Vol. 296, No. 1, July, pp. 81-87, 1992
Characteristics of NADPH-Dependent Thymidylate Synthetase Purified from Streptomyces aureofaciens Marta Gaov6,*,’
Peter Racay,? and Marta Koll6rov6t
*Slovak Academy of Sciences, Institute of Molecular Biology, Dribravsti cesta 21, 842 51 Bratisluva and TDepartment of Biochemistry, Faculty of Natural Sciences of Komensky University, 842 15 Bratislava, Mlynskb Dolina CH-1, Czechoslovakia
Received September 26, 1991, and in revised form January
thymidylate synthetase from Streptomyces aureofaciene has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, K,,, values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 PM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. azweofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity -a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaeiens thymidylate synthetase show that it is a monofunctional bacterial enzyme. 0 1992 Academic Press, Inc.
Synthesis of deoxyribonucleotides (dNTP)2 from the corresponding ribonucleotides is a stepwise process including several enzymes: ribonucleotide reductase, thioredoxin reductase, thioredoxin, and thymidylate synthetase (TS). The process of dNTP synthesis has been studied most thoroughly in Eschrichia coli (1). Unlike E. co& Streptomyces are subjected to distinct morphological * To whom correspondence should be addressed. ’ Abbreviations used: dNTP, 2’-deoxyribonucleoside 5’-triphosphate; dUMP, P’-deoxyuridine 5’-monophosphate; FdUMP, 5-Auoro-2’-deoxyuridine 5’-phosphate; FdU, 5-fluoro-2’-deoxyuridine; DHF, dihydrofolic acid; THF, tetrahydrofolic acid; mTHF, N6p”-methylenetetrahydrofolic acid; DHFR, dihydrofolate reductase; MTX, methotrexate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction
in any form reserved.
and physiological differentiation. Consequently, differences in the regulation of the dNTP synthesis in Streptomyces are to be expected. The components of the enzyme complex of ribonucleotide reduction have already been studied in the chlortetracycline producing strain of Streptomyces aureofaciens (2-4). No attention has been paid so far to another component of the dNTP synthesisthymidylate synthetase of S. aureofaciens. TS (EC 22.214.171.124), which gives rise to thymine 2’-deoxyribonucleotides (5) has been purified to homogenity from different sources (6-11) and its properties are relatively well studied. In this paper purification and characterization of TS from S. aureofaciens is described. It was found that it is a monofunctional enzyme with an absolute requirement for NADPH in TS activity. NADPH functions as an effector in TS reactions in S. aureofaciens. MATERIALS
Chemicals. The chemicals and radiochemicals used were purchased from the following producers. dUMP, NADPH, Triton X-100, Norit A, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide * HCI-Serva, Heidelberg, Germany; [5-3H]dUMP (sp act 943 TBq * mol-I)-Techsnabexport, USSR, folic acid-Merck, Darmstad, Germany; DEAE-cellulose DE-52-Whatman, England; Sepharose 4B, Superose 12 HR lo/ 30-Pharmacia, Sweden; methotrexate-Lachema, Czechoslovakia; (+)L-tetrahydrofolate was prepared as described (12); dihydrofolate was prepared as described (13). 4B Synthesis of affinity carrier. Methotrexate-aminoethyl-Sepharose was synthesized by published procedures (8, 14). S. aureofaciens cultivation. An industrial strain of S. aureofaciens BMK was used. Cultivation of the cells was carried out as previously described (15). Solutions. Buffer A was 25 mM imidazol-HCl, pH 7.0, 2 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 10 mM 2-mercaptoethanol; buffer B was buffer A with 10% glycerol; buffer C was buffer A with 30% glycerol;
