Agric. Biol. Chem., 53 (5), 1313-1319, 1989 1313

Purification and SomeProperties of Deaminase from Bakers' Yeast Tohoru Katsuragi, Toshihiro Sonoda, Kin'ya Matsumoto, Takuo Sakai and Kenzo Tonomura Laboratory of Fermentation Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka 591, Japan Received November 24, 1988

Cytosine deaminase (EC 3.5.4.1) was extracted from commercial compressed bakers' yeast and purified to an almost homogeneous state. The activity was more than 200U/mg of protein, which was several times higher than reported before. The molecular weight was 41,000 by gel permeation. The pi was at pH4.7. 5-Fluorocytosine, 5-methylcytosine, and creatinine were other substrates for the enzyme.An experiment with inhibitors suggested that the enzyme was an SH- enzyme. The enzyme was unstable to heat, with a half-life of about 0.5hr at 37°C. Characteristics of the enzyme, especially its specificity, were compared with those reported earlier for other cytosine deaminases from bacteria and a mold.

Local chemotherapy of cancer with the com- (5MC), a 5-substituted cytosine.4) 5FC, an- bined use of 5-fluorocytosine (5FC) given oral- other 5-substituted cytosine, is deaminated to ly and a cytosine deaminase capsule implant- 5FU in Saccharomyces cerevisiae.5) So, cy- ed locally may be possible.1} However, al- tosine deaminase of bakers' yeast should con- though this approach is successful in animal vert 5FCto 5FU, and could be used in place of experiments,1'2) there are problems when we E. coli cytosine deaminase. Although the yeast use the enzyme from Escherichia coli,3) which enzyme is unstable to heat (at 37.5°C),4) which is thermostable,1'3) and which can deaminate would prevent its use in long-term therapy in 5FC to 5-fluorouracil (5FU).1>3) First, it is the body, it might be stabilized by immobili- difficult to culture the bacteria on a large scale zation or other techniques. to obtain enough activity.3) As often occurs Anextract from compressed yeast deami- during purification of , E. coli cytosine nated 5FC to form 5FUin a preliminary ex- deaminase was obtained only in low yield periment, so we prepared the enzyme from when highly purified.3) Second, because of the yeast. possible pyrogenic reactions, it is preferable to The enzyme from a commercial bakers' use a highly purified preparation of the enzyme yeast has been reported to be purified, 340-fold or else to use an enzymefromother, safer at first,6) and "completely" in a second re- sources than bacteria. port,7) although only a few times more pure Cytosine deaminase can, however, be ob- than the first preparation. We purified this tained in large quantities from commercial enzyme much more, and describe the pro- compressed bakers' yeast.4) Yeast has been cedure and some characteristics of the enzyme used as an agent of alcoholic fermentation preparation here. from ancient times, and also as an agent for bread leavening in the Western world; so, it Materials and Methods seems to be a safe source. Yeast cytosine deaminase deaminates 5-methylcytosine General. Unless otherwise noted, all experiments were 1314 T. Katsuragi et al.

