©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glutathionylspermidine Metabolism in Escherichia coli
PURIFICATION, CLONING, OVERPRODUCTION, AND CHARACTERIZATION OF A BIFUNCTIONAL GLUTATHIONYLSPERMIDINE SYNTHETASE/AMIDASE (*)

(Received for publication, February 22, 1995; and in revised form, March 24, 1995 )

J. Martin BollingerJr. (1) David S. Kwon Gjalt W. Huisman (2)(§) Roberto Kolter (2) Christopher T. Walsh (1)(¶)

From the  (1) Department of Biological Chemistry and Molecular Pharmacology and the (2) Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and P(i)) with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (-Glu-Cys-Gly). In the pathogenic trypanosomatids, this reaction is the penultimate step in the biosynthesis of the antioxidant metabolite, trypanothione (N^1,N^8-bis(glutathionyl)spermidine), and is a target for drug design. In this study, GSP synthetase was purified to near homogeneity from E. coli B, the gene encoding it was isolated and sequenced, the enzyme was overexpressed and purified in quantity, and the recombinant enzyme was characterized. The 70-kDa protein was found to have an unexpected second catalytic activity, glutathionylspermidine amide bond hydrolysis. Thus, the bifunctional GSP synthetase/amidase catalyzes opposing amide bond-forming and -cleaving reactions, with net hydrolysis of ATP. The synthetase activity is selectively abrogated by proteolytic cleavage 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg.

A series of analogs was used to probe the spermidine-binding site of the synthetase activity. The activity of diaminopropane as a substrate, inactivity of the C(4)-C(8) diaminoalkanes, and greater los s of specificity for analogs modified in the 3-aminopropyl moiety than for those modified in the 4-aminobutyl moiety indicate that the enzyme recognizes predominantly the diaminopropane portion of spermidine and corroborate N-1 (the aminopropyl N) as the site of glutathione linkage (Tabor, H. and Tabor, C. W.(1975) J. Biol. Chem. 250, 2648-2654). Trends in K and k for a set of difluoro-substituted spermidine derivatives suggest that the enzyme may bind the minor, deprotonated form of the amine nucleophile.


INTRODUCTION

The tripeptide, glutathione (-Glu-Cys-Gly, GSH), and the polyamine, spermidine (N-3-aminopropyl-1,4-diaminobutane), are present at high concentrations (0.1-10 mM) in both eukaryotic and bacterial cells (see, for example, Refs. 1, 2 for reviews). Among the many functions of GSH is detoxification of xenobiotics and reactive oxygen species, which it serves by virtue of its nucleophilic and redox-active cysteine sulfhydryl group. As an antioxidant, GSH quenches oxygen radicals (O, HO) by direct reduction and acts as a cofactor in the scavenging of peroxides via the GSH peroxidase-GSH reductase couple. The polyamines have also been ascribed a variety of roles, including charge neutralization and complexation of the anionic phosphodiester backbone of DNA. An intersection of GSH and spermidine metabolism, in the form of the amide-linked conjugate, N^1-glutathionylspermidine (see for structure), was first observed in Escherichia coli(3, 4) but has not been characterized with regard to its physiological significance. More recently, it was shown that glutathionylspermidine and the corresponding bis-amide, N^1,N^8-bis(glutathionyl)spermidine (trivial name trypanothione, see for structure), are present in the pathogenic protozoa of genera Trypanosoma and Leishmania(5, 6) . Several observations suggest that the GSH-spermidine conjugates are physiologically important to these organisms: first, the parasites appear to lack typical catalase and peroxidase hemoproteins (7, 8) , enzymes which function in oxidant defense in other organisms; second, a considerable portion of their GSH (a critical antioxidant) is conjugated with spermidine (6) ; and third, they lack the typical glutathione peroxidase-glutathione reductase enzyme couple (9) , but instead utilize an analogous system based on trypanothione (10, 11, 12) . On the basis of these observations, it has been suggested that the parasites are dependent on trypanothione metabolism for oxidant defense, and, in view of their inherent sensitivity to compounds which can induce oxidative stress (i.e. redox cyclers), that trypanothione metabolism might be a fruitful target for new antiparasitic drugs (5) . Indeed, several existing drugs (e.g. organic arsenicals, nitrofurans, and difluoromethylornithine) have been proposed to exert their trypanocidal activity by interfering with some aspect of trypanothione metabolism (5, 13-16).

Earlier work in our laboratory was directed toward molecular characterization of trypanothione reductase and identified this enzyme as a new target for antitrypanosomal drug design (10, 17, 18, 19, 20, 21) . More recently, we have turned our attention to the ATP-hydrolyzing, amide-bond-forming (ATP-amide) enzymes, glutathionylspermidine synthetase (GSP^1(^1) synthetase), and trypanothione synthetase (T(SH)(2) synthetase), which catalyze the biosynthesis of trypanothione from glutathione and spermidine () (22, 23) . We purified both synthetases to homogeneity in minute quantities from the insect trypanosomatid Crithidia fasciculata(23) , but to date have not succeeded in isolating the genes that encode them. As noted above, the intermediate in trypanothione synthesis, glutathionylspermidine, was first identified in E. coli(3, 4) , and a GSP synthetase activity was partially purified (24, 25) . With the aim of understanding in detail the () bacterial and parasitic enzymes that conjugate GSH and spermidine, we have now purified E. coli GSP synthetase to near homogeneity, isolated and determined the sequence of the gene which encodes it, overproduced it in quantities sufficient for characterization, and used a series of analogs to examine features of the spermidine binding site. In addition, we have shown that the 70-kDa protein surprisingly possesses an opposing GSP amidase activity and that the two reactions are most likely catalyzed at distinct active sites.


EXPERIMENTAL PROCEDURES

General Materials

The ordered library of the E. coli genome (26) was kindly provided by Prof. Kohara. Oligonucleotides were purchased from Oligos, Etc. (Guilford, CT) or from the Biological Chemistry and Molecular Pharmacology departmental biopolymer facility. Restriction endonucleases and DNA ligase were purchased from New England Biolabs. NADH, phospho(enol)pyruvate, trypsin, leupeptin, and dithiothreitol were purchased from Sigma. Pyruvate kinase and lactate dehydrogenase (an equi-unit suspension in 3.2 M ammonium sulfate) were purchased from Boehringer Mannheim. Protein native molecular weight standards for size exclusion chromatography were purchased from Pharmacia LKB Biotechnol.

GSP Synthetase/Amidase Substrates and Analogs

ATP, glutathione, and spermidine were purchased from Sigma. AMP-PNP was purchased from Boehringer Mannheim. [S]Glutathione (5 times 10^5 Ci/mol) was purchased from New England Nuclear. N^1-glutathionylspermidine disulfide was purchased from Bachem. The C(3)-C(8) diaminoalkanes were purchased from Sigma. Bis-(3-aminopropyl)amine was purchased from Fluka. Other spermidine analogs were generously provided by the following: 2,2-, 6,6-, and 7,7-difluorospermidines by Marion Merrell Dow; N-(3-aminopropyl)-1,5-diaminopentane, N-(2-cyanoethyl)-1,4-diaminobutane, and N-(3-cyanopropyl)-1,3-diaminopropane by Dr. J. Folk at the National Institutes of Health; and bis-(4-aminobutyl)amine by Prof. T. Oshima at the Tokyo Institute of Technology.

