preventing dUTP from being incorporated into DNA and may have a significant role in both thesynthesis of ... synthesize dUMP from cytidine, uridine, or deoxycytidine, ... They suggested that the deoxyribose-1-phosphate .... The ribo- and deoxyribonucleoside triphosphates ATP, UTP, dCTP, and dTTP were not hydro-.
JOURNAL OF BACTERIOLOGY, July 1984, p. 278-282
Vol. 159, No. 1
0021-9193/84/070278-05$02.00/0 Copyright X) 1984, American Society for Microbiology
Purification and Characterization of a dUTPase from Acholeplasma laidlawii B-PG9 of Medical
WILLIAMS' 2 AND J.
D. POLLACK'* Microbiology and Immunology' and Comprehensive Cancer Center,2 The Ohio State University, M. V.
Department
Columbus, Ohio 43210 Received 6 December 1983/Accepted 13 April 1984
dUTP was purified 120-fold from extracts of Acholeplasma laidlawii B-PG9 by Blue-Sepharose, PhenylSepharose, hydroxyapatite, and DEAE-Sephacel chromatography techniques. The only substrate for the enzyme was dUTP with an apparent Km of 4.5 ,uM. The only reaction products were dUMP and PP;. The dUTPase did not exhibit any specific divalent cation requirement, but it was inhibited by EDTA. The enzyme was not inhibited by Pi or p-hydroxymercuribenzoate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 48,000, and its isoelectric point was 5.3. The enzyme was thermostable at 55°C for 1 h. A. laidlawii dUTPase was distinguishable from KB (human epidermoid carcinoma) dUTPase by differences in electrophoretic migration, isoelectric point, and thermostability. The enzyme is important in preventing dUTP from being incorporated into DNA and may have a significant role in both the synthesis of thymidine- and PPi-dependent phosphorylations. MATERIALS AND METHODS Materials. Nonradioactive nucleotides and Sephadex G-75 were purchased from Sigma Chemical Co., St. Louis, Mo. Blue-Sepharose CL-6B, Phenyl-Sepharose CL-6B, Sephadex G-25 PD-10 columns, DEAE-Sephacel, Polybuffer exchanger PBE-94, and Polybuffer 74 were purchased from Pharmacia Fine Chemicals, Piscataway, N.J. Hydroxyapatite (Bio-Gel HPT) and Coomassie blue protein dye reagent were obtained from Bio-Rad Laboratories, Richmond, Calif. The radionucleotide [5-3H]dUTP (11 Ci/mmol) was purchased from Moravek Biochemicals, Inc., Brea, Calif., whereas [5-3H]ATP (29 Ci/mmol), [5-3H]UTP (14.5 Ci/ mmol), [5-3H]dCTP (21 Ci/mmol), and [5-methyl-3H]dTTP (46 Ci/mmol) were purchased from Amersham Corp., Arlington Heights, Ill. DE-81 filter disks were obtained from Whatman, Inc., Clifton, N.J. Organisms. A. laidlawii B-PG9, Acholeplasma hippikon Cl, and Acholeplasma morum S2 were grown in a modified Edward medium containing 1% (vol/vol) heat-reinactivated horse serum (2). A cytoplasmic fraction from hypotonically shocked washed cells was prepared as described by Pollack (17). dUTPase assay. The enzyme was assayed by a modification of the procedure described by Williams and Cheng (29). The assay mixture contained (in a total volume of 0.1 ml) 0.1 mM [5-3H]dUTP (50 ,uCi/,imol), 2 mM 2-mercaptoethanol, 1 mM MgCI2, 0.1% (wt/vol) bovine serum albumin, 50 mM Tris-hydrochloride (pH 8.0), and the enzyme preparation (0.06 to 22 ,ug of protein). Al reactions were conducted at 37°C. Reactions were stopped by spotting 50 RI of the reaction mixture on a DE-81 filter disk. The disks were processed as described previously (29). Products of the reaction were determined by thin-layer chromatography (3). By using this disk assay, dUTP remains bound to the disk, whereas the deoxyuridine derivitives (deoxyuridine, dUMP, dUDP) are eluted. Products of the reaction were determined by thin-layer chromatography (3). A unit of dUTPase activity was defined as the amount of enzyme which converted 1 nmol of dUTP to dUMP and PP1 per min at 37°C. Purification of A. laidlawii B dUTPase. The cytoplasmic fraction of A. laidlawii B-PG9 after ultracentrifugation
