrophilic compounds of both endobiotic and xenobiotic origin [2,3]. Much is known about cyto-
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 1 5 - 9
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solic mammalian glutathione transferases which are composed of a number of isoenzymes with some overlapping substrate speci¢cities [2]. On the basis of primary structure and immunological properties, they have been grouped into four classes designated alpha, mu, pi and theta. Another class, sigma, composed mainly of cephalopod glutathione transferases [4], has also been suggested. In prokaryotes, glutathione transferase activity is very low or undetectable, but it seems to be very important in the processes involved in the biodegradation of xenobiotics, including antibiotics [5], although other roles of this enzyme have not yet been well de¢ned. In recent years, several bacterial glutathione transferases which appeared to be homodimers of about 22 kDa have been puri¢ed [6^9]. Bacterial glutathione transferases do not cross-react with antisera from mammals and plants. Moreover, they show low cross-reactivity among themselves [5]. At present it is not clear whether bacterial glutathione transferases can be classi¢ed, as is the case with mammalian enzymes, into several distinct classes. For this reason it is important to understand the structural, kinetic and immunological properties of the glutathione transferases of a wide number of bacterial strains. Here we report the puri¢cation and N-terminal sequence of a novel glutathione transferase from O. anthropi, a bacterium able to degrade atrazine [10]. We have studied the immunological properties of the enzyme of O. anthropi and compared the N-terminal sequence with known glutathione transferases puri¢ed from other bacterial strains.
2. Materials and methods 2.1. Microorganism isolation and identi¢cation The microorganism was isolated from activated sludge contaminated with atrazine. Culture morphology and biochemical reaction in the API ID 32 E and ID 32 GN (Biomeèrieux) system were consistent with the identi¢cation of O. anthropi [10]. 2.2. Assay of glutathione transferase activity The activity was assayed by the spectrophotomet-
ric method [11] using 1 mM GSH and 1 mM 1chloro-2,4-dinitrobenzene (CDNB) as substrates. Protein concentration was determined by the bicinchoninic acid assay (BCA, Pierce) with bovine serum albumin as standard. 2.3. Puri¢cation of glutathione transferase from O. anthropi Cells were grown overnight at 37³C in 2 l of LB broth. All operations described below were performed at 4³C. The bacteria were harvested by centrifugation at 13 000Ug for 20 min suspended in 30 ml of 10 mM potassium phosphate bu¡er (pH 7), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl £uoride (PMSF) (bu¡er A), and disrupted by sonication. The particulate material was removed by centrifugation at 100 000Ug for 1 h and the supernatant applied to a GSH-agarose a¤nity column (1U10 cm) (Pharmacia). The column was exhaustively washed with buffer A and supplemented with 200 mM KCl. The enzyme was eluted with 50 mM Tris-HCl (pH 9.6) containing 10 mM GSH. The GST fractions were dialyzed and concentrated using Centriprep-10 (Amicon), with 10 mM potassium phosphate bu¡er (pH 7) and stored at 380³C. SDS-PAGE was carried out and proteins were detected by silver staining. 2.4. N-terminal amino acid sequencing SDS-PAGE of the puri¢ed enzyme was performed. The protein band was then transferred from the unstained gel onto a polyvinylidenedi£uoride membrane (PVDF, ProBlott Applied Biosystem), and analyzed by a protein sequencer (470A Applied Biosystem model). 2.5. Preparation of anti-glutathione transferase antibodies A speci¢c antiserum against glutathione transferase antigen was prepared in our laboratory using the puri¢ed enzyme. Three di¡erent lanes were loaded with 10 Wg of protein and run on 12.5% polyacrylamide gels. After electrophoresis, the protein bands were located by staining with copper chloride. The three gel slices were excised and fragmented by repeated passages through a syringe, and then injected
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83
subcutaneously into a New Zealand white rabbit. After primary immunization, the animal was given additional injections on days 21 and 42, it was then bled 14 days later and the antiserum was used for immunoblotting. 2.6. Immunoblot analysis Puri¢ed enzymes from di¡erent sources (O. anthropi, Serratia marcescens and Proteus mirabilis) were analyzed by SDS-PAGE on 12.5% polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose (for 3 h at 1 A) using an immunoblot transfer apparatus (Hoefer). After transfer, the nitrocellulose was incubated for 2 h at 40³C in 5% dry milk powder (Merck) in Tris 20 mM, 500 mM NaCl, pH 7.5 (milk-TBS) to block nonspeci¢c binding. The blot was incubated overnight at 4³C with milk-TBS containing antiserum at a dilution of 1:500. After three 20-min washes with TBS, containing 0.25% dry milk powder and 0.1% Tween20 (milk-T-TBS), the blot was incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit immunoglobulin (Sigma) diluted 1:1000 in milk-TBS. The blot was again washed three times with milk-T-TBS and twice with TBS. Antibodies were visualized using the chemiluminescence detection system (ECL, Amersham).