[S]Glutathionylspermidine was prepared enzymatically from [S]GSH by using pure, recombinant GSP synthetase. The reaction contained in a final volume of 1.76 ml: 39 µM [S]GSH (1.5 Ci/mmol), 2.0 mM spermidine, 2.0 mM ATP, 2.6 mM MgCl(2), 5.1 mM dithiothreitol, 41 mM Na-PIPES buffer, pH 6.8, and 3.1 µM GSP synthetase. The reaction was allowed to proceed at 37 °C for 11 min and was quenched by a 2.5-min incubation at 90 °C. The solution was centrifuged briefly to pellet denatured protein and was loaded onto a 1.5-ml SP-Sepharose (Pharmacia) column (NH(4) counter ion) equilibrated in H(2)O. The column was washed with 2 times 1 ml aliquots of 40 mM Na-PIPES, pH 6.8, containing 4 mM EDTA and 4 mM dithiothreitol (to wash through [S]GSH, which does not bind to the column, and to elute bound Mg), followed by 3 times 1 ml of 150 mM ammonium acetate, pH 5.2. Glutathionylspermidine was then eluted with 4 times 1 ml of 300 mM ammonium acetate, pH 5.2 (spermidine elutes at >500 mM ammonium acetate). Fractions 2 and 3 of the 300 mM ammonium acetate wash, which contained >98% of the glutathionylspermidine and 79% of the total radioactivity were evaporated to dryness in vacuo. The material was redissolved in 250 µl of 10 mM dithiothreitol. Analysis by thin layer electrophoresis on a cellulose plate (as described below for the radiometric GSP synthetase assay) revealed that >90% of the isolated radioactivity co-migrated with authentic glutathionylspermidine, and could be converted by treatment with GSP synthetase/amidase to a species which co-migrated with GSH.

Protein Assays

Protein concentrations were determined by the method of Bradford (27) , with bovine serum albumin as standard.

Enzyme Activity Assays: Radiometric GSP Synthetase Assay

During purification of the enzyme from E. coli B and for analysis of dilute enzyme samples, GSP synthetase activity was determined by a radiometric assay employing [S]GSH as labeled substrate. Reactions were carried out at 37 °C in 50 mM Na-HEPES at pH 7.5 (adjusted at room temperature) in a final volume of 20-50 µl. Assays contained 2 mM ATP, 5 mM MgCl(2), 10 mM spermidine, 50-100 µM [S]GSH (15-40 Ci/mol), and 5 mM dithiothreitol. Reactions were initiated by addition of the enzyme (cell extract or column fraction), allowed to proceed for the desired time (generally 30-120 min), and terminated by incubation at >90 °C for 2-3 min. Dithiothreitol and unlabeled GSH and glutathionylspermidine disulfide were added to final concentrations of 50, 5, and 1.6 mM, respectively, and the solutions were incubated at >90 °C for 2-3 min to ensure complete reduction of GSH and glutathionylspermidine. Assay solutions were analyzed by thin layer electrophoresis. Aliquots (3-4 µl) were spotted along the center of a 20 times 20-cm cellulose thin layer chromatography plate (Kodak), which was subjected to electrophoresis for 20-30 min at 1000-1500 V in 50 mM Na-MES buffer, pH 6.5, containing 1 mM dithiothreitol. The plate was dried and stained with ninhydrin, and the spots corresponding to GSH (migrating toward the anode) and glutathionylspermidine (toward the cathode) were cut out and counted with 7 ml of scintillant.

Spectrophotometric GSP Synthetase Assay

Activity measurements on partially purified wild-type and recombinant GSP synthetase and kinetic analysis (K and k determination) and substrate specificity studies on the pure recombinant enzyme were carried out by using a continuous spectrophotometric assay, in which ATP hydrolysis is coupled to oxidation of NADH through the activities of pyruvate kinase and lactate dehydrogenase. Assays were carried out at 37 °C in 50 mM Na-PIPES, pH 6.8, in a final volume of 400 µl. They contained 0.19 mM NADH, 1 mM phospho(enol)pyruvate, 5 mM dithiothreitol, 15 µg of pyruvate kinase, 5 µg of lactate dehydrogenase, 40 mM (NH(4))(2)SO(4), 2-20 µg of GSP synthetase, and varying amounts of ATP, GSH, and spermidine. When saturating concentrations of ATP, GSH, and spermidine were desired, their concentrations were 2 (2.5 mM MgCl(2)), 10, and 10 mM, respectively. The K value for each substrate/analog was determined with saturating concentrations of the other substrates. For determination of the pH optimum for k, a mixed buffer system containing 20 mM each of Bicine, MES, Bis-Tris Propane, and HEPES (adjusted to the desired pH with HCl or NaOH) was used. At pH 8.0 and 6.0 (above and below the observed optimum of 6.8), it was verified that the activities of the coupling enzymes were not rate-limiting.

Radiometric GSP Amidase Assay

Reactions were carried out at 37 °C in 50 mM Na-HEPES, pH 7.5, in a final volume of 20-50 µl. Routine assays contained 4 mM [S]glutathionylspermidine (1-2 Ci/mol) and 15-20 mM dithiothreitol (from prereduction of glutathionylspermidine disulfide). Reactions were initiated by addition of the enzyme, incubated for the desired time (typically 10-30 min), and quenched by heating as described above for the radiometric synthetase assay. Analysis was carried out by thin layer electrophoresis as described for the synthetase assay.

Partial Purification of GSP Synthetase

GSP synthetase was purified from E. coli B grown in LB at 37 °C and harvested in late log phase by centrifugation. Wet cell paste was frozen in liquid N(2) and stored at -80 °C until use.