dUTPase (E.C. 3.6.1.23) catalyzes the hydrolysis of dUTP was first reported in Escherichia coli (8) and subsequently has been purified from a number of bacterial (19, 26) and mammalian (29) cells. The only known function of dUTPase is to prevent dUTP from being incorporated into DNA while providing the cell with dUMP (4, 16). dUMP by the action of thymidylate synthetase is the major precursor of dTMP and, ultimately, dTTP (4, 16, 28). Mollicutes (mycoplasmas), although capable of autonomous growth, have complex nutritional requirements (21, 23, 24) which are related to their decreased biosynthetic capabilities (15). A number of the mycoplasmas have an absolute growth requirement for thymidine (15, 27). Although only Acholeplasma laidlawii B has been reported to synthesize dUMP from cytidine, uridine, or deoxycytidine, it cannot synthesize dTMP from dUMP (27). This is due to the fact that it apparently has nonfunctional thymidylate synthetase activity, which is activated by folate (14, 20). Recently, Neale et al. (15) reported that the synthesis of dTMP by Mycoplasma mycoides subsp. mycoides primarily occurs through the salvage-synthesis of dTMP from thymine. They suggested that the deoxyribose-1-phosphate required for the phosphorylation of thymine to deoxythymidine was derived in the initial steps of the sequence from the deamination of dCMP to dUMP. The dUMP was dephosphorylated to deoxyuridine which was then converted to uracil and deoxyribose-1-phosphate. They further suggested that dUTP, as well as dCTP, could serve as a source of dUMP, although, heretofore, dUTPase activity has not been demonstrated in mycoplasmas. In this report, we describe the purification and characterization of a dUTPase activity from A. laidlawii B-PG9 and demonstrate that it can be distinguished from a eucaryotic form of the enzyme. Also, we discuss the possible metabolic role of this enzyme in A. laidlawii and report that dUTPase activity is present in other species in the genus. to dUMP and PP,. This enzyme
*
Corresponding author. 278
VOL. 159, 1984
DEOXYPYRIMIDINE METABOLISM IN ACHOLEPLASMA LAIDLAWII
(100,000 x g for 60 min) (17) served as the starting material for purification of the enzyme. All procedures were conducted at 4°C, and all buffers contained 0.2 mM phenylmethylsulfonyl fluoride to reduce proteolytic digestion. The cytoplasmic fraction was dialyzed overnight against TMG buffer (0.01 M Tris-hydrochloride [pH 7.5], 2 mM 2mercaptoethanol, 1 mM MgCl2, 10% [vol/vol] glycerol) and chromatographed on a Blue-Sepharose column (0.9 by 10 cm) equilibrated in the same buffer. The dUTPase was not bound to the Blue-Sepharose matrix, but there was a statistically significant (P < 0.05) enhancement of its activity after chromatography on this matrix when compared to the units of dUTPase applied to the column. This was presumably due to the removal of an inhibitor which was present in the cell extract.
