Agric. Biol. Chem., 52 (1), 169-175, 1988 169

TV-Methyl Nucleosidase from Tea Leaves Osamu Negishi,* Tetsuo Ozawa and Hiroshi Imagawa** Institute of Applied Biochemistry, University of Tsukuba, Sakura-mura, Niihari-gun, Ibaraki 305, Japan Received August 10, 1987

N-Methyl nucleoside (TV-methyl nucleosidase, N-MeNase), which hydrolyzes 7- methylxanthosine to produce 7-methylxanthine, was detected in tea-leaf extracts and separated from nucleosidase (ANase, EC 3.2.2.7) by DEAE-cellulose column chromatography. The optimum pH for the N-MeNase ranged from 8.0 to 8.5. The was strongly inhibited by EDTA. Inhibition by the hydrolysis products of 7-methylxanthosine and 7-methylinosine was also observed. The molecular weight was estimated to be about 55,000 by gel-filtration. Amongpurine and Af-methylpurine nucleosides, 3- and 7-methylpurine nucleosides were hydrolyzed preferentially by N-MeNase. On the other hand, ANase could not hydrolyze 7-methyl- xanthosine, although the enzyme showed high activity toward 7-methyladenosine. As a result, it is suggested that N-MeNasecatalyzes the hydrolysis reaction of 7-methylxanthosine in the pathway of caffeine biosynthesis, whereas ANaseis not directly concerned with it.

In the previous papers we suggested the adenosine nucleosidase (EC 3.2.2.7) from bar- presence of a nucleosidase or nucleoside phos- ley leaves,7) whereas reports on nucleoside phorylase, which degrades 7-methylxanthosine phosphorylase in plants are limited.8) to produce 7-methylxanthine, in cell-free ex- Adenosine nucleosidase (ANase) was iso- tracts of tea1} and coffee2) leaves (Fig. 1). The lated from tea leaves and characterized.9) presence of a nucleosidase was also suggested However, it remains to be determined whether in cell-free extracts of coffee fruits3A) and this nucleosidase hydrolyzes 7-methylxantho- coffee callus cultures.4'5) However, detailed sine. Recently, we observed that the deg- studies on the enzyme have not been per- radation of 7-methylxanthosine is caused by formed hitherto. another enzyme. Several nucleosidases have been isolated This paper deals with the partial purification from various plants, e.g., nucleosidase and characterization of the enzymedegrading (EC 3.2.2.2) from yellow lupin seeds6) and 7-methylxanthosine, TV-methyl nucleosidase

Fig. 1. Biosynthesis of Caffeine from 7-Methylxanthosine in Tea and Coffee Plants. (A) nucleosidase; (B) nucleoside phosphorylase; R-l -P, -1-phosphate; SAM,S-adenosylmethionine. * Present address: Tokyo Research Laboratory, Takasago International Corporation, 5-36-31 Kamata, Ota-ku, Tokyo 144, Japan. ** To whomcorrespondence should be addressed. Abbreviations: PCMB, />-chloromercuribenzoate; Tricine, Tris(hydroxymethyl)methylglycine; HEPES, N-2- hydroxyethylpiperazine-A^-2-ethanesulfonic acid; PIPES, piperazine-A^,A/^/-bis(2-ethanesulfonic acid). 170 O. Negishi, T. Ozawa and H. Imagawa

