A chemo-enzymatic hydrolysis of calcium salt of RNA to nucleosides

BIOTECHNOLOGY

TECHNIQUES

Volume 10 No. 1 I (November 1996) pp.875878 Received as revised 13 September A CHEMO-ENZYMATIC

HYDROLYSIS OF CALCIUM NUCLEOSIDES

SALT OF RNA TO

V.N. Barai*, A.I. Zinchenko, LA. Bravsevich, and I.A. Mikhailopulo# Institute of Microbiology and #Institute of Bioorganic Chemistry, Academy of Sciencesof Belarus, 220141 Minsk, Republic of Belarus SUMMARY Ribonucleosides can be produced by calcium-catalyzed cleavageof RNA followed by dephosphorylation of calcium salts of mononucleotides by intact mycelium of Spicurio violucea as a biocatalyst. Under optimal conditions the efficiency of degradation of baker’syeast RNA to a mixture of ribonucleosides is 96% of the theoretical value. INTRODUCTION Naturally occurring ribonucleosidesare valuable starting materials for the chemical and enzymatic synthesis of pharmaceutical agents(Wang and Whitesides, 1995). These compoundscan be obtained by both chemical synthesis and fermentation, however the most convenient mode of their large-scale production is still isolation from yeastRNA (Sowa, 1976;Teshiba and Furuya, 1989). Preparation of ribonucleosidesfrom RNA may be regardedas two-stageprocesswhere RNA is split into a mixture of four constituent nucleosidemonophosphatesfollowed by their dephosphorylation to the corresponding nucleosidesand inorganic phosphate. Chemical decomposition of RNA can be achieved by several procedures,e.g., treatment of RNA with concentrated aqueous ammonia at 175-178 “C; with aqueous pyridine for a few days at room temperature; with aqueous formamide; or with metallic catalysts such as lead hydroxide, calcium hydroxide or lanthanum hydroxide in aqueoussolution under reflux for 40-50 h (Dimroth et al., 1959; Hayes, 1960; Sowa, 1976; Brown et al., 1985).Degradation of RNA to nucleotides proceedsmuch more easily than their subsequentdephosphorylation. Enzymatic cleavage of RNA by the consecutive action of exonuclease and phosphatase offers obvious advantagesover chemical proceduresincluding the mild operating conditions and the specificity of the reactions (Nakao, 1976; Shakhova et al., 1988). Nevertheless, it is difficult to obtain ribonucleosides by enzymatic digestion of RNA in good yields due to product inhibition causedby the inorganic phosphate generated during the reaction (Dam and Harsanyi, 1972; Wiegand et al., 1975; Arnold et al., 1986; Kliment et al., 1987). In the present paper we have demonstratedthat the efficiency of RNA cleavageto ribonucleosides can be enhanced by coupling chemical and enzymatic processes,viz., hydrolysis of RNA to nucleoside monophosphatesunder the action of Ca2+ ions followed by dephosphorylation of formed calcium salts of mononucleotidesemploying a fungal phosphatase.