Solid (NH4)2SO4 was added to the pooled active fraction from Blue-Sepharose until a final concentration of 1 M was obtained. The solution was centrifuged at 30,000 x g for 30 min to remove any precipitate, and the supernatant was applied to a Phenyl-Sepharose column (0.9 by 10 cm) equilibrated in TMN buffer (10 mM Tris-hydrochloride [pH 6.0], 1 mM MgCl2, 2 mM 2-mercaptoethanol, 1 M (NH4)2SO4). The dUTPase activity was eluted with a linear gradient of decreasing (NH4)2SO4 concentration and increasing concentration of ethylene glycol monomethyl ether by using TMN buffer and TMM buffer (10 mM Tris-hydrochloride [pH 6.0], 1 mM MgCl2, 2 mM 2-mercaptoethanol, 50% [vol/vol] ethylene glycol monomethyl ether). The dUTPase activity eluted as a single peak at a (NH4)2S04 concentration of 0.29 M. The fractions containing dUTPase activity were pooled and dialyzed against TMG buffer. The dialysate was chromatographed on a hydroxyapatite column (0.9 by 10 cm) equilibrated in KGM buffer (10 mM KH2PO4 [pH 8.0], 10% [vol/voll glycerol, 1 mM MgClI, 2 mM 2-mercaptoethanol). Although the dUTPase activity was not bound to the matrix, it was retarded and eluted as a single peak. The fractions containing the enzyme activity were pooled and chromatographed on a DEAE-Sephacel column (0.9 by 10 cm) equilibrated in TGM buffer (10 mM Tris-hydrochloride [pH 7.5], 10% [vol/vol] glycerol, 2 mM 2mercaptoethanol). The dUTPase was eluted from this column with a linear gradient of Tris-hydrochloride (pH 7.5; 0.01 to 0.5 M). The dUTPase activity eluted as a single peak at a Tris-hydrochloride concentration of 0.25 M. Other methods. Protein was assayed by the Coomassie blue method as described by Bio-Rad Laboratories, using bovine serum albumin as the standard. The methods for the growth and purification of the dUTPase from KB cells have been described previously (M. V. Williams, manuscript in
preparation). Nondenaturing polyacrylamide tube gel electrophoresis was performed in a Bio-Rad model 150A apparatus at 4°C. The gel consisted of acrylamide (%T = 5.2) methylenebisacrylamide (%C = 2.7), 384 mM Tris-hydrochloride (pH 8.8), 0.06% (wt/vol) ammonium persulfate, and 0.07% (vol/ vol) N,N,N',N'-tetramethylenediamine. The gels were polymerized in siliconized tubes (0.5 by 12 cm) at 25°C. The running buffer consisted of 25 mM Tris (free base) and 200 mM glycine (pH 8.4). Samples (3 to 50 [Lg of protein) were applied to the top (cathode) of the gel along with bromphenol blue as the tracking dye. Electrophoresis was performed for 30 min at 1.5 mA per tube and then for 3 h at 3 mA per tube. After electrophoresis, the gels were removed from the tubes and cut into 3-mm cross-sectional slices. Each fraction was immersed in 150 pd of the standard radioactive reaction mixture and incubated at 37°C for 12 h. Fifty microliters of
279
the supernatant was removed and assayed for dUTPase activity. The isoelectric point of the A. laidlawvii dUTPase was determined by chromatofocusing. The purified enzyme was equilibrated in 0.025 M imidazole-hydrochloride (pH 7.4) by passage over a Sephadex G-25 PD-10 column preequilibrated in the same buffer. The sample was loaded on a PBE-94 (1 by 27 cm) equilibrated in 0.025 M imidazole-hydrochloride (pH 7.4). The molecular weight of the A. laidlawvii B dUTPase was determined by gel filtration chromatography, using a Sephadex G-75 column (1.5 by 45.5 cm) which was equilibrated in TMG buffer containing 0.2 M KCI. The column was calibrated with blue dextran, bovine serum albumin (Mr, 67,000), ovalbumin (Mr, 43,000), chymotrypsinogen A (Mr, 25,000), and ribonuclease A (Mr, 13,700). One milliliter of the purified enzyme (4 U, 6 pg of protein) was applied to the column. Fractions of 2.3 ml each were collected and assayed by the filter disk assay. For the divalent cation and thermostability studies, the purified enzyme was equilibrated in 10 mM Tris-hydrochloride (pH 8.0) containing 10% (vol/vol) glycerol and 2 mM 2mercaptoethanol by chromatography on a Sephadex G-25 PD-10 column equilibrated in the same buffer. For the divalent cation studies, MgCl2 was deleted from the standard reaction mixture. The thermostability of the dUTPase was determined by incubating the enzyme preparations at 55°C. At various times, samples were removed and assayed for dUTPase activity. The percent activity remaining was determined by comparing the residual activity in the test sample to a control maintained at 4°C. RESULTS Purification of dUTPase. The purification of dUTPase from A. laidlauii B-PG9 is summarized in Table 1. A purification of 120-fold was routinely achieved, and there was no loss of activity from these preparations after storage at -20°C for 1 month. Biochemical studies. The ribo- and deoxyribonucleoside triphosphates ATP, UTP, dCTP, and dTTP were not hydrolyzed (+ (1 mM) concentration. Over the concentration range employed, dUTP behaved as a Michaelis-Menten-type substrate (Fig. 1A). A double-reciprocal plot of the initial velocities demonstrated that the Km for dUTP was 4.5 F.M and that the Vmax was 0.625 nmol for dUTP hydrolyzed min-' mg of protein-' (Fig. IB). Physical properties. Although the A. laidlawvii B and KB dUTPases could not be distinguished by their biochemical