All steps in the purification were performed at 4 °C. All buffers contained (in addition to the buffering component) 1 mM EDTA and 5 mM dithiothreitol, and the pH of each was adjusted at room temperature. In the most successful purification, 20 g of cell paste was resuspended in 55 ml of 50 mm Na-HEPES, pH 7.5 (buffer A). Phenylmethylsulfonyl fluoride, benzamidine, and trypsin inhibitor were added to concentrations of 500 µM, 6 mM, and 0.04 mg/ml, respectively. The cells were lysed by passage through a French pressure cell at 14,000-16,000 pounds/square inch. The lysate was centrifuged at 17,000 times g for 20 min, and the supernatant was brought to 1% (w/v) in streptomycin sulfate by dropwise addition of a 6% solution in buffer A with stirring. The solution was centrifuged at 17,000 times g for 20 min, and the supernatant was brought to 33% of saturation in ammonium sulfate by addition of the solid (0.18 g/ml) with stirring. This solution was centrifuged at 17,000 times g for 20 min, and the supernatant was brought to 65% of saturation in ammonium sulfate by addition of the solid (0.17 g/ml) with stirring. The sample was centrifuged at 17,000 times g for 20 min, and the pellet was redissolved in 10 ml of buffer A. This solution was centrifuged briefly at 17,000 times g to pellet undissolved material and then was desalted through a 2.6 times 26-cm Sephadex G-25 column equilibrated in 50 mM Bis-Tris propane-HCl, pH 7.15 (buffer B). The fractions containing protein (116 ml) were pooled and loaded onto a 2.6 times 20-cm DEAE-Sepharose column equilibrated in buffer B. The column was developed with a 50-ml gradient of 0-160 mM NaCl in buffer B, then with a 300-ml gradient of 160-400 mM NaCl in buffer B. Fractions containing GSP synthetase activity (47 ml eluting at 170-210 mM NaCl) were pooled. The pool was dialyzed against buffer B, then loaded onto a mono-Q HR 10/10 column (Pharmacia), equilibrated in buffer B. The column was developed with a 20-ml linear gradient of 0-180 mM NaCl in buffer B, then with a 100-ml linear gradient of 180-360 mM NaCl in buffer B, and fractions containing enzyme activity (10 ml eluting at 150-190 mM NaCl) were pooled. At this stage, an attempt was made to bind the enzyme to Mono-S resin, as follows. The Mono-Q pool was dialyzed against 25 mM Na-MES buffer, pH 6.35 (buffer C). The pool was then injected onto a Mono-S HR 10/10 column (Pharmacia) equilibrated in buffer C, and the column was washed with buffer C prior to development with a NaCl gradient. All enzyme activity was found in the wash fractions, and these were pooled and loaded onto a 2.6 times 12-cm hydroxylapatite column equilibrated in 20 mM potassium phosphate buffer, pH 6.8 (buffer D). The column was developed with a 20-ml gradient of 20-68 mM potassium P(i), then with a 300-ml gradient of 68-212 mM potassium P(i). Fractions containing activity (60 ml eluting at 130-160 mM P(i)) were pooled, and the pool was concentrated to 15 ml in a Centriprep 30 concentrator (Amicon). The pool was diluted with H(2)O to 30 ml, and 5.2 g of solid ammonium sulfate was added over several minutes with stirring. This solution was centrifuged at 2,000 times g for 10 min and then loaded onto a phenyl-Superose HR 10/10 column (Pharmacia) equilibrated in buffer D containing 1.2 M ammonium sulfate. The column was developed with a 30-ml linear gradient of 1.2-0.78 M ammonium sulfate in buffer D, then with a 100-ml linear gradient of 0.78-0.36 M ammonium sulfate in buffer D. Fractions containing activity (9 ml eluting at 0.64-0.60 M ammonium sulfate) were pooled and dialyzed against buffer C. This solution was loaded onto a Mono-S HR 5/5 column, which was developed with a 2-ml linear gradient of 0-100 mM NaCl in buffer C followed by a 40-ml linear gradient of 100-500 mM NaCl in buffer C. Fractions containing enzyme activity (5 ml eluting at 210-260 mM NaCl) were pooled, and the pool was concentrated to 0.25 ml. This sample was analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on a 7.5% gel, and the gel was electroblotted to a polyvinylidene fluoride membrane. The two bands detected by Ponceau S staining of the membrane (70 and 90 kDa) were excised and submitted to the Harvard Microchemistry Facility for endoproteinase lys C (which proteolyzes after Lys residues) digestion and sequence determination of proteolytic peptides.

Isolation and Mapping of gsp, the Gene Encoding GSP Synthetase/Amidase

From the sequence of one endoproteinase lys C peptide from the 70 kDa band (DPLQNAYVQANGQVINQDPYHYYTITESA), two degenerate oligonucleotides were designed (probe 1: 5`-CC(A,C,T,G)-TA(C,T)-CA(C,T)-TA(C,T)-TA(C,T)-AC-3`, and probe 2: 5`-CA(A,G)-GCI-AA(C,T)-GGI-CA(A,G)-GTI-ATI-AA(C,T)-CA(A,G)-GA-3`, where I is inosine). These were used as hybridization probes against the ordered library of the E. coli genome (26) . Labeling and detection of the probe were accomplished with the Amersham ECL 3`-Oligolabeling and Detection System, according to the manufacturer's instructions. For probe 1, hybridization was carried out for 24 h at 37 °C in the recommended buffer. Washing was carried out at 37 °C in 1 times SSC (150 mM NaCl, 15 mM sodium citrate, pH 7.0) containing 0.1% (w/v) SDS. For probe 2 hybridization was carried out for 6 h at 44 °C in 1 times SSC, 0.02% SDS, with the recommended concentrations of blocking agent and hybridization buffer component. Washing was carried out at 50 °C in 1 times SSC, 0.1% SDS.

DNA from the Kohara -phage clones (26) was isolated from infected cultures of E. coli strain ZK126 (28) by using a kit from Qiagen and following the manufacturer's instructions.

DNA Sequencing

Sequencing of double-stranded DNA was accomplished by the dideoxynucleotide chain termination method (29) as described in the Sequenase kit from United States Biochemical Corp.

Construction of Plasmid for Overexpression of GSP Synthetase

A standard expression casette polymerase chain reaction approach (30) was employed to generate a vector to overproduce GSP synthetase. Primer 1 (5`-AAGGTAAACATATGAGCAAAGGAACGACCAG-CCAGGATGC-3`) was designed to anneal to the antisense strand at the 5` end of gsp and included an NdeI site (CAT-ATG) immediately 5` of the initiator ATG. Primer 2 (5`-ATATTGAATTCTTTGATTAATCCCCGTACTGATTATTC-3`) was designed to anneal to the sense strand 3` of gsp and included an EcoRI site. A portion of the 1.9-kilobase polymerase chain reaction fragment was digested with NdeI and BamHI, and a second portion was digested with BamHI and EcoRI. The resulting 740-base pair NdeI-BamHI fragment (the 5` end of gsp) and 1180-base pair BamHI-EcoRI fragment (the 3` end of gsp) were circularized in a three-piece ligation with NdeI-EcoRI-digested pET22b (Novagen) to give pGSP, which contains gsp under the control of the T7 RNA polymerase promoter. Transformation of E. coli strain BL21DE3 (Novagen), which contains in its chromosome the T7 RNA polymerase gene behind the pLAC promoter, with pGSP gave the desired overexpressing strain (BL21DE3/pGSP). The sequence of gsp in pGSP was verified to confirm that no mutations were introduced during polymerase chain reaction amplification.

Purification of GSP Synthetase from Overexpressing Strain

BL21DE3/pGSP was grown at 37 °C with vigorous aeration in LB containing 150 µg/ml ampicillin. When cultures reached an OD of 0.5, IPTG was added to a concentration of 0.5 mM to induce expression of gsp. Cultures were grown an additional 3-5 h (to early stationary phase) and then harvested by centrifugation. A typical yield was 1.5 g of wet cell paste/liter of culture. The cell paste was frozen in liquid N(2) and stored at -80 °C until use.