done as described before,1} and at below 10°C, with a scribed before.3) Densitograms for stained disc gels were 50mMpotassium phosphate buffer, pH 7.0. recorded at a wavelength of 550nmwith a densitometer (model 39433; Gelman Sciences Japan, Ltd., Tokyo). Materials. Compressed bakers' yeast was obtained from Oriental Yeast Co. (Tokyo). DEAE-Sephacel, Octyl- Results Sepharose CL-6B, LKBAmpholine carrier ampholytes, and a pi caliberation kit of marker proteins (range pH 3~10) for isoelectrofocusing were obtained from Purification of enzyme Pharmacia KK (Tokyo). Toyopearl HW-50S was from Cytosine deaminase was extracted from Tosoh Corp. (Tokyo). Other chemicals used were pur- compressed yeast by plasmolysis. The enzyme chased from WakoPure Chemical Industries (Osaka), was purified from the extract by fractional and were of guaranteed reagent grade. precipitation with ammoniumsulfate, anion- exchange chromatography (with DEAE- Enzymeassay. The enzyme activity was assayed in a cellulose or DEAE-Sephacel), hydrophobic mixture of an adequate amount of the enzyme and 3mM chromatography (with Octyl-Sepharose CL- cytosine in 0.20 m potassium phosphate buffer (pH 8.0) as described previously.1} After 30 min of incubation at 37°C, 4B), and gel-permeation chromatography the mixture was diluted with 0. 1 n HC1to stop the reaction (with Toyopearl HW-50S). A typical pro- andto measurethe conversionof the substrate cytosine to cedure was as follows, and the results are the spectrophotometrically (at 280 nm). For the study of the substrate specificity, the substrates and the summarized in Table I. products were assayed by dual-wavelength spectropho- Cell extract. Compressed yeast (2.0 kg) was tometry also described there1}: cytosine and uracil at 250 broken into flakes with the fingers, mixed with and 280nm; 5FC and 5FU, or 5MC and thymine, at 255 ethyl acetate (200 ml) with a spatula, stirred for and 290nm. The creatinine specificity was established 30min with a magnetic stirrer, and further through the enzymatic assay of the ammonia that had stirred for a few hours with the addition of formed as described before,3) and compared with the 2.00 1 of buffer that had been 15% saturated cytosine value by the same method for the reference. with ammoniumsulfate and adjusted to pH Ultrafiltration and diafiltration. Concentration and 7.0, so that the cells were plasmolyzed. To the diafiltration of a small volume of enzyme solution were autolysate obtained, 8.00 1 of buffer was add- done with an ultra filter with a molecular-weight cutoff of ed; the mixture was left in a cold room and about 10,000 (UK-10 filter, fitted in a UHP-25 holder stirred for a few minutes every day. Cell debris equipped with a spinner; Toyo Roshi Co., Tokyo) under was removed by centrifugation after 3 days. pressure with nitrogen gas. The clarified lysate (1 1 1) was referred to as the Electrophoreses. Discontinuous (disc) and isofocusing cell extract. electrophoreses on polyacrylamide gels were run as de- Ammoniumsulfatefractionation. The extract

Table I. Purification of Cytosine Deaminase Specific Activity activity Purification Yield Step (U) (U/mg (fold) (%) (mg)(A280 x ml) protein) Autolysis (2.0 kg 98,000 3,600 0.037 1 100 of compressed yeast) 1st ammoniumsulfate 39,000 3,000 0.077 2.1 82 (50 - 73% saturation) 2ndammoniumsulfate 1 1,000 1,500 0.14 3.8 42 (56 - 70% saturation) DEAE-Sephacel 78 420 5.5 150 12 Octyl-Sepharose CL-4B 4.9 210 42 1,100 5.7 Toyopearl HW-50S 0.41" 1 00 250a 6,800" 2.9