(N-MeNase). We also discuss the role of N- methylxanthosine by high performance liquid chroma- MeNase and ANase in the biosynthesis of tography (HPLC). The standard assay mixture contained 50/il each of caffeine and . 0. 1 m Tricine-NaOH buffer (pH 8.5), 7-methylxanthosine (3.35 mM)and enzyme solution. The reaction was carried MATERIALS AND METHODS out at 37°C for 1 hr and terminated bythe addition of20fA of 0.5m HC1O4. As the internal standard, 30/A of theo- Plants. Fresh tea leaves {Camellia sinensis (L.) O. bromine (1.34mM) was added to the reaction mixture. Kuntze, cv. Yabukita) were plucked at a tea garden near The reaction was analyzed with a HPLC(TRI the University ofTsukuba in May and stored at -20°C in ROTAR; Japan Spectroscopic Co., Ltd.). After centri- a freezer. fugation of the reaction mixture at 1,600 x g for 5 min, 2/u\ of the supernatant was injected into the ODS column, Chemicals. 7-Methyixanthosine, 7-methylinosine, 7- 4mm i.d.x300mm in size (LS-410; Toyo Soda methylguanosine, 1-methyladenosine, 1-methylinosine, 1- Manufacturing Co-., Ltd.) and elution was carried out with methylguanosine, 1-methyladenine and 3-methylxan- 5% AcOH-MeOH(80:20) at the flow rate of 1 ml/min. thine were purchased from Sigma Chemical Co. 7-Meth- Theeluate was monitoredas the absorbanceat 270nm yladenosine, 3-methyladenosine, 3-methylguanosine, 3- with a UVspectrophotometer (UVIDEC-100-III; Japan methylinosine and 3-methylxanthosine were kindly pro- Spectroscopic Co., Ltd.). Under these conditions, vided by Professor T. Fujii, Faculty of Pharmaceutical 7-methylxanthosine (tR: 2.7 min), 7-methylxanthine (tR: Sciences, KanazawaUniversity. 1-Methylxanthosine was 4.5min) and theobromine (tR: 7.3min) were completely generously provided by Dr. A. Yamazaki, Central Re- separated fromeach other. Peak areas werecalculated by search Laboratories, Ajinomoto Company Inc. The 1-, the internal standard method with an integrator (Model 3- and 7-methylpurine bases except those described 5000E; System Instruments Co., Ltd.). above were prepared in our laboratory through hydrol- ANaseactivity of the eluates from the DEAE-cellulose ysis of the corresponding nucleosides. and Sephadex G-100 columns was assayed by the Somogyi-Nelson method, as described previously.9) Assay for enzyme activity. The assay for N-MeNaseis One unit was defined as the amount of enzyme which based on the separation of 7-methylxanthine from 7- hydrolyzes 1 //mol of per 1 minute under the

Fig. 2. Retention Times of Purine Nucleosides and Bases on HPLCAnalysis. Column, UNISIL PACK5C18-250A (4.6 mmi.d. x 250 mm); solvent, H2O-acetonitrile-AcOH-triethylamine (95 : 3 :0.3 :0.3); flow rate, 1 ml/min; temperature, 22°C. Detection: A250 for Guo, Gua, lMeGuo, lMeGua, Ino, Hyp, lMelno, lMeHyp, 7MeIno, 7MeHyp; A260 for Ado, Ade, lMeAdo, lMeAde, 3MeIno, 3MeHyp; A270 for 3MeAdo, 3MeAde, 7MeAdo, 7MeAde, 3MeGuo, 3MeGua, 7MeGuo,7MeGua, Xao, Xan, lMeXao, lMeXan, 3MeXao, 3MeXan, 7MeXao,7MeXan. Abbreviations for nucleosides and bases: Ado, adenosine; Ino, inosine; Guo, guanosine; Xao, xanthosine; Ade, ; Hyp, ; Gua, guanine; Xan, xanthine. TV-Methyl Nucleosidase from Tea Leaves 171 standard assay conditions. Table I. Degradation of Ribonucleosides by In the experiment on substrate specificity, the assaying the Crude Enzyme of both was carried out by the HPLCmethod. The assay conditions were changed as follows because N- Substrate Activity* (nmol/min) methylated nucleosides are unstable under acidic and alkaline conditions.lo'n) The buffers used were 0.1 m Adenosine 376,000 HEPES-NaOH(pH 7.5) for N-MeNase and 0.1 m sodium Guanosine 510 acetate (pH 5.5) for