3. Results 3.1. Puri¢cation Bacterial lysates, obtained as described in Section 2, showed a very low GSH-conjugating activity toward CDNB; a similar value was detected when bacteria were cultivated in succinate and atrazine (data not shown). After one-step chromatography on a GSH-agarose a¤nity column, the speci¢c activity, evaluated as described in Section 2, was 1.54 U/mg, corresponding to a 1400-fold puri¢cation, as reported in Table 1. Eluates from this GSH-agarose a¤nity column produced a single band of about 24 kDa on SDS-PAGE (Fig. 1). This molecular mass re£ects the range of those reported in the literature and is compatible with that of bacterial glutathione transferases.
Fig. 1. SDS-PAGE of GST puri¢ed from O. anthropi. Lane 1, crude extract. Lane 2, puri¢ed GST through a GSH-agarose column. Lane 3, molecular mass standards: myosin (200 kDa), phosphorylase L (97.4 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.3 kDa).
3.2. Immunological properties The speci¢city of the antiserum, raised against puri¢ed enzyme, was assayed by immunoblotting experiments. These antibodies recognized only the glutathione transferase puri¢ed from O. anthropi when in the presence of two other glutathione transferases puri¢ed from S. marcescens and P. mirabilis, as shown in Fig. 2. The antiserum raised against glutathione transferase from P. mirabilis did not recognize puri¢ed enzyme from O. anthropi (data not shown). 3.3. N-terminal amino acid sequencing Fig. 3 shows the N-terminal amino acid sequence
Fig. 2. Western blot analysis using an antiserum raised against glutathione transferase from O. anthropi. Lane 1, 1 Wg S. marcescens glutathione transferase ; lane 2, 1 Wg P. mirabilis glutathione transferase; lane 3, 1 Wg O. anthropi glutathione transferase.
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Fig. 3. Comparison of the N-terminal amino acid sequence of O. anthropi glutathione transferase with other bacterial, plant, insect and mammalian glutathione transferases. N-terminal sequencing was performed by Edman degradation. Dots indicate deletions. The black squares indicate invariant or conservatively replaced residues among glutathione transferases from di¡erent species. The gray boxes indicate the invariant serine in bacteria and plant/theta glutatione transferases. Pseudomonas sp. LB400 GST [14]; S. marcescens GST [7]; P. mirabilis GST [16]; X. campestris GST [8]; E. coli K-12 GST [9]; maize GST III [23]; A. thaliana GST [22]; theta, Lucilia cuprina GST [17]; Pi, human GST P1 [15]; mu, rat M1 [18]; alpha, human A1 [19]; theta, rat Yrs [24].
determined for the glutathione transferase from O. anthropi. The corresponding structures of the other glutathione transferases are also provided. Comparison with known N-terminal amino acid sequences of bacteria, plant, insect and mammalian glutathione transferases demonstrates that plant, insect and mammalian enzymes are distantly related (less than 24% identity) to O. anthropi, whereas those of bacteria are more similar (68% identity). This analysis
indicates that some amino acid residues, such as Tyr5 , Leu18 and Gly22 , are highly conserved among the glutathione transferases of prokaryotes and eukaryotes (plants, insects and mammals).
4. Discussion Glutathione transferases have been found in in-
Table 1 Puri¢cation of GST from O. anthropi
Crude extract GSH-agarose
Protein (mg)
Total activity (units)
Speci¢c activity (units mg31 )
Puri¢cation (fold)
924 0.022
1.02 0.034
0.0011 1.54
1 1400
One unit of enzyme produces 1 Wmol of S-(2,4-dinitrophenyl)glutathione per min at 30³C.