buffer D was buffer A with 10% glycerol, 0.1 mM dUMP, buffer E was buffer A with 10% glycerol, 0.1 mM dUMP, 20 mM KCl. Enzyme assay. The standard reaction mixture in a total volume of 40 pl contained 5 PM [5-3H]dUMP (58.3 GBq/mol), 62.5 pM (*)-L-t&rahydrofolate, 625 PM formaldehyde, 12.5 mM 2-mercaptoethanol, 6.25 mM potassium phosphate buffer, pH 7.5, 1.25 mM ascorbic buffer, pH 7.5,375 PM NADPH, and the enzyme. In the kinetic measurements the 81
Purification of Thymidylate Synthetase from S. aureofaciens
Crude homogenate Streptomycin sulfate precipitation DEAE-cellulose DE 52 MTX-Sepharose 4B
71 67 40 20
Protein (md 1469.2 676.7 136.0 0.6
concentrations of respective components varied as indicated under Results. The reaction was started by addition of the enzyme and terminated after 30 min incubation at 30°C by introducing 200 pl of charcoal suspension (Norit A, 100 mg/ml) in 2% trichloracetic acid. After 2 min centrifugation at 12,OOOg,a loo-p1 aliquot from the supernatant was added to 4 ml of scintillation cocktail (50 g of naftalene, 6 g 2,5diphenyloxazol, and 0.1 g of 1,4-di-2-(5-phenyloxazoyl)-benzene dissolved in 1 liter of dioxan) and measured in a Packard 3320 liquid scintillation counter. All assays were performed in duplicate. The activity of the enzyme is expressed in nanomoles per minute per milligram. Protein determination. The spectrophotometrical method described in (16) was used for determination of protein in crude homogenate. The Lowry method (17) was used for all other measurements. Purification procedure. All operations were performed at 0-5°C. Thirty grams of the thawed cells was suspended in 75 ml of buffer A and sonicated in two portions six times for 30 s, with 1-min breaks. The suspension was clarified by 15 min centrifugation at 20,OOOg.Eight milliliters of 10% streptomycin sulfate was added to 100 ml of the cell free extract. The suspension was stirred for 30 min and the precipitate was removed by centrifugation at 40,OOOgfor 30 min. DEAE-cellulose column chromatography. The supernatant from the preceding step was applied to a DEAE-cellulose DE-52 column (3.5 X 12.5 cm) equilibrated with buffer A. The column was washed with 100 ml of equilibration buffer followed by 0.1 M KC1 in buffer A until no protein could be detected in the effluent. The enzyme was eluted with a 500-ml linear gradient of 0.1-0.35 M KC1 in buffer B. Fractions containing TS activity were pooled and dialyzed 3 h against buffer C. Methotrexate-Sephurose 4B afjinity chromatography. dUMP (100 pM) was added to the dialyzed enzyme preparation from the previous purification step. The sample was loaded on a MTX-Sepharose 4B column (1.0 X 6.5 cm) equilibrated with buffer D. The column was washed with 200 ml of buffer D followed by 30 ml of buffer E. The enzyme was eluted with buffer B. A4okcu/ur weight determination. SDS-PAGE was performed in 10% polyacrylamide gels (18). A Pharmacia calibration kit of low molecular weight proteins was used for molecular weight determination. Proteins in the gels were stained with silver (19). The molecular weight of the native TS was estimated by FPLC gel filtration on a Superose 12 HR IO/30 column. One-hundred microliters of the enzyme obtained after the MTX-Sepharose purification step was applied to the Superose column. Gel filtration was performed at 4°C using 0.05 M sodium phosphate buffer, pH 7.0, 0.15 M NaCl as the eluent. A Calibration kit of low molecular weight proteins (Boehringer) was used for calibration of the column. TS was detected in the effluent enzymatically. DHFR actiuity determination. The enzyme activity was determined spectrophotometrically according to (20). Kinetic measurements. Kinetic parameters of the enzyme were determined from the Cleland equation for a bisubstrate ordered sequential reaction mechanism (21). The initial velocity was determined from TS activity values measured at 0, 3, 6, 9 and 12 min of incubation. Linear plots were fitted using a linear regression program. Measurement of NADPH level. The reaction mixtures were analyzed by multicomponent spectrophotometrical analysis at 345 and 360 nm