ANase. The reaction mixture com- Inosine 113 prised 40[A of the buffer, 40/^1 of 3.75mM substrate and Xanthosine 2,060 7-Methylxanthosine 6,560 20ji\ of enzymesolution. The reaction was performed at 37°C for 1 hr and stopped by freezing the reaction mixture immediately in a cold MeOHbath (-50°C). The column The assay conditions were the same as in the experi- and solvent used for HPLCwere an ODScolumn, 4.6mm ment on substrate specificity. The buffers used were i.d.x250mm in size (UNISIL PACK 5C18-250A; 0.1 m Tricine-NaOH (pH 8.5) for 7-methylxan- Gasukuro Kogyo Inc.) and H2O-acetonitrile-AcOH- thosine and 0.1 m sodium acetate (pH 5.0) for the triethylamine (95 : 3 :0.3 :0.3), respectively. The nucle- other substrates. osides and the corresponding bases were detected at the wavelengths indicated in the legend to Fig. 2. They were all separated from each other (Fig. 2), and the bases were 30,000 x g and then dissolved in the extraction quantitatively determined. solvent used above. After centrifugation, the supernatant was dialyzed for 20hr against Identification of the degradation products of 7-methyl- 0.01 m Na-phosphate buffer (pH 7.5). xanthine. One of the reaction products, 7-methylxanthine, was identified by HPLC as described above. The other Five kinds of purine ribonucleosides (aden- one, derived from the sugar moiety, was characterized by osine, guanosine, inosine, xanthosine and 7- paper chromatography. The standard reaction mixture methylxanthosine) were incubated with the incubated at 37°C for 4hr was chromatographed using crude enzyme solution at 37°C for 1 hr, and the Whatman No. 1 filter paper in «-BuOH-AcOH-H2O bases released were assayed by HPLC. As (4 : 1 : 5), and then reducing sugar was detected by spraying with aniline hydrogen phthalate followed by heating for shown in Table I, besides adenosine nucleo- 5min at 105°C. sidase activity, degrading activity toward 7- methylxanthosine was also detected. The other RESULTS AND DISCUSSION nucleosides can also serve as substrate, but their cleavage rate were relatively low. Preparation andproperties of the crude enzyme All the enzymepurification procedures were Purification of N-methyl nucleosidase performed at 4°C. Step 1. DEAE-Cellulose column chroma- Tea leaves (1 kg) were homogenized in sev- tography. The dialyzed enzyme solution was eral separate batches with cold acetone for applied to a DEAE-cellulose column (2.5 x 3 min. The homogenate was filtered through a 28cm) equilibrated with 0.01m Na-phos- filter paper with suction and the residue was phate buffer (pH 7.5) and then the column washed three times with a mixture of acetone was washed with the same buffer. Elution was and water (4 : 1) containing 0.1% ascorbic acid performed successively with 0.01 m, 0.05 mand to removethe polyphenols. Theacetone pow- 0.1 m Na-phosphate buffer (pH 7.5). A typical der thus obtained was extracted for 1 hr with enzyme activity elution pattern is shown in 0.1 m disodium hydrogen phosphate solution Fig. 3. A peak showing 7-methylxanthosine (pH 7.0) containing 0.6% ascorbic acid. The degrading activity was eluted at 0.05m Na- solution was squeezed through cheesecloth phosphate and thus separated from ANase and then centrifuged for 30min at 3,300 xg. activity, which was eluted at 0.1m Na- Solid ammoniumsulfate was added to the phosphate. A minor peak of another purine crude extract to 70% saturation, followed by nucleosidase was found in the 0.01m Na- stirring for 30min. The precipitate was col- phosphate fraction. lected by centrifugation for 20min at Step 2. Hydroxylapatite colunm chroma- 172 O. Negishi, T. Ozawa and H. Imagawa