Purification of GSP synthetase from BL21DE3/pGSP cell paste was accomplished by a procedure modified from that used to purify the enzyme from E. coli B. Lysis of the cells and streptomycin sulfate and ammonium sulfate precipitation steps were carried out as described, except protease inhibitors (phenylmethylsulfonyl fluoride, benzamidine, and trypsin inhibitor) were not added to the lysis buffer. Desalting following ammonium sulfate precipitation was accomplished by dialysis against buffer B. DEAE-Sepharose chromatography was as described. Following this step, ammonium sulfate was added to the pool to a concentration of 1.2 M. This solution was loaded on the phenyl-Superose HR 10/10 column, which was developed as described. Fractions of highest specific activity were pooled. At this stage, GSP synthetase was estimated by SDS-PAGE to be 95% pure. When greater purity was desired, a portion of the phenyl-Superose pool was dialyzed against buffer B. This solution was loaded onto a Mono-Q HR 5/5 column (Pharmacia) equilibrated in buffer B. The column was developed with a 10-ml linear gradient of 0-100 mM NaCl in buffer B, then with a 45-ml gradient of 100-250 mM NaCl in buffer B. Fractions with highest specific activity were pooled.

Limited Trypsin Proteolysis of GSP Synthetase

In a typical experiment, a solution containing 3.2 mg/ml GSP synthetase, 0.025 mg/ml trypsin, 15 mM potassium P(i), pH 6.8, 0.75 mM EDTA, 3.7 mM dithiothreitol, and 450 mM ammonium sulfate (from phenyl-Superose chromatography) was incubated at 37 °C. In experiments to determine time courses of proteolysis and loss of GSP synthetase activity, aliquots (33 µl) were withdrawn at various times following addition of trypsin, and proteolysis was inhibited by addition of 0.11 volumes (3.7 µl) of 1 mM leupeptin (100 µM final concentration). Each aliquot was diluted 6-fold by addition of 50 mM Na-PIPES, pH 6.8, containing 1 mM EDTA and 5 mM dithiothreitol, and each was analyzed for synthetase activity by the spectrophotometric assay (described above) and for extent of proteolysis by SDS-PAGE on a 15% gel. In substrate protection experiments, MgCl(2), ATP, GSH, and/or spermidine were included, either individually or in combination, at concentrations of 2.5, 0.5, 4, and 0.3 mM, respectively (5 K ), and proteolysis was allowed to proceed at 37 °C for 30 min prior to quenching with leupeptin. In an experiment to determine the site of trypsin cleavage, a sample of GSP synthetase/amidase was digested to completion, the trypsinized protein and the native protein were subjected to SDS-PAGE on a 7.5% gel, and the gel was blotted to polyvinylidene fluoride membrane. The protein bands, visualized by Ponceau S staining, were excised and submitted to the Dana Farber Cancer Institute Core Facility (DFCICF) for N-terminal sequence analysis. Solution samples of the trypsinized and native proteins were also submitted to DFCICF for laser desorption mass spectral analysis.

Chromatographic Separation of Native and Trypsin-cleaved GSP Synthetase/Amidase

Native and trypsin-cleaved GSP synthetase/amidase were chromatographed separately on Mono-Q, as follows. For native GSP synthetase, 2 mg of protein in 20 mM potassium P(i), pH 6.8, containing 5 mM dithiothreitol was injected on a Mono-Q HR 5/5 column equilibrated with buffer B, and the column was developed with a 5-ml linear gradient of 0-120 mM NaCl in buffer B, then with a 30-ml linear gradient of 120-240 mM NaCl in buffer B. Fractions were assayed for protein concentration and for GSP amidase activity as described above. Trypsin-cleaved GSP synthetase/amidase was prepared as described above and was exchanged into buffer B containing 100 µM leupeptin by four cycles of concentration-dilution in a Centricon 30 Microconcentrator (Amicon). An aliquot of the protein (containing 0.8 mg) was chromatographed on Mono-Q as described above for the native enzyme, and fractions were analyzed for protein concentration and GSP amidase activity.

To prove chromatographic resolution of the native and trypsin-cleaved proteins, a solution containing 0.5 mg of each was injected on the Mono-Q HR 5/5 column. The column was developed with a 5-ml linear gradient of 0-150 mM NaCl in buffer B, then with a 30-ml linear gradient of 150-250 mM NaCl in buffer B. Fractions were analyzed for protein concentration, amidase activity, and synthetase activity (by the spectrophotometric assay) and were characterized by SDS-PAGE.

Estimation of Native Molecular Mass of GSP Synthetase/Amidase

GSP synthetase/amidase was chromatographed on a Superose 6 10/30 column (Pharmacia) as follows. A 100-µl aliquot containing 0.2-0.5 mg of protein was injected, and protein was eluted at 0.4 ml/min with 50 mM Na-HEPES, pH 7.5, containing 5 mM dithiothreitol and 150 mM NaCl. In some injections, 1 mM CHAPS was included in the elution buffer but had no effect. Fractions containing protein were assayed for GSP amidase activity. A standard curve of elution volume versus molecular mass was constructed by using thyroglobulin (m = 669 kDa, elution volume 12.0 ml), ferritin (440 kDa, 14.1 ml), catalase (232 kDa, 15.35 ml), aldolase (158 kDa, 15.63 ml), bovine serum albumin (67 kDa, 16.27 ml), ovalbumin (43 kDa, 16.9 ml), and ribonuclease A (13.7 kDa, 18.8 ml) as standards.


RESULTS

Assay for GSP Synthetase Activity in E. coli B Extracts

Incubation of [S]GSH with spermidine, ATP, Mg, and E. coli B cell extract resulted in the time-dependent production of a radioactive species which co-migrated with authentic glutathionylspermidine during thin layer electrophoresis. Omission of spermidine, ATP, or cell extract from the reaction reduced the radioactivity in the glutathionylspermidine spot to near background levels. On the basis of these results and previous work of Tabor and Tabor (4, 24, 25) , it was concluded that the radioactive species produced from [S]GSH is glutathionylspermidine and that the thin layer electrophoresis method would provide a useful assay for purifying GSP synthetase.