Total activity (nmol * min-‘) 9.3 12.1 7.0 4.1
Specific activity (nmol * min-’ . mg-r)
0.006 0.017 0.049 6.52
100 58 34
1.0 2.7 7.8 1025.5
(22) using a Beckman DU-65 spectrophotometer. The mixtures were bubbled 15 min with nitrogen prior to the reaction and continuously during the reaction to suppress NADPH autooxidation. The absorbancy of the reaction mixtures was measured at competent wavelengths at 0, 15,45,60, and 90 min. The absorbancy of NADPH solution under the reaction conditions in the absence of the enzyme was simultaneously measured. RESULTS
Purification of TS. Under the conditions used, the TS activity of S. aureofaciens cells was highest at the 20th hour of cultivation when the culture reached a maximal growth rate (23). Therefore, the cells from 20th hour of cultivation were used in all experiments. Table I shows that TS was purified from S. aureofaciens cells 1025fold with a 34% yield. DEAE-cellulose proved to be very efficient in removing considerable amounts of substances prior to the affinity chromatography. Rapid and efficient final purification of S. aureofaciens TS was achieved by affinity chromatography. TS was bound to MTX-Sepharose only in the presence of dUMP. Most of the contaminating proteins were already removed during the application of the sample. However, subsequent washing of the column with a large volume of buffer D was necessary for a satisfactory purification. Biospecific elution of the enzyme with buffer B (Fig. 1) released pure active enzyme in a single sharp peak. The pooled active fractions from the MTX-Sepharose step contained 0.6 mg protein (6.52 nmol. min-’ * mg-l), i.e., 58% of the activity applied to the column. Stability of TS. Total enzyme activity decreased during the purification. For better yields it was necessary to perform the purification without interruption. TS from S. aureofmiens was rather stable in the crude homogenate; after DEAE-cellulose chromatography TS activity remained unchanged for 1 month of storage at -20°C. The purified TS lost about 50% of its activity within 3-4 weeks under these conditions. Glycerol and Triton X-100 were added to the buffers to stabilize the enzyme (10). Molecular weight of TS. Purity of TS was tested by SDS-PAGE. Purified TS showed a single band (Fig. 2) corresponding to a molecular weight of 28,000. The molecular weight of the native enzyme was determined by gel filtration on Superose 12 HR lo/30 (Fig. 3). In three separate experiments the molecular weights of
FIG. 1. Elution profile of thymidylate synthetase from S. aureofaciens on MTX-Sepharose 4B (1 X 6.5 cm), flow rate 12 ml/h, l-ml fractions collected. The arrows indicate application of buffers D, E, and B. Protein (0); activity (@); n, fraction number. Experimental details are under Materials and Methods.
the native TS from S. aureofuciens was estimated to represent 80,000, 76,000, and 75,000, respectively. The molecular weight of S. aureofmiens native TS was determined to be 77,000. Properties of TS. Studies on the dependence of the quantity of the formed product on the time of incubation revealed that the amount of the product increases practically linearly up to 15 min of the incubation. Some TSs
FIG. 2. SDS-PAGE of S. aureofaciens thymidylate synthetase. Lane 1, TS after MTX-Sepharose 4B (5 pg protein). Lane 2, standard proteins (phosphorylase b, M, 94,000, albumin, i&f, 67,ooO, ovalbumin, M, 43,000; carbonic anhydrase, M, 3O,ooO, trypsin inhibitor, A4, 20,100, ol-lactalbumin M, 14,000). Lane 3, TS after DEAE-cellulose (10 pg protein).
FIG. 3. Molecular weight determination of native thymidylate synthetase from S. aureojczciem by gel filtration on Superose 12 HR 10/30. Standard proteins: aldolase, M, 158,000; albumin R, J4,68,000; albumin H, M, 45,000; myoglobin, M, 17,800, cytochrome c, M, 12,500. V,, void volume, V, elution volume. Experimental details are under Materials and Methods.