The amount of protein was calculated with the assumption that the concentration was proportional to A280. Cytosine Deaminase from Bakers' Yeast 1315 was brought to 70% saturation. The pre- Instruments, Inc., Osaka). The active fractions cipitate that formed overnight was collected by were combined (12ml). They contained 100U centrifugation, dissolved in 3.50 1 of buffer, ofenzyme activity, corresponding to a yield of and dialyzed thoroughly against the buffer. 2.9%, and had an absorbance of 0.043 at The dialyzed solution (3.99 1) was fractionally 280nm, from which we calculated that there precipitated with ammoniumsulfate from 50 was about 0.41mg of protein, with the as- to 73% saturation. The precipitate was col- sumption that absorbance and protein con- lected with 1.00 1 of 20mMbuffer, dialyzed as above, and diluted to 4.001 with the same centration were proportional. buffer. Fractionation was again done between Purity 56 and 70%saturation, and the precipitate was The fraction above had a specific activity of dissolved in 350ml of 10mMbuffer, followed over 200 U/mg (Table I). This corresponds to a by dialysis. 6800-fold purification over the cell extract, Anion-exchange chromatography. The above calculated from the purification factors from fraction (350ml) was put onto a column of step to step. Whenthe gel was stained, discon- DEAE-Sephacel (4.4x 14cm) previously tinuous gel electrophoresis gave one dense equilibrated with buffer. The column was main band, and two very faint bands on both washed thoroughly with buffer, and the en- sides of the main band. Figure la shows a zymewas chromatographed with a linear gra- densitogram. To identify which of the three dient of potassium chloride from 0 to 100 bands was the enzymeprotein, another gel rod mMin 1000ml of buffer at a flow rate of was cut lengthwise into two almost equal about 50ml/hr. Active fractions were collect- parts. One piece was studied by dye-staining, ed and combined (40ml). and the other was studied by activity-staining, Hydrophobic chromatography. The above as follows. This piece was further cut crosswise 40ml was dialyzed against 3.1 m ammonium into 1.7-mmslices. Every slice was transferred sulfate solution buffered at pH 7 with 100mM potassium phosphate, centrifuged to remove the precipitates formed, and put onto a col- umn of Octyl-Sepharose CL-4B (2.2x 12.5 cm). Elution was forced by application of a linear gradient of ammoniumsulfate from 3.1 to 0m in 200ml of 100him.buffer at a flow rate of about 25ml/hr. The active fractions (18 ml) were combined, concentrated by ultra- filtration to about 1.5ml, and diafiltrated with 50mMbuffer containing 100mMpotas- sium chloride. Gel-permeation chromatography. The dia- lyzed solution was chromatographed on a column ofToyopearl HW-50S (1.3 x 6.3 cm) at a flow rate of about 14ml/hr. The active fractions were collected (8ml; 130U; 0.130 absorbance at 280 nm), concentrated by ultra- filtration to about 1.4ml, and passed again Fig. 1. Polyacrylamide Gel Electrophoresis of the through the same column in the same way, Purified Cytosine Deaminase. but with a recycling operation of two pas- (a) Dye-staining. Densitogram is shown. sages. The chromatography was monitored (b) Activity-staining. Histogram indicates the activity. by a UV-recorder (M&S-Altex 150B; M & S Approximate position of the stained band is shaded. 1316 T. Katsuragi et al. into a test tube, ground with a microspatula, of the enzymegave the isoelectric point of pH and extracted with 1 ml of buffer. This extract was assayed for enzyme activity. The main band was accompanied by the enzyme activity Enzymechemistry (Fig. lb). Substrate specificity. Besides cytosine and 5FC, the enzyme deaminated 5MCand erea- Protein chemistry 4.7. tinine. The ratio of the activities toward these Molecular weight. The column used in the substrates at 3-mMconcentration wasabout fractionation by gel-permeation chromatog- 1:0.8:0.8:0.3. raphy was calibrated with standard proteins, Michaelis constants. The effects of concen- and the molecular weight of the enzyme pro- tration of cytosine or 5FCas substrate were tein was found from a graph to be 41,000. examined. Lineweaver and Burk double- Isoelectric point. Isofocusing electrophoresis reciprocal plots led to the calculation of the apparent Michaelis constants, Km,of 3.1 and 1.2 mM, respectively.

Other characteristics In the following experiments, partially pu- rified enzyme preparations were used. These