Purification of Wild-type GSP Synthetase

Fractionation of E. coli B extracts by a sequence of streptomycin sulfate and ammonium sulfate precipitation steps followed by five chromatographic steps (DEAE-Sepharose, Mono-Q, hydroxyapatite, phenyl-Superose, and Mono-S) resulted in an estimated 2000-5000-fold purification of GSP synthetase activity. In the last step of the purification, Mono-S chromatography resulted in resolution of enzyme activity from the main protein peak (Fig. 1A). SDS-PAGE analysis of the pooled, active fractions (24, 25, 26, 27, 28, 29, 30) revealed a 1:1 m ixture of a 90-kDa species and a 70-kDa species (Fig. 1B). In this and other attempts at purification, a 90-kDa species was persistent and prevalent throughout the chromatographic sequence and did not correlate with GSP synthetase activity. Indeed, the main protein peak in Fig. 1A, which lacked activity, revealed predominantly a 90 kDa band when analyzed by SDS-PAGE (data not shown). It was therefore considered likely that the 70-kDa species was associated with GSP synthetase activity. From a different preparation, it was estimated that the pure 70-kDa species would have a specific activity of 4.5 units/mg, corresponding to k = 300 min, which is comp arable to the value of 600 min measured for the GSP synthetase from C. fasciculata(23) . The 70-kDa species was therefore targeted as GSP synthetase.


Figure 1: Mono-S chromatography as final step in purification of GSP synthetase from E. coli B. A, absorbance at 280 nm (filled circles) and enzyme activity (open circles) of column fractions. B, SDS-PAGE analysis of pooled fractions 24-30.



Sequence of Endoproteinase lys-C Peptides

In the preparation described, approximately 25 µg of protein (corresponding to 12.5 µg of the 70-kDa species) was obtained after concentration of the Mono-S pool. Following resolution of the 70- and 90-kDa species by SDS-PAGE and blotting to polyvinylidene fluoride, the sequences of four endoproteinase lys-C peptides from the 70-kDa species were determined. None of the peptide sequences was identical with a known protein sequence nor with one predicted by a known DNA sequence.

Isolation and Mapping of gsp, the Gene Encoding GSP Synthetase

Two oligonucleotide probes were designed from one of the above peptide sequences (see ``Experimental Procedures'' for probe sequences), and these were found to hybridize specifically to -503 in the Kohara et al. (26) miniset, which corresponds to 67.35-67.63 min on the physical map of the chromosome (Fig. 2). Digestion with the eight Kohara endonucleases and Southern blot analysis identified the 0.5-kilobase EcoRV-BglI fragment at 67.5 min as the site of hybridization of probe 2. With the expectation that a 1.8-kilobase gene would be required to encode a 70-kDa protein, the 5.8-kilobase PstI-EcoRI fragment of -503, which brackets the site of hybridization by >1.8 kilobases on either side, was excised and ligated into the multicopy vector pBluescript KS(+) to give pJMB1. Crude lysates from E. coli strain DH5alpha transformed with pJMB1 exhibited 11-fold greater GSP synthetase activity than lysates from DH5alpha transformed with pBluescript KS(+). This enhancement was interpreted as evidence that the cloned fragment contained the functional gsp gene.


Figure 2: Physical map of the region of the E. coli chromosome containing gsp (26).



Sequence of gsp

The plasmid pJMB1 was subjected to the m-1 (a Tn1000 derivative) transposon mutagenesis procedure of Berg et al.(31) . A set of mutant plasmids with insert positions spanning the 5.8-kilobase fragment of pJMB1 was identified by restriction analysis, and these plasmids were reisolated from E. coli strain DH5alpha for sequencing. By reading out from the m-1 inserts, the sequence (both strands) of a 3.2 kilobase fragment spanning gsp was determined. Within this sequence, an open reading frame of 1857 bases, coding counterclockwise on the physical map (Fig. 2), was identified. This open reading frame encodes a 619-amino-acid protein with a molecular mass of 70,539 Da (Fig. 3). Each of the four peptide sequences determined from the 70-kDa species (underlined in Fig. 3) is found in the predicted protein product. The predicted sequence has no significant similarity with any protein in the data base of known function. The sequence does, however, have significant similarity (25-28% identity) in its C-terminal 370 amino acids with two hypothetical 45-kDa E. coli proteins, YgiC (32) and YjfC.


Figure 3: Sequences of gsp and the predicted GSP synthetase/amidase protein. The dots above the nucleotide sequence mark the positions to which the nucleotide and amino acid numbering (to the left and right of the dots, respectively) refer. Peptide sequences obtained from the 70-kDa protein purified from E. coli B are underlined.



A second partial open reading frame of 651 bases was identified 300 bases downstream of gsp. The protein sequence predicted is 85% identical over 217 amino acids with the E. coli pit gene product, which is involved in low affinity phosphate transport. As pit has been mapped to 77 min of the E. coli chromosome (33) , this sequence may represent a second pit gene.

Overexpression and Purification of GSP Synthetase

An expression cassette polymerase chain reaction approach (30) was used to create a vector (pGSP) for overproduction of GSP synthetase. Growth of E. coli strain BL21DE3 transformed with pGSP in the presence of IPTG resulted in marked overproduction of a 70-kDa protein. It was estimated by SDS-PAGE that the 70-kDa species constituted 60% of the total cellular protein (Fig. 4, lane 2). GSP synthetase activity (as determined by both radiometric and coupled assays) was high in the lysates of these cells (see Table I), confirming that the functional enzyme is encoded by pGSP. Purification of GSP synthetase approximately 1.7-fold from these lysates to greater than 95% homogeneity (see ``Experimental Procedures,'' Table I, and Fig. 4) typically yielded 70 mg/liter of culture.


Figure 4: SDS-PAGE to monitor purification of recombinant GSP synthetase/amidase from overproducing strain BL21DE3/pGSP. Lanes 1 and 7, molecular weight markers; 2, total cell lysate; 3, lysate supernatant; 4, 1% streptomycin sulfate supernatant; 5, 33% saturation ammonium sulfate supernatant; 6, 66% saturation ammonium sulfate pellet after dialysis; 8, DEAE-Sepharose pool; 9, phenyl-Superose pool; 10, Mono Q pool.



Kinetic Characterization of GSP Synthetase

By using the coupled assay, the pH optimum (for k) of GSP synthetase was determined to be 6.8. Steady-state kinetic parameters were determined at this pH. For K determinations, all substrates except the variable substrate were saturating (>20K ). K values for ATP, GSH, and spermidine were found to be 100 ± 20, 800 ± 150, and 60 ± 10 µM, respectively. K for spermidine is noticeably pH dependent, decreasing from 60 µM at pH 6.8 to 20 µM at pH 7.5. The optimum k is 7 s, comparable to the 10 s observed for C. fasciculata GSP synthetase (23) .

Use of Spermidine Analogs to Characterize Binding Site of GSP Synthetase

To delineate features of the spermidine-binding site of GSP synthetase, analogs were tested by the coupled assay as substrates/inhibitors. (It should be noted that for no analog (other than spermidine itself) has formation of a glutathione-amide product yet been explicitly demonstrated. Instead, it has been assumed that ADP production in all cases correlates with amide formation.) Among the C(3)-C(8) diaminoalkanes, only diaminopropane is a good substrate (Table II), consistent with an important role in recognition for this portion of spermidine and with N-1 (see Table II I for numbering of spermidine atoms), rather than N-8, being the site of acylation (glutathionylation) (4) . None of the diaminoalkanes from C(6)-C(8) detectably inhibits at 5 mM. The surprising inactivity of diaminooctane both as substrate and inhibitor suggests that N-4 of spermidine is a crucial recognition determinant.