280
WILLIAMS AND POLLACK
J. BACTERIOL.
TABLE 1. Purification of dUTPase activity from A. laidlawii B-PG9" Procedure
Cytoplasmic fraction Blue-Sepharose Chromatography Phenyl-Sepharose Chromatography Hydroxyapatite chromatography DEAE-Sephacel chromatography a
dUTPase activity was purified from 1 x
Total U
(mg/ml)
Sp act U/ng of protein
Yield
(ml) 9 19 17
119.7 135.5 83.3
2.22 0.83 0.17
6.05 8.6 28.5
100 113 70
1 1.4 4.7
13 14
63.6 57.2
0.022 0.006
53 48
36.8 122.3
Total vol
Protein
Purification fold
(%)
222.9 740.3
103' to 3 x 10i3 cells.
properties, these enzymes differed in a number of physical properties. The A. laidlawii B dUTPase exhibited an electrophoretic mobility (Rf) of 0.52 on polyacrylamide gels, whereas the KB enzyme exhibited an Rf of 0.61 under the same conditions. Coelectrophoresis of a mixture of both enzymatic activities resulted in separation and Rf s which were identical to those of the separately electrophoresed purified enzymes. Similarly, the two enzymes could be distinguished by their isoelectric points (pI). The KB dUTPase exhibited a pl of 6.4, which is similar to the value previously reported for this enzyme (5), whereas the A. laidlawii B. dUTPase had a pl of 5.3. The molecular weight of the A. laidlawii and KB dUTPases was determined by gel filtration chromatography. The molecular weight was determined by comparing the partition coefficient (Kav) of the dUTPases to the standards. A semilogarithmetic plot of molecular weight versus Kav was constructed, and the molecular weight of the A. laidlawii dUTPase was estimated to be 48,000, whereas the KB dUTPase was 43,000 (Fig. 2). The difference in the protein structure of the enzymes was further supported by thermostability studies. The dUTPase activity from A. laidlawii B was stable at 55°C for at least 60 min, whereas the KB dUTPase activity was thermolabile at this temperature, exhibiting less than 1% of its original activity after 10 min of incubation. The decrease in activity of the KB dUTPase was not due to any detectable contaminating protease activity in the purified enzyme preparation (data not shown). dUTPase activity in acholeplasmas. We examined the unpurified cytoplasmic fractions of other mycoplasmas for dUTPase activity. All Acholeplasma species we examined had dUTPase activity (average units per milligram of protein + standard deviation): A. laidlawii B-PG9 (6.41 + 2.34, n = 6), A. hippikon Cl (8.30), and A. morum S2 (7.05 ± 0.64, n =
structure of these enzymes. The ability to distinguish the A. laidlawii B and KB dUTPases is important since numerous studies have been conducted in eucaryotic cells (7, 9, 25) to determine the possible role of dUTPase in normal cellular functions and since many cultured eucaryotic cells are
.6 r
A.
.5
7.4 V. E
-tg .3 0
E
c
-.2 .1
10
30
20
50
40
60
uuM
3). DISCUSSION Our studies demonstrated that A. laidlawii B-PG9 possesses a dUTPase activity. The A. laidlawii B dUTPase is similar to the dUTPases purified from E. coli (26), Bacillus subtilis (19), human leukemic cells (29), and KB cells (M. V. Williams, unpublished data), with respect to its inability to hydrolyze ATP, UTP, dCTP, or dTTP; an apparent Km of 4.5 ,uM for dUTP; and its molecular weight. However, the A. laidlawii dUTPase differs from the KB dUTPase in a number of physical and biochemical properties. For example, these enzymes are easily distinguished based upon differences in their isoelectric points, electrophoretic mobilities, thermostabilities, and sensitivity to p-hydroxymercuribenzoate. These results suggest that differences exist in the protein
2
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
[1,S] (lo 6) FIG. 1. Initial velocity studies of dUTPase. (A) Rate of dUTP
hydrolysis as a function of dUTP. Initial velocities were determined by the standard dUTPase assay as described in the text, but the concentration of dUTP was varied from 2 to 50 ,uM. The units of purified enzyme used per assay was 0.029, and the reaction mixture was incubated at 37°C for 10 min. (B) Double-reciprocal plot of initial velocities.