Fig. 2. Effects of Temperature on Reactivity and Stability of Gytosine Deaminase. Fig. 3. Effects of pH on Reactivity and Stability of (a) Optimumtemperature. The assay mixture with 0.016 U of enzyme activity in 0.50ml was incubated under the Cytosine Deaminase. standard conditions except for temperature and pH. The (a) Optimum pH. The assay mixture with 0.017 U of activity (meanof two measurements) is expressed relative activity/ml was incubated under the standard conditions to that of the control (37°C; pH8.0). Buffers used: , except for the different buffer, at 50min. The activity 0.20m potassium phosphate (pH 8.0); , 0.20m pot- (mean of two to four measurements) is expressed relative assium phosphate (pH 7.0). to that at pH 8.0 with 50mMphosphate. (b) Thermostability. The enzyme with 0.01 1 U of activity (b) pH stability. The enzyme with 0.012 U of activity was was incubated at the indicated temperature for 15 min in incubated at 37°C for 15min in 0.20ml of the indicated 0.25ml of 40mMpotassium phosphate buffer, pH 7.4. It buffer at the final concentration of 50mM.Then 0. 10 mmol was immediately cooled in an ice bath, and 0.15ml of of pH 8.0 phosphate buffer and 3/miol of cytosine in 0.67m potassium phosphate buffer, pH 8.0, was added. 0.30ml of water were added (final volume: 0.50ml), and After several minutes of incubation at 37°C, the assay the assay was done as usual (but for 60min). The residual reaction was started by the addition of 0.10ml of pre- activity (mean of two or three measurements) is expressed incubated 15 him cytosine solution. The residual activity relative to the activity of the untreated enzyme. (meanof two measurements)was expressed relative to Buffers used: O, sodium acetate; #, potassium phosphate; that r»f the* iintrp»atf»H carrmif aesavpH at r»I-T 8 0 A, Tris-HCl; A, sodium borate. Cytosine Deaminase from Bakers' Yeast 1317 mainly consisted of the fraction that was elut- incubated at various temperatures for 15 min, ed after the main fraction in the hydrophobic activity decreased at over 30°C (Fig. 2b). After chromatography step and had the specific ac- 30min of incubation at 37°C, the enzyme tivity of about 10 U/mg of protein, which was retained about half of its activity. about 4%of the highest purity appearing in Effects of pH. The activity of the enzymeat this study. different pHs was studied (Fig. 3a). The op- Effects of temperature. The enzyme was in- timum for the reaction was at around pH 7.5. cubated under the standard conditions but at The effects of pHon the stability of the enzyme various temperatures. The optimumtempera- were examined by measurementof the activity ture for the reaction was at around 30°C when after incubation at different pHs at 37°C for the pH was 8.0, and at around 40°C when the 15min (Fig. 3b). It seemed that the activity pH was 7.0 (Fig. 2a). Whenthe enzyme was was most stable at around pH 7.5. Effectors. Some metal salts that may be Table II. Effects of Various Classes of General general inhibitors for enzymes and some Effectors on Cytosine Deaminase Activity amino acids were tested to see if they affected Enzyme with 0.016U of activity was incubated for the reaction of this enzyme from bakers' yeast 60min in 1.0ml of the assay mixture containing 0.1 or (Table II). The enzyme was inhibited strongly 1 mMof possible effectors in three categories: (a) metal by low concentrations (0.1 and 1 him) ofAg+, salts, (b) general inhibitors, and (c) amino acids. Activity Hg+, and Hg2+, and weakly by high con- was expressed relative to that of the standard assay as control. No substance affected the enzyme activity by centrations (lmM) of Fe2+, Fe3+, and Pb2+, raising it to more than 1.2-fold at either concentration. suggesting that this enzyme was an SH- The other substances that gave enzyme activity not smaller than 0.8 are not shown. They included (a) enzyme,8) like the enzyme from E. coli.3) p- AgNO3, BaCl2, CaCl2, CdCl2, CoCl2, CuSO4, FeCl2, Chloromercuribenzoic acid, /?-chloromercuri- FeCl3, KC1, MgCl2, MnCl2, NaCl, NiCl2, SnCl2 and phenylsulfonic acid, and mersalyl at 1- and tetrasodium ethylenediaminetetraacetate (as chelator); (b) NaNO2, NaF, NaN3, a,a'-dipyridyl, 2-mercapto- 0. 1-mMconcentrations strongly inhibited the ethanol, 7V-ethylmaleimide, iodoacetic acid, and reaction, and o-phenanthroline at 1 mMdid so weakly, suggesting again that the enzymewas Na2HAsO4; (c) l- and D-alanine, jS-alanine, L-arginine, l- an SH-enzyme.8) The 36 amino acids, in- asparagine, l- and D-aspartic acid, L-cystein, L-cystine, l- and D-glutamic acid, L-glutamine, glycine, L-histidine, l- cluding five d- and DL-isomers, listed in the hydroxyproline, l- and DL-isoleucine, L-leucine, L-lysine, l- and D-methionine, L-ornithine, L-phenylalanine, l- headnote of Table II, did not affect the en- proline, l- and D-serine, l- and DL-threonine, l- and dl- zyme activity much (from 0.9 to 1.2 relative tryptophan, L-tyrosine, L-valine, DL-homoserine, dl- to the control; not shown). norvaline, DL-a-amino-H-butyric acid, and jS-amino-«- butyric acid. Discussion Relative activity Compound added Wehere obtained an almost pure cytosine 1.0HIM 0.1 HIM deaminase, the purest preparation from bak- ers' yeast reported so far. The enzyme from 0 (a)AgNO3Metal salts bakers' yeast was previously reported to be FeCl2 0.7 purified partially6) or completely,7) so we start- Hg(CH3COO) 0 ed the purification of the enzyme from autol- HgCl2 0 Pb(CH3COO) 0.6 ysates of the yeast, following the reported (b) General inhibitors procedures. Our enzyme soon reached the 0-Phenanthroline 0.4 same purity (specific activity) as these prepara- /7-Chloromercuribenzoic acid 0 tions reported to be highly or completely /7-Chloromercuriphenylsulfonic 0 acid purified. However, our preparation was still 0 Mersalyl .1 0.1 very impure by the criterion of disc gel elec- trophoresis at that stage (unpublished results). 1318 T. Katsuragi et al.