A series of spermidine analogs, previously used by Folk and co-workers (34) to probe the spermidine-binding site of deoxyhypusine synthase, was tested with GSP synthetase (Table II). In this series, either the 3-aminopropyl or the 4-aminobutyl substituent on the central amine (N-4) is modified. Addition-subtraction of a methylene unit to/from the 4-aminobutyl moiety (1 or 2) or conversion of the -CH(2)-NH(2) unit to a cyano- substitutent (4) has a much smaller effect on the substrates' specificity constant (k/K ) than the equivalent modification of the 3-aminopropyl moiety (compare 1-3 and 4-5). These results, especially the observation that 4 is a substrate while 5 is completely inactive, again corroborate N-1 as the site of acylation (4) .

The effects on recognition and catalysis of perturbing the pK values of the spermidine amine groups were assessed by measuring catalytic parameters for 2,2- 6,6-, and 7,7-difluorospermidine derivatives (6-8), a series of analogs previously used by the Merrell Dow group to probe the active site of spermine synthase (35) . As indicated in Table III, difluoro-substitution drastically decreases the pK value of the nearest amine group(s) (35) . With GSP synthetase, the difluorospermidine derivatives all have k values identical with that of spermidine. In contrast, K is markedly affected by difluoro-substitution. The modest (3-fold) increase observed in 8 may reflect a binding preference for the N-8-protonated species, of which the concentration in solution at pH 6.8 is reduced 2.4-fold by difluoro-substitution at position 7. Likewise, the more than 20-fold increase in K seen in 7 most likely reflects a binding preference for the N-4-protonated species, of which the concentration is 13-fold lower than for spermidine. The largest effect observed is for substitution at position 2 (6), which improves (i.e. decreases) K so that it becomes immeasurable by the coupled assay. In order to quantify this effect, K values for 6 and for spermidine were determined in 50 mM Bis-Tris propane-HCl, pH 6.8. This buffer contains a diaminopropane moiety and inhibits with respect to spermidine, thereby increasing the apparent K for 6 into the concentration range where it can be accurately measured with the coupled assay. The K values for 6 and spermidine determined in the presence of this buffer (Table III, in parentheses) indicate that difluoro-substitution at position 2 improves K 25-fold relative to spermidine. Substitution at this position decreases the pK values of both N-1 and N-4, and the effect of decreasing the N-4 pK (as observed for 7) opposes the net increase in specificity observed for 6. Thus, the data suggest that the intrinsic N-1 effect is a large increase in specificity, which in turn suggests that the enzyme may have a preference for binding the N-1-deprotonated form of spermidine. For 6, this form is present at 330-fold higher concentration than for spermidine. The aforementioned opposing N-4 effect may prevent the preference for N-1-free amine binding from being fully expressed in 6. The proposal that GSP synthetase binds the N-1-deprotonated form of spermidine is consistent with the inverse pH dependence of the spermidine K.

Discovery of GSP Amidase Activity Associated with GSP Synthetase

During characterization of pure, recombinant GSP synthetase, it was observed that less radiolabeled glutathionylspermidine (and more GSH) was detected following prolonged incubation of the enzyme with its substrates than following shorter incubations. The possibility was considered that the 70.5-kDa GSP synthetase also possesses GSP amidase activity. A qualitative assay employing thin layer electrophoresis and ninhydrin staining was used to demonstrate enzyme-dependent, catalytic hydrolysis of glutathionylspermidine to GSH and spermidine, and to examine the pH dependence of this activity (Fig. 5). A series of experiments was then undertaken to distinguish whether the observed GSP amidase activity is associated with the GSP synthetase polypeptide or arises from a contaminating species.


Figure 5: Qualitative thin layer electrophoresis and ninhydrin staining assay to demonstrate GSP amidase activity and its pH dependence. Assays were carried out in a mixed buffer system containing 25 mM each of Bicine, MES, Bis-Tris propane, and HEPES adjusted to the indicated pH with HCl or NaOH. They contained 6.2 mM glutathionylspermidine, 20 mM dithiothreitol, and 7 µM GSP synthetase/amidase. Analysis was carried as described under ``Experimental Procedures.''



Preliminary Kinetic Characterization of GSP Amidase Activity

By using [S]glutathionylspermidine as substrate and thin layer electrophoresis for analysis, k and K for the amidase activity at pH 7.5 were found to be 2.1 ± 0.5 s and 0.9 ± 0.2 mM. Based on the measured k and the estimated purity of the GSP synthetase (>98%), it was estimated that a contaminant would require a k in excess of 100 s to account for the activity.

Limited Trypsin Proteolysis of GSP Synthetase

Conditions were sought for proteolysis of the protein into resolvable synthetase and amidase activities. Treatment with trypsin in the presence of 240-450 mM ammonium sulfate resulted in rapid cleavage of the 70.5-kDa protein into a soluble, metastable species of approximately 60 kDa (Fig. 6A). (The ammonium sulfate was found to be important, as trypsin treatment in its absence gave a completely different cleavage pattern and resulted in insoluble fragments.) A 10-kDa fragment corresponding to the portion of the native enzyme that is removed was not detected, presumably because it is further proteolyzed. Loss of the 70.5-kDa native protein correlated with loss of GSP synthetase activity (Fig. 6B). A qualitative assay (thin layer electrophoresis with ninhydrin staining) after complete conversion to the 60-kDa species indicated that GSP amidase activity was unaffected by trypsin cleavage of the protein (data not shown).


Figure 6: Trypsin proteolysis of native GSP synthetase/amidase into a stable 60- kDa species devoid of synthetase activity. A, time course of cleavage monitored by SDS-PAGE. B, time course of loss of GSP synthetase activity.



Determination of Trypsin Cleavage Site

The position of trypsin cleavage which results in the metastable 60-kDa species was defined. The N-terminal sequences of native and 60-kDa species were determined and found to be identical (SKGTTS). This sequence is the predicted N terminus of the native protein, after the initiator Met is removed post-translationally (Fig. 3). A minor (10%) sequence (XDAPFG) was detected for the native protein, which corresponds to cleavage of the N-terminal 7 amino acids from the translated protein. The identity of the N termini of native and trypsin-treated species indicates that trypsin cleavage to generate the 60-kDa species occurs near the C terminus of the native protein. To more precisely define the site, laser desorption mass spectra of the two species were obtained. The molecular mass of 70,846 Da measured for the native protein agrees well (0.6% error) with the calculated value of 70,408 Da. The molecular mass of the trypsin-treated species was found to be 61,606 Da. Two Arg or Lys residues were identified which might represent the site of trypsin cleavage: proteolysis after Lys would give a protein of molecular mass 60,913 Da, while cleavage after Arg would give a protein of molecular mass 61,409. The latter agrees more closely with the experimental value (0.3% compared to 1.1% error); therefore, Arg is tentatively assigned as the C terminus of the trypsin-inactivated GSP synthetase.