DEOXYPYRIMIDINE METABOLISM IN ACHOLEPLASMA LAIDLAWII
VOL. 159, 1984
8 r -
fructose-6-phosphate (unpublished data) and in other metabolic reactions in this organism. Although we found dUTPase activity in other Acholeplasma spp., in preliminary experiments we detected no dUTPase activity in cytoplasmic fractions from Mycoplasma pillmonis JB, Mycoplasma pnelimoniae FH, or Mycoplasma arthritidis 07. Further studies are necessary to demonstrate the specific enzymes necessary for the synthesis of dUTP and the degradation of dUMP and to determine what role, if any, dUTP or dUMP or both may have in regulating deoxypyrimidine metabolism in A. laidlawvii B-PG9 and other mollicutes.
BSA
6 5 0
4-
4
A. Iaidlowii B-PG9 dUTPase KB dUTPase Ovalbumin
m
3 0
Chymotrypsinogen
L.
281
0
2 Ribonuclease A
0.1
0.2 Kav
0.3
0.4
FIG. 2. Molecular weight determination. Sephadex G-75 gel filtration chromatography was performed as described in the text with blue dextran, bovine serum albumin (BSA; Mr = 67,000), ovalbumin (Mr = 43,000), chymotrypsinogen A (Mr = 25,000), and ribonuclease A (Mr = 13,700) as standards. One milliliter of the purified enzyme (4 U, 6 p.g of protein) was applied to the column. Fractions of 2.3 ml each were collected and assayed by the disk assay. Kav, Partition coefficent.
contaminated with different mycoplasmas, including A. laidlawii (1, 13). Presumably, the major physiological role of the dUTPase in A. /aidlawii B is to prevent dUTP from being incorporated into DNA. Studies in eucaryotic cells have demonstrated that disruption of normal deoxyuridine metabolism results in an increase in dUTP pools relative to dTTP pools and an increased incorporation of dUTP in DNA (9, 25). This results in a cyclical repair process of dUMP removal of uracil-DNA glycosylase (11, 12) that ultimately results in the fragmentation and degradation of the DNA. Hochhauser and Weiss (10) suggested that a similar process of dUTP incorporation into DNA with its subsequent removal was responsible for thymidineless death. Smith and Hanawalt (27) reported that thymidine deprivation of Mycoplasma (Acholeplasma) laidlawii resulted in the phenomenon of thymineless death (6). Although a similar cyclical repair process probably occurs in thymidine-starved mycoplasmas, we have not been able to detect a specific uracil-DNA glycosylase in A. laidlawii B-PG9 (5) (unpublished data). However, the presence of a number of DNA nucleases has been reported in this organism, and it is possible that a nonspecific nuclease would remove uracil residues from the DNA (2, 18, 22). Our results also infer that A. laidlawii B possesses the biochemical mechanisms necessary for the synthesis of dUTP either through the phosphorylation of dUMP or through the reduction and phosphorylation of UDP. Neale et al. (15) suggested that M. mycoides subsp. mycoides had a ribonucleoside diphosphate reductase that was similar to the enzyme from E. coli, and it has been reported that the E. coli ribonucleoside diphosphate reductase reduces UDP but at a lower rate than the other nucleoside diphosphates (4). The dUMP produced by the action of dUTPase might be used in a reaction sequence similar to that proposed by Neale et al. for M. mycoides subsp. mycoides (15) in which dUMP serves as a source of deoxyribose-1-phosphate for dTTP synthesis. Also, the PPR that is generated by the dUTPase could be employed in the phosphorylation of
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