exhibit linearity in this process up to 60 min (20). The activity increase was proportionate to enzyme concentration in the enzyme assay. TS of S. aureofaciens exhibited a sharp pH optimum at 7.0 which was identical to that of Saccharomyces cereuisiue (24), E. coli K 12 (ll), and calf thymus (25). The temperature dependence of TS activity was examined within the temperature range 15-60°C. The temperature optimum of the enzyme was around 30°C. This finding is in agreement with the temperature optimum for the majority of microbial TSs (11, 24). Similar to other TSs, Effectsof sulfhydryl compounds. mercaptoethanol was essential for maximum TS activity of S. aureofaciens. Within the concentration range tested (up to 30 mM) the activating effect of mercaptoethanol was higher than that of dithiothreitol and dithioerythritol. Effect of cations. Many reactions in the biosynthesis of nucleic acids require the presence of metal ions. The activity of the majority bacterial TSs is stimulated by Mg2+ (l&26). We examined the effect of mono-, di-, and trivalent metal cations on TS activity. Mg2+ was without effect on the enzyme activity. TS from S. aureofuciens was stimulated by Na+ ions. The optimum Na2+ concentration in assay was 2 mM. Inhibition of TS. Deoxyuridylate analogs are well known inhibitors of TS. Table II illustrates the inhibitory effect of 5FdUMP on S. aureofuciens TS activity. TS activity was completely inhibited by 0.2 mM 5-FdUMP. 5-FdU neither inhibited the TS reaction nor affected the
Effect of 5-FdUMP on S. aureofaciem TS Relative Activity (4)
2 x 10-s
2 x 1o-7
2 x 10-G
2 x 10-h
increase in the cellular TS level when added to cultivation media (27, 28). Similarly, hydroxyurea, a potent ribonucleotide reductase inhibitor (27), did not affect TS activity. Methotrexate, a specific inhibitor of dihydrofolate reductase (DHFR), was without any inhibitory effect on S. aureofacims TS. Similarly, human leukemic TS was reported not to be affected by MTX (29). Kinetic characteristics. We assumed that in the reaction catalyzed by S. aureofaciens TS, dUMP binds to the enzyme before the coenzyme, since the monoglutamyl form of coenzyme mTHF was present (12, 30, 31). This assumption is strongly supported by the fact that the enzyme binds to MTX-Sepharose 4B only in the presence of dUMP. The reaction of S. aureofuciens TS thus follows an ordered sequential mechanism. The Michaelis constant for substrate and coenzyme was determined from double reciprocal plots. In these experiments substrate concentrations of 0.4, 0.66, 1.18, and 5.13 PM were used repeatedly for five different mTHF concentrations (5, 10,15,20, and 62.5 PM); NADPH was
2 x 1o-3
kept constant at a saturating concentration of 375 PM (Fig. 4, Fig. 5). K,,, for NADPH was determined in experiments where the substrate concentrations 0.34, 0.58, 1.09, and 5.09 j.bM were used with four different NADPH concentrations (15, 30, 45, and 60 PM, respectively); mTHF was kept at a saturating concentration of 250 PM (Fig. 6). Km for dUMP was determined to be 3.78 + 0.38 PM, K,,, for mTHF was 21.1 f 4.4 PM and Km for NADPH was 38.9 + 7.8 PM. K,,, for dUMP of S. aureofaciem TS is comparable with the K,,, value for the S. cerevisiae enzyme (24) and for the cells overproducing TS, such as dichloromethotrexate-resistant Lmtobacillus casei (32). Bifunctional TS-DHFR from protozoa Crithidiu fasciculatu has a very similar Km for dUMP, 3.3 PM (6). K,,, for mTHF of TSs from various sources varies from 12.5 to 400 PM. The estimated K,,, value for S. aureofaciens TS, 21.1 PM, is similar to that of TS from FdU-resistant Ehrlich ascites tumor cells, 25.3 PM (31). The estimated dissociation constant (K, = 3.64 f 0.72) indicates that the dissociation velocity of the E-S complex is negligible to the velocity of its formation.
2 x lo-’
CmfHFl-‘(jhll-’ CdUMPJ-’ (/AM)-’ FIG. 4. Plot of the reciprocal of initial reaction velocity (u) versus the reciprocal of the concentration of dUMP at different concentrations of mTHF: (0) 5, (A) 10, (A) 15, (a) 20, and (0) 62.5 pM.
FIG. 6. Plot of the reciprocal of initial reaction velocity (u) versus the reciprocal of the concentration of mTHF at different concentrations of dUMP: (A) 0.4, (A) 0.66, (0) 1.18, and (0) 5.13 pM.