Chromatographic Separation of Native and Trypsinized GSP Synthetase

The 61.6-kDa tryptic fragment of GSP synthetase (hereafter designated S2-R538) has a predicted pI of 4.98 compared to 5.09 for the native protein. Resolution of the two species by anion-exchange (Mono-Q) chromatography was therefore attempted. In agreement with the prediction, S2-R538 eluted from the Mono-Q column at slightly higher NaCl concentration (Fig. 7A, filled squares) than the native protein (filled circles) when the species were chromatographed separately. In each case, GSP amidase activity coeluted with the protein (open squares and circles). To prove that the differential elution of native and S2-R538 species seen in Fig. 7A is real, the two species were coinjected (Fig. 7B). Resolution of the proteins was observed in the chromatogram (filled circles, dashed line) and by SDS-PAGE analysis of column fractions (inset below x axis). As expected, GSP synthetase activity (open circles, solid line) was observed only in fractions containing the native protein. In contrast, GSP amidase activity (open squares, dotted line) exhibited peaks corresponding with both native and S2-R538 species. The proteolysis of GSP synthetase into a chromatographically separable form and the coelution of both forms with GSP amidase activity is compelling evidence that GSP synthetase also catalyzes glutathionylspermidine hydrolysis. The fact that the synthetase activity can be selectively abrogated by proteolysis suggests that the enzyme possesses distinct sites for the two activities.


Figure 7: Mono-Q chromatography (as described under ``Experimental Procedures'') of native and trypsin-cleaved GSP synthetase/amidase. A, superimposed chromatograms from separate injections of species: protein concentration (filled symbols and solid lines) and GSP amidase activity (open symbols and dashed lines) for native protein (circles) and trypsin-cleaved protein (squares). The values for the trypsin-cleaved protein have been multiplied by 2.5 to normalize for the amount of protein injected. B, chromatogram from coinjection of species in 1:1 ratio: protein concentration (filled circles and dashed line), GSP synthetase activity (open circles and solid line) and GSP amidase activity (open squares and dotted line). The inset below the x axis shows SDS-PAGE analysis of the fractions.



Estimation of Native Molecular Mass of GSP Synthetase

To provide further evidence for the bifunctionality of the GSP synthetase polypeptide, the native molecular masses of the protein and of the species responsible for the observed amidase activity were estimated by size exclusion (Superose 6) chromatography. Comparison of the elution volume for GSP synthetase with that of standard proteins gave a molecular mass of 170 ± 20 kDa, which implies that the native protein is a dimer or trimer. (The former is more likely, as the deviation of the estimated molecular mass from the 141 kDa of a dimer might result from the dimer's having a non-globular shape.) GSP amidase activity coeluted with the protein, consistent with the proposal that the enzyme is bifunctional.

Assay for Coinduction of GSP Amidase with GSP Synthetase

A final test of the bifunctionality hypothesis involved growing cultures of the overexpressing strain, BL21DE3/pGSP, in the presence and absence of the inducing agent, IPTG, and assaying for coinduction of GSP amidase activity with GSP synthetase. Dialyzed cell extract from an induced culture (IPTG added to 1 mM at OD = 1, followed by 3 h of additional growth at 37 °C) was found to have 21-fold greater GSP synthetase activity (per milligram of protein) and 39-fold greater GSP amidase activity than dialyzed extract from an otherwise identical, uninduced culture. This coinduction of GSP amidase activity with GSP synthetase, together with the proteolysis and chromatography results, demonstrate that the 70.4-kDa protein is a bifunctional GSP synthetase/amidase.

Substrate Protection against Trypsin Cleavage

The rapid trypsin cleavage of GSP synthetase into a relatively stable 61.6-kDa fragment suggested a potential probe for protein conformational changes induced by substrate binding. Among ATP-amide enzymes, precedent exists for protection against proteolysis at a labile site by binding of substrates or inhibitors (36, 37) . Combinations of GSP synthetase substrates were therefore tested for their ability to inhibit proteolysis of this ATP-amide enzyme (Fig. 8). The presence of Mg, ATP, and GSH, with or without spermidine, markedly protects the enzyme from cleavage by trypsin (lanes 9 and 13). In both cases, Mg is required for protection (compare to lanes 8 and 12). ATP and Mg in the absence of GSH protect to a much lesser extent (compare lanes 3 and 9). In combination with GSH and Mg, the non-hydrolyzable ATP analog AMP-PNP protects to a much lesser extent than does ATP (compare lanes 9 and 11), and protection by this analog is GSH-independent (compare lanes 7 and 11).


Figure 8: Test for protection against trypsin cleavage of GSP synthetase by substrates and/or substrate analogs. Proteolysis was carried at as described under ``Experimental Procedures,'' with the indicated substrates and/or analogs included. The designation - or + above the gel indicates the absence or presence of 2.5 mM MgCl(2).




DISCUSSION

Catalytic Properties of Recombinant GSP Synthetase

The overexpression of E. coli GSP synthetase has allowed this homolog of the trypanosomatid drug target to be purified in quantities sufficient for characterization. The size, pH optimum, and k of the E. coli enzyme are quite similar to those reported for the C. fasciculata homolog (23), consistent with the anticipated similarity of the enzymes.

As an initial step in molecular characterization of GSP synthetase, the nature of the spermidine-binding site was probed. The enzyme appears to recognize predominantly, though not exclusively, the diaminopropane moiety of spermidine. The C(3) diaminoalkane itself is a substrate (albeit with elevated K ), and various modifications of the 4-aminobutyl substituent are tolerated. Conversely, modifications of the 3-aminopropyl substitutent abrogate activity (except in the case of bis(4-aminobutyl)amine). These results are consistent with the conclusion of Tabor and Tabor (4) that glutathionylation occurs predominantly at N-1, although the activity of bis(4-aminobutyl)amine raises the possibility that modification of N-8 can also occur. Interestingly, spermine (N,N`-bis(3-aminopropyl)-1,4-diaminobutane) is a good substrate (K<20 mM, k 6 s), and the ratio of moles of NADH consumed in the coupled assay/mole of spermine added suggests that both aminopropyl substituents are glutathionylated.

Results with the difluorospermidine derivatives give insight into the charge complementarity of the spermidine-binding site. Not surprisingly, it appears that protonation of N-4 and N-8 is favored for binding. In contrast, the simplest interpretation of the improved K of 2,2-difluorospermidine is that the enzyme has a preference for binding the N-1-deprotonated species. Given the evidence that N-1 is the site of glutathionylation (4) , this observation is of mechanistic interest. If GSP synthetase is, as expected, mechanistically similar to other ATP-amide enzymes such as glutamine synthetase (38, 39) , glutathione synthetase (40) , and D-ala:D-ala ligase (41) , N-1 acts as nucleophile to attack a glutathionylphosphate intermediate (). One might expect that the enzyme would possess an active site base to deprotonate the ammonium form of N-1. In contrast to this expectation, the data suggest that the enzyme selects from solution the minor N-1-free amine species. This tentative conclusion is reminiscent of work on glutamine synthetase (42) and D-alanine:D-alanine ligase (43), for which binding of the free amine nucleophile has also been proposed. Thus, this catalytic strategy may prove to be a general feature of ATP-amide enzymes ().