Fig. 3. Elution Profiles of N-Methyl Nucleosidase and Adenosine Nucleosidase on a DEAE-Cellulose Column. Column size, 2.5 x 28cm; flow rate, 32ml/hr; fraction volume, 16ml/tube. Enzyme activities: 7MeXao, #-#; Ado, O-O; Xao, O-O- Absorbance at 280nm, tography. The fractions containing N-MeNase Table II. Purification of tV-Methylnucleosidase activity obtained on DEAE-cellulose column from Tea Leaves chromatography were applied to a hydroxyl- Total Total Specific apatite column (2.0 x 12cm) previously equi- Step protein activity activity Yield librated with 0.01 m Na-Phosphate buffer (mg) (mU) (mU/mg) (pH 7.5). After washing the column with the Ammonium sulfate 1 1 13 6560 5.9 100 same buffer, the enzyme was eluted successive- DEAE-Cellulose 355 4150 1 1.7 63 ly with 0.04m, 0.08m and 0.16m Na-phos- Hydroxylapatite 61.5 1570 25.5 24 phate buffer (pH 7.5). N-MeNase activity Sephadex G-100 12.6 370 29.4 5.6 and a small amount of xanthosine degrad- ing activity were eluted in the 0.08m buffer fraction, and no other peak of nucleosidase II. The N-MeNasefractions were pooled and activityStep 3. beingGel filtrationdetected. on Sephadex G-100. concentrated to 134mU/ml, and used for the The enzyme solution from step 2 was con- subsequent experiments. centrated to 1ml by ultra filtration with an Amicon PM-10 membrane and then chroma- Properties of N-methyI nucleosidase tographed on a Sephadex G-100 column Molecular weight. The molecular weight was (2.0x82cm) equilibrated with 0.01m Na- estimated to be about 55,000 by Sephadex G- phosphate buffer (pH 7.5). N-MeNaseactivity 100 column chromatography, through com- was eluted as a single peak and a small amount parison with the elution volumes of standard of contaminating adenosine degrading activity proteins (aldolase, 168,000; bovine serum al- was eluted separately. Xanthosine degrading bumin, 68,000; ovalbumin, 45,000; chymotryp- activity was.not detected. The results of the sinogen A, 25,000; and cytochrome c, 12,000). overall purification are summarized in Table Mode of action. The sugar portion of the A^-Methyl Nucleosidase from Tea Leaves 173 reaction products was detected as a pink spot sayed. The enzyme was unstable in acidic on the chromatogramafter spraying with the media (Fig. 5). reagent. Its Rfvalue corresponded with that of Effects of temperature. The effects of tem- an authentic sample of D-ribose. This indicates perature on the enzyme activity and stability that the present enzyme cleaves 7-methyl- were examined. Maximumactivity was ob- xanthosine hydrolytically and not phospho- served at temperatures ranging from 40 to rolytically. 45°C and the activity rapidly decreased above Effect of pH. The optimum pH, from 8.0 to 50°C. The enzyme solution was kept at vari- 8.5 (Fig. 4), was considerably different from ous temperatures for 15min. After cooling, that for adenosine nucleosidase, i.e., pH 4.5.9) the remaining activity was assayed under After keeping the enzyme at various pHs for the standard conditions. The enzyme rapid- 24hr at 4°C, the remaining activity was as- ly lost its activity above 40°C. Effects of metal ions and other compounds. Among the various substances tested, EDTA was a potent inhibitor (Table III). 7- Methylxanthine and 7-methylhypoxanthine at 5niM inhibited about 50% of the enzyme activity. Substrate specificity. Enzyme activity was measured using 16 purine nucleosides, includ- ing four free purine nucleosides and their TV- methylated derivatives, as substrates. The specificity of ANase (specific activity: 4.0 units/mg), which was partially purified by col- umn chromatography on DEAE-cellulose and Sephadex G-100, was also examined. Fig. 4. Effect of pH on the Enzyme Activity. The reaction was carried out under the standard assay Table III. Effects of Metal Ions and Reagents conditions. The buffers used were PIPES-NaOH å (à"), on the N-Methyl Nucleosidase Activity Tricine-NaOH (O) and glycineTNaOH (A). The enzymeactivities were measuredunder the stan- dard assay conditions, after incubating the enzymeso- lution with various metal ions or reagents at 37°Cfor 15min.