Then, as described here, we purified this en- The yeast cytosine deaminase deaminated zyme 6800-fold, up to the specific activity of 5MC,5FC, and creatinine as well as cytosine. over 200U/mg of protein, which is probably 5MCwas also deaminated by other cytosine several-fold higher than that of the "pure" deaminases, ones from bakers' yeasts,4'5) As- enzyme already reported.7) Our enzyme was pergillus (unpublished results), Pseudomonas,12) different from the other(s)6'7) in many of its and Escherichia,3) but not by that of Serra- properties. tia,10) or Salmonella}5) Moreover, it inhibited Enzyme activity was as labile (Fig. 2b) as the activity toward cytosine in Flavobacte- when the enzyme was crude or partially pu- rium.16) The conversion of 5FC to 5FU by rifed.4) This would cause difficulties if it were cytosine deaminase in fungi5'20) explains used in long-term cancer chemotherapy. It the antifungal activity of 5FC.20) 5FC was must be stabilized by immobilization or the also deaminated by the other cytosine de- like before use. In fact, it could be much aminases, those from Aspergillus (unpublish- stabilized by immobilization on many ad- ed results), Salmonella,l5) Escherichia,1 ' 3) Flavo- sorbents.9) bacterium,ll) the two strains of Arthrobac- Wehave purified five cytosine deaminases: ter ii,i8) pseudomonas (unpublished results), this one from yeast, one from the mold Alcaligenes (both enzymes),18) and many Aspergillus fumigatus IFO 5840 (unpublished other bacteria.21) results), and three from the bacteria Serratia E. coll K-12 cytosine deaminase1'3* de- marcescens IFO 3054,10' n ) Pseudomonas aureo- aminates 5MCand 5FC, besides cytosine (and faciens IFO 3521,12'13) and E. coli K-12 IFO also creatinine; unpublished results). In an 3301.1'3) They were different from each other early report, 5MC and 5-hydroxymethylcy- in molecular weight, substrate specificity, and tosine were found to be inert to the cytosine thermal stability. We also examined cell ex- deaminase of this bacterium.22) So E. coll tracts from manybacteria for enzymeactivities cytosine deaminase has been considered not to involved in the metabolism of cytidine, and deaminate 5-substituted . In fact, in found that half of the extracts had cytosine unpublished observations, cytosine de- deaminase activity.14) Half of the cytosine aminases from two derivative strains of E. coll deaminases are thermostable, and the others K-12, HB101 and C600, did not deaminate unstable. 12) 5FC or 5MC at all, and the activities of In addition to the purified enzymes men- cytosine deaminase toward 5MC and 5FC in tioned above, cytosine deaminases of Sal- comparison to the cytosine values varied from monella typhimurium,15) Flavobacterium fila- strain to strain in some stock strains of E. coll mentosum (as creatinine deiminase),16) and K-12 that had been preserved in different strains Arthrobacter sp. JH-13 (extracellu- laboratories. lar),1^ Arthrobacter sp. Jll,18) and Alcali- genes denitrificans ssp. denitrificans J9 (con- Acknowledgments.Wethank Ms. MakikoShibata and taining two enzymes)18) have been studied in Mr. Tatsuji Sakamoto,undergraduates studying at our laboratory, for technical assistance. Thanks are due to detail. All cytosine deaminases with rare ex- Professor Tae-Shick Yu of Keimyung University, Taegu, ceptions16'18) deaminate (deiminate) creati- Korea, and Professor Hong-Ki Jun of Pusan National nine (unpublished results), which can be call- Univesity, Pusan, Korea, for helpful discussions. The authors thank Dr. S. S. Cohen, Distinguished Professor ed creatinine deiminase activity. Emeritus at the State University of NewYork, for dis- The outstanding feature of yeast and mold cussions and interest. cytosine deaminases is the molecular weight of the protein, which is of about 40,000 and 30,000, different from that of the bacterial References enzymes reported, which are 200,000~ 1) T. Sakai, T. Katsuragi, K. Tonomura, T. Nishiyama 600,000.3'10'12'15'16'18) and Y. Kawamura, J. Biotechnol., 2, 13 (1985). Cytosine Deaminasefrom Bakers' Yeast 1319

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