The proteolysis and substrate protection experiments also provide some mechanistic insight. In both D-ala:D-ala ligase (37, 44) and glutathione synthetase (36, 45, 46) , a flexible -loop provides a labile site for proteolysis in the unliganded protein (47) . Addition of the proper combination of substrates and/or inhibitors partially protects this site (36, 37) , presumably by causing the loop to close over the active site, as seen in the crystal structure of D-ala:D-ala ligase inhibited by a phospinate substrate analog (44) . Similarly, certain combinations of GSP synthetase substrates inhibit its trypsin cleavage. Maximum protection is observed only when ATP, Mg, and GSH are present, under which conditions the first step of the anticipated catalytic mechanism, formation of the glutathionylphosphate intermediate, is possible. It is likely that, as in the other ATP-amide enzymes, protection from proteolysis results from closing of a flexible loop over the active site to protect the reactive acylphosphate intermediate from hydrolysis.

Discovery and Implications of Bifunctionality

A puzzle was initially posed by the relatively high molecular mass of GSP synthetase. Although the enzyme is smaller than its C. fasciculata homologs (reported molecular masses for GSP synthetase and T(SH)(2) synthetase are 90 and 82 kDa, respectively (23)), it is considerably larger than the mechanistically cognate 30-35-kDa glutathione synthetase and D-ala:D-ala ligase. The three-dimensional structures of these ATP-amide enzymes have recently been solved (44, 45) , and, despite little sequence similarity, their folds are nearly identical (47) . These structures may thus define the fold of a bacterial ATP-amide module, and it was not immediately apparent why the GSH-spermidine coupling enzymes should require more than twice the number of amino acids as this module. A solution to this puzzle is suggested by our discovery that E. coli GSP synthetase possesses a second catalytic activity, namely, glutathionylspermidine hydrolysis. This observation leads to the current hypothesis that the protein has two distinct domains/active sites, with the amidase domain in the N terminus and the synthetase domain in the C terminus. In support of this hypothesis, cleavage 81 amino acids from the C terminus abolishes synthetase but not amidase activity. Expression of each domain as a separate polypeptide with a single activity (currently being attempted) would provide proof of distinct active sites.

The presence of two domains in GSP synthetase/amidase may resolve the puzzle of its high molecular mass, but it raises a second question: why has the protein evolved with opposing catalytic activities? Precedent exists for a bifunctional enzyme with opposing activities. In the case of the glycolytic enzyme, 6-phophofructo-2-kinase/fructose-2,6-bisphosphatase, the activities are reciprocally regulated by protein phosphorylation at a serine residue (48). The human ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (CD38) synthesizes and degrades cyclic ADP-ribose (49) , which serves as a signaling molecule in pancreatic islets to induce the cells to secrete insulin in response to glucose (50) . In this case, the hydrolase activity is inhibited by ATP (a product of glycolysis) providing a mode for differential regulation (49) . In addition, the opposing activities in this case may provide a mechanism to terminate the signal.

These two precedents suggest that a key feature of GSP synthetase/amidase may be differential regulation of its activities. One factor which might effect regulation is the intracellular pH: the pH optimum of the synthetase activity is 6.8, while our preliminary data suggest that the pH optimum of the amidase activity is higher (see Fig. 5). Indeed, Tabor and Tabor (51) reported that the accumulation of glutathionylspermidine in stationary cultures of E. coli B is favored by a lower medium pH.

With regard to the function of glutathionylspermidine metabolism in E. coli, the discovery of linked synthesizing and degrading activities emphasizes possible regulatory roles (e.g. modulation of levels of free spermidine and/or GSH in response to growth conditions, sparing of GSH and/or spermidine from degradation). It is clear that glutathionylspermidine metabolism is not essential, as mutants lacking either GSH or spermidine grow on minimal media (52-55). Nevertheless, the metabolism may be beneficial under conditions of environmental stress. To test this possibility, the phenotypes of gsp mutants exposed to different forms of stress (oxidant, osmotic, and nutrient) will need to be examined. Identification of conditions under which glutathionylspermidine metabolism confers a selective advantage would provide a clue as to the function of glutathionylspermidine and might allow the trypanoso-matid homologs of E. coli GSP synthetase to be cloned by complementation.

Whatever the function of this metabolism, the presence of both activities in GSP synthetase/amidase implies that the growth phase-dependent redistribution among levels of glutathionylspermidine, spermidine, and GSH observed by Tabor and Tabor (51) may be effected by a single protein. Redistribution among trypanothione, glutathionylspermidine, GSH, and spermidine has also been observed in C. fasciculata grown in culture and has been proposed as a mechanism of growth regulation in trypanosomatids (56). Given these observations and the high molecular masses of C. fasciculata GSP and T(SH)(2) synthetases (90 and 82 kDa), it is tempting to speculate that the trypanosomatid enzymes may also have both amidase and synthetase activities and that they may effect the observed growth phase-dependent redistribution among the various glutathione-spermidine conjugates.

  
Table: Summary of purification of GSP synthetase/amidase from BL21-DE3/pGSP

The reason for the apparent loss and regain of activity in the streptomycin sulfate step is not known.


  
Table: Steady-state kinetic constants for spermidine analogs

In 1-5, the portion of the molecule that is modified from spermidine is boxed. The designations + and - indicate whether the compound detectably inhibits at a concentration of 5 mM, and nd means not determined.


  
Table: Steady-state kinetic parameters and pK values (35) for difluorospermidines

The values in parentheses were determined in Bis-Tris propane buffer, which inhibits with respect to spermidine, thereby increasing the apparent K of (6) into the range where it can be measured accurately with the coupled assay.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM20011 (to C. T. W.), National Science Foundation Grant DMB 9207323 (to R. K.), National Institutes of Health Post-doctoral Award GM15477 (to J. M. B.), a grant from the Lucille P. Markey Charitable Trust (to D. S. K.), and a grant from the Medical Foundation, Inc. of Boston (to G. W. H.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U23148.

§
Present address: Metabolix, Inc., Cambridge, MA 02139.

To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1715; Fax: 617-432-2452.

(^1)
The abbreviations used are: GSP, glutathionylspermidine; T(SH)(2), trypanothione; ATP-amide, ATP-hydrolyzing, amide bond-forming; AMP-PNP, adenylyl-imidodiphosphate; PIPES, piperazine-N,N`-bis(2-ethanesulfonic acid); MES, 2-(N-morpholino)ethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; Bis-Tris propane, 1,3-bis[tris-(hydroxymethyl); PAGE, polyacrylamide gel electrophoresis; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Jack Folk, Tairo Oshima, and Marion Merrell Dow for gifts of spermidine analogs, and Elaine Joseph for technical assistance.


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