o , , . Relative Substance mM .. /o/. activity (%)

None 100 MgCl2 (1) 105 CaCl2 (1) 69 FeSO4 (1) 104 CoCl2 (1) 101 CuSO4 (l) 83 ZnSO4 (1) 90 NaF (1) 104 EDTA (l) ll ICH2COOH (1) 107 Fig. 5. pH-Stability. PCMB (0.5) 19 7MeXan (1) 95 The enzyme solution was kept at 4°C for 24hr at various (5) 60 pHs (A, sodium acetate; A, PIPES-NaOH; #, Tricine- 7MeHyp (1) 103 NaOH; O, glycine-NaOH). After adjustment of the pH to (5) 50 8.5, the remaining activity was assayed. 174 O. Negishi, T. Ozawa and H. Imagawa

The results are shown in Table IV. N- substrates tested, the 3- and 7-methylpurine MeNase hydrolyzed the nucleosides except nucleosides were hydrolyzed preferentially by guanosine and 1-methylguanosine. Amongthe this enzyme. These facts indicate that a methyl group attached at the 3- or 7-position of the nucleosides is important for the enzyme ac- Table IV. Substrate Specificities of TV-Methyl NUCLEOSIDASE AND ADENOSINE NUCLEOSIDASE tivity. On the other hand, ANase showed high activity toward adenosine and 7-methyladen- The final concentrations of substrates were 1.5mM. The incubation was carried out at 37°C for 1 hr, with osine, but the activity toward other substrates 1.5 mUof TV-methyl nucleosidase in 0.1 MHEPESbuffer was very low or nil. It had already been (pH 7.5) or 0.5mU of adenosine nucleosidase in 0.1 m reported9) that ANase hydrolyzes only aden- acetate buffer (pH 5.5). osine amongfree ribonucleosides. Amongthe Relative activity (%) methylated nucleosides tested here, 7-methyl- Substrate" - adenosine was hydrolyzed five times faster TV-MeNase AN ase than adenosine, whereas 7-methylxanthosine Ado 6 100e was not hydrolyzed at all. l MeAdo 22 1 3MeAdofe 1 7.8 7 Role of nucleosidases in caffeine biosynthesis 7MeAdoc 13 515 In the previous papers1'2'12) we indicated Guo 0 0 l MeGuo 0 0 that the pathway for caffeine biosynthesis is as 3MeGuo 32 0 follows: xanthosine-åº7-methylxanthosine -åº7- 7MeGuo 189 0 methylxanthine ^ theobromine -^caffeme. This Ino 5 0 IMelno 9 0 pathway involves the degradation reaction for 3MeIno 128 0 the iV-glycosyl bond of methylated nucleoside. 7MeIno 121 0 Based on the results as to the substrate speci- Xao 8 0 ficities of the nucleosidases it is evident that lMeXao 12 0 the enzyme responsible for the hydrolysis of 3MeXao 198 0 7-methylxanthosine is N-MeNase. Thus 7MeXao 100d 0 ANase does not directly catalyze the reac- a See Fig. 2 for abbreviations for substrates. tion in the pathway of caffeine biosynthesis. b 3-Methyladenosine /7-toluenesulfonate. In plants, ANase is regarded as a member of c 7-Methyladenosine perchlorate. the adenine salvage pathway.7) The same role d 73.8 nmol of 7-methylxanthine was released into the is considered for ANase from tea leaves. reaction mixture. e 46.9nmol of adenine was released into the reaction Suzuki and Takahashi13) demonstrated that mixture. the radioactivity of [8-14C]adenine is incor-

Fig. 6. Adenine Salvage Pathway and Caffeine Biosynthesis. a) Adenosine nucleosidase; b) N-methyl nucleosidase; c) purine nucleosidase. N-Methyl Nucleosidase from Tea Leaves 175

porated into RNA purine nucleotides and REFERENCES caffeine. This indicates the presence of the adenine salvage pathway in tea plants. Re- O. Negishi, T. Ozawa and H. Imagawa, Agric. Biol. Chem., 49, 887 (1985). cently, Suzuki and Waller14) indicated that O. Negishi, T. Ozawa and H. Imagawa, Agric. Biol. caffeine was mainly derived from the nucle- Chem., 49, 2221 (1985). otides produced through the purine salvage M. F. Roberts and G. R. Waller, Phytochemistry, 18, pathway rather than from those synthesized 451 (1979). de novo. T. Suzuki and G. R. Waller, Abstracts of Papers, Besides the caffeine biosynthetic pathway, 23rd Ann. West Cent. States Biochem. Conf., Columbia, Mo., Paper No. 53, 1980. there is the catabolic pathway from xanthosine G. R. Waller, C. D. MacVean and T. Suzuki, Plant to allantoin and other metabolites.2) In the Cell Reports, 2, 109 (1983). present study, the presence of an enzyme A. Guranowski, Plant Physiol., 70, 344 (1982). A. Guranowski and Z. Schneider, Biochim. Biophys. which hydrolyzes xanthosine was detected in Acta, 482, 145 (1977). the crude extracts of tea leaves. Charac- C-M Chen and B. Petschow, Plant Physiol., 62, 871 terization of the enzyme responsible for the (1978). degradation of xanthosine remains to be per- H. Imagawa, H. Yamano, K. Inoue and Y. Takino, formed. Agric. Biol. Chem., 43, 2337 (1979). The roles of nucleosidases in tea leaves, T. Itaya and H. Matsumoto, Tetrahedron Lett., 1978, 4047. related to purine metabolism and caffeine bio- T. Fujii, T. Itaya and T. Saito, Yuki Gosei Kagaku synthesis, are illustrated in Fig. 6. Kyokaishi, 41, 1193 (1983). Acknowledgments. The authors wish to express their O. Negishi, T. Ozawa and H. Imagawa, Agric. Biol. Chem., 49, 251 (1985). thanks to Professor Tozo Fujii, Faculty of Pharmaceutical Sciences, Kanazawa University, and Dr. Akihiro T. Suzuki and E. Takahashi, Phytochemistry, 15, 1235 (1976). Yamazaki, Central Research Laboratories, Ajinomoto Company Inc., for kindly supplying the methylated T. Suzuki and G. R. Waller, Nippon Doj'yo- nucleosides. Hiryogaku Zasshi, 57, 70 (1986).