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sects, plants, parasites and fungi, and they are involved in the mechanism of resistance against agricultural medicines and insecticides [12]. In prokaryotes, glutathione transferase activity is often very low or completely undetectable, as described for Bacillus subtilis and Pseudomonas putida [13]. Although glutathione transferases have been puri¢ed from several bacterial species [6^9], little is known about their biological functions, structures and regulation. Di Ilio et al. have demonstrated that glutathione transferases from several bacterial species have distinct antigenic determinants. In fact, an antiserum against glutathione transferase from O. anthropi did not cross-react with bacterial enzymes, such as P. mirabilis and S. marcescens. These results reinforce the hypothesis of immunological speci¢city among bacterial glutathione transferases. The sequence of our enzyme compared to other known bacterial glutathione transferases showed a homology of more than 64%. The highest level of similarities was found in the enzyme from Pseudomonas LB 400. In this strain, a glutathione transferase-encoding gene was located within the bph locus that contains genes encoding polypeptides of polychlorinated biphenyl-degrading enzymes [14]. O. anthropi was isolated from activated sludge contaminated with atrazine, and was able to grow on plates containing atrazine as the only source of carbon [10]. Therefore, we hypothesize that the glutathione transferase may be involved in the metabolism of this type of herbicide. The N-terminal sequence of glutathione transferase from O. anthropi, as compared to mammalian enzymes, showed similarities of less than 24%; although identities between glutathione transferases from prokaryotes and eukaryotes were low, several amino acid residues were highly conserved. One of the conserved residues was Tyr5 , which aligned with Tyr7 of the human glutathione transferase P1 [15] and with corresponding Tyr residues in other species [7,9,14,16^19]. In mammalian enzymes of the classes alpha, mu and pi, this tyrosyl residue was shown to be spatially located adjacent to the thiol group of GSH by X-ray crystallography [20]. This tyrosyl residue is considered to be a catalytic residue in mammalian glutathione transferases for the activation of the thiol group of GSH [21]. Although several bacterial N-terminal glutathione transferase sequences, including ours, have conserved this residue, it is un-
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likely that this amino acid has the same function in bacteria. In fact, site-speci¢c mutagenesis studies in glutathione transferase from Escherichia coli showed that the conserved tyrosyl residue was not essential for catalysis [9]. Moreover, the three-dimensional structure of glutathione transferase from the herbicide-conjugating plant Arabidopsis thaliana indicates the lack of a tyrosine residue in the active site [22], while a Ser9 residue is reported to be essential to the catalytic activity of class theta glutathione transferase from Lucilia cuprina pupae [17]. Although the Nterminal sequence of class theta glutathione transferases is not similar to those from bacteria, the sequence of O. anthropi contains a serine residue that is highly conserved among bacterial glutathione transferases. Since only the Ser9 residue of the Nterminal sequence from P. mirabilis aligns to the conserved Ser9 from plant/theta enzymes [17,22^24], the highly conserved Ser11 residue in bacterial glutathione transferases could be the counterpart of the `catalytic' serine residue of class theta. The knowledge of the three-dimensional structure of bacterial glutathione transferases will be a useful tool to classify bacterial enzymes as either theta or belonging to a speci¢c class.
Acknowledgments The authors wish to thank A. Tamburro for his valuable suggestions, R. Bertazzi for her expert assistance in the preparation of the ¢gures, D. Spadano, M.G. Mencuccini, P. Di Nardo and M.P. De Simone for helping prepare the manuscript.
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[16] Mignogna, G., Allocati, N., Aceto, A., Piccolomini, R., Di Ilio, C., Barra, D. and Martini, F. (1993) The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes. Eur. J. Biochem. 211, 421^425. [17] Wilce, M.C.J., Board, P.J., Feil, S.C. and Parker, W. (1995) Crystal structure of a theta-class glutathione transferase. EMBO J. 14, 2133^2143. [18] Ding, G.J.-F., Lu, A.Y.H. and Pickett, C.B. (1985) Rat liver glutathione S-transferase. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit. J. Biol. Chem. 260, 13268^ 13271. [19] Board, P.G. and Webb, G.C. (1987) Isolation of a cDNA clone and localization of human glutathione S-transferase 2 genes to chromosome band 6p12. Proc. Natl. Acad. Sci. USA 84, 2377^2381. [20] Sinning, I., Kleywegt, G.J., Cowan, S.W., Reinemer, P., Dirr, H.W., Huber, R., Gilliland, G.L., Armstrong, R.N., Ji, X., Board, P.G., Olin, B., Mannervik, B. and Jones, T.A. (1993) Structure determination and re¢nement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. J. Mol. Biol. 232, 192^212. [21] Kolm, R.H., Sroga, G.E. and Mannervik, B. (1992). Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1. Biochem. J. 285, 537^540. [22] Reinemer, P., Prade, L., Hof, P., Neuefenind, T., Huber, R., Zettl, R., Palme, K., Schell, J., Koelln, I., Bartunik, H.D. and Bieseler, B. (1996). Three-dimensional structure of glutathione î resolution: S-transferase from Arabidopsis thaliana at 2.2 A structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J. Mol. Biol. 255, 289^309. [23] Grove, G., Zarlengo, R.P., Timmermann, K.P., Li, N.-Q., Tam, M.F. and Tu, C.-P. (1988) Characterization and heterospeci¢c expression of cDNA clones of genes in the maize GSH S-transferase multigene family. Nucleic Acid Res. 16, 425^ 438. [24] Ogura, K., Nishiyama, T., Okada, T., Kajita, J., Hiroyuki, N., Watabe, T., Hiratsuka, A. and Watabe, T. (1991) Molecular cloning and amino acid sequencing of rat liver class theta glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters of carcinogenic arylmethanols. Biochem. Biophys. Res. Commun. 181, 1294^1300.
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