FIG. 7. Dependence of S. aureofaciens thymidylate synthetase activity on NADPH concentration at standard assay conditions. 0
FIG. 6. Plot of the reciprocal of initial reaction velocity (u) versus the reciprocal of the concentration of NADPH at different concentrations of dUMP: (A) 0.34, (A) 0.58, (0) 1.09, and (0) 5.09.
The role of NADPH. The requirement of NADPH for TS activity of S. aureofaciens TS is a unique feature of the enzyme among all known TSs. Our results showed that the TS reaction did not proceed in the absence of NADPH (Fig. 7). The reaction was specific for NADPH; neither NADH nor FADH could substitute for this compound. These facts support the idea that NADPH acts as an effector or as a second coenzyme in the TS reaction. Therefore, it was desirable to test whether NADPH serves as a coenzyme of other enzyme reactions besides the TS reaction. DHFR activity of the purified TS. If our purified TS contained some other proteins not detected by PAGE, some other NADPH-dependent enzyme reactions might have proceeded under our assay conditions. Since bifunctional TS-DHFR enzymes were found in protozoa (33) and in green algae (20), S. aureofaciens TS was checked for DHFR activity. No DHFR activity could be detected in the TS preparation after the MTX-Sepharose 4B purification step. Other NADPH requiring enzymes. We have examined possible changes in NADPH concentration in the presence of the purified enzyme within 1 h at 30°C while dUMP and mTHF were omitted. Autooxidation of NADPH caused a slight decrease in NADPH concentration. Besides this change no additional NADPH consumption was observed. Consumption of NADPH during TS reaction, We measured the decrease in NADPH during the TS reaction
spectrophotometrically at 340 nm. DHF formed during the TS reaction, however, interferes with the measurement. A multicomponent spectrophotometrical analysis was used to measure the reaction mixture at two wavelengths (22). The resulting absorbances were analyzed to determine the concentrations of each component. Figure 8 shows that, while DHF was formed under the assay conditions, the NADPH level remained unchanged. NADPH was not consumed in the TS reaction. DISCUSSION
Thymidylate synthetase from species of the genus Streptomyces has been purified to homogenity for the first
m i nubs
Formation of DHF and the level of NADPH during S. aureofaciem thymidylate synthetase reaction. (0) DHF formed, (0) NADPH; the reaction contained 30 pg * ml-’ of enzyme, 100 pM NADPH, saturating concentrations of dUMP and mTHF. Experimental details are under Materials and Methods.
time. Purification of TS from S. aureofaciens was repro- the most advanced group of bacteria and are an example ducible, resulting on average in a lOOO-fold purification of convergent evolution between prokaryotic bacteria and and indicating a low level of this enzyme in S. aureofaciens eukaryotic fungi (39,40). The NADPH dependence of TS cells. has additional evolutionary significance. Streptomyces are As presented above, chromatography on MTX-Sephfacultative anaerobes. The requirement of NADPH for arose was a very useful step in the purification of S. au- TS activity might have developed as a consequence of a reofaciens TS. This adsorbent has been used for the prep- primeval reducing atmosphere under which Streptomyces aration of a variety dihydrofolate reductases (14) and a originated (41). The oxygen-sensitive ribonucleotide refew thymidylate synthetases (6, 7). The specific activity ductase in anaerobic E. coli, which is stimulated by of the enzyme was compared with that of other TSs. The NADPH (42), may be analogous. relatively low specific activity of S. aureofaciens TS was due to a wild bacterial strain where overproduction of the ACKNOWLEDGMENTS enzyme was not observed. thank Dr. Eva KutejovL for performing of FPLC filtration and The purified protein migrated as a single band on SDS- Dr.WeFrank Maley for his interest and helpful suggestions in the course PAGE with an apparent molecular weight of 28,000. The of this work. estimated molecular weight of the native enzyme, 77,000 f lo%, most likely reflected the enzyme conformation, REFERENCES since the enzyme subunit size determined by the more accurate SDS-PAGE was reproducibly found to be 28,000. 1. Mathews, C. K., Moen, L. K., Want, Y., and Sargent, R. G. (1988) Trends Biochem. Sci. 13,394-397. On the basis of the molecular weight values estimated for 2. 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