MATERIALS AND METHODS Reagents.RNA (from baker’syeast) and its calcium salt were preparedas reported previously (Barai et al., 1995). Standard Silufol UV254 plates usedfor thin layer chromatography(TIC) and DNA from calf thymus were purchasedfrom Serva.p-Nitrophenyl phosphatedisodium salt was obtained from Fluka. Microorganisms. A fungal strain usedfor RNA hydrolysis to ribonucleosideswas Spicaria violacea BM105D. The mycelium showing exonucleaseand phosphataseactivities was preparedas described earlier (Zinchenko et al., 1990). Exonucleaseand phosphataseactivities exhibited by mycelium at pH 5.0 were equal to 27 and 90 units/mg of dry wt. At pH 9.0 the same activities were respectively 23 and 22 units/mg of dry wt. Enzvme U.WJVS. The acid and alkaline phosphataseactivities were assayedat 50 “C for I5 min in a medium containing intact mycelium (1 mg/mL, calculated as dry wt.) and 10 mM disodium pnitrophenyl phosphate in 0.1 M sodium acetate (pH 5.0) or 0.1 M Tris-HCl (PH 9.0) respectively. Eiberatedp-nitrophenol was quantified from the absorbanceat 400 nm. For the exonucleasedetermination, the mixture containing mycelium (3 mg/mL), DNA as substrate (10 mg/mL), 5 mM MgC12 and 0.1 M sodium acetate(pH 5.0) or 0.1 M Tris-HCl (pH 9.0) was incubated at 50 “C for 15 min. The reaction was terminated by addition of two volumes of cold ethanol containing 20 mM MgCl2. AEtera short time in the cold the mixture was centrifuged and amount of mononucleotides in the supernatantwas estimatedspectrophotometricallyat 260 nm. All enzyme activities were expressedas nanomoles of product formed per minute per milligram of mycelium. Enzvmatic hvdrolvsis ofRALA.A standardreaction mixture (20 mL) containing 400 mg RNA (calculated as free acid) and intact mycelium of Spicaria violucea (160 mg, calculated as dry wt.) was incubated at 50 “C with gentle mixing. Other parametersof the reaction varied in accordancewith below itemized variants. Variant I. The reaction mixture contained sodium salt of RNA (Na-RNA). The mixture was kept at pH 9.0 by the pH stat-controlled addition of 1 M NaOH. Variant II. The reaction mixture contained RNA in the form of its calcium salt (Ca-RNA). The pH was maintained at 9.0 by addition of 10% Ca(OH)2 suspension. Variant IV. The reaction mixture contained Na-RNA and 50 mM sodium acetatebuffer @H 5.0). Variant V. The reaction mixture contained Ca-RNA and 50 mM sodium acetatebutfer @H 5.0). Chemo-enzvmatic hvdrolvsis of RNA. For the chemical transformation of RNA into 2’(3’)mononucleotides, Ca-RNA (400 mg, calculated as free acid) was suspendedin 15 mL of water. The pH of the mixture was adjustedto 12.0 with Ca(OH)2, and then it was incubated with stirring at 85 “C! for 45 min. Under these conditions the yield of 2’(3’)-mononucleotidesattained >98% relative to the initial RNA amount. Upon cooling to 50 “C, the reaction mixture was brought to pH 9.0 (variant III) or to pH 5.0 (variant VI) with 1 M H3P04. Moreover, in caseof variant VI, 1.0 mL of 1 M sodium acetate buffer (pH 5.0) was added to the reaction mixture. Intact mycelium of Spicuria violacea (160 mg, calculated as dry wt.) was addedand the volume was adjustedto 20 mL. The mixture was incubated at 50 “C with gentle mixing. The pH of the medium corresponding to variant III, was kept during the reaction near 9.0 by addition of Ca(OH)2. Products formed in the courseof RNA hydrolysis were monitored by TLC. Before TLC, aliquots of reaction mixtures containing Ca2+ ions were diluted two times with 0.2 M EDTA (pH 7.0). The solvent used was n-butanol - acetic acid - water (5:2:3; v/v). Products were eluted from the plates with 5 mM potassium phosphate buffer (PH 7.0) and quantified spectrophotometrically. For estimation of overall amounts of mononucleotidesand oligonucleotides one optical unit at 260 nm was taken to correspond to 0.1 pmol of free nucleotides, and 0.11 umol of nucleotidesarrangedin oligonucleotides. RESULTS AND DISCUSSION Earlier we selecteda fimgal strain Spicuria viofucea BM-105D whose intact mycelium displays high exonucleaseand phosphataseactivities and can effectively hydrolyze DNA to 2’deoxynucleosides at 50 “C (Zinchenko et al., 1990).Further studies revealedthat the mycelium of Spicaria violacea BM105D shows ability to hydrolyze both DNA and RNA to nucleosidesover a broad pH range (from 4 to

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lo), and these processesare considerably suppressedby orthophosphategeneratedas an end product of mononucleotide dephosphorylation. We have attemptedto eliminate the negative effect of inorganic phosphateon the processof RNA hydrolysis by meansof removal of orthophosphatefrom the reaction medium using Ca2+ precipitation. Calcium was chosenbecauseof its low toxicity and poor solubility of calcium orthophosphatein water. Experiments on RNA hydrolysis were carried out both in alkaline @H 9.0) and acid (pH 5.0) media. Four variants of the procedurewere initially compared,namely: I and IV - hydrolysis of RNA in the form of sodium salt; II and V - hydrolysis of RNA in the form of calcium salt. The variants 11and V were set up to test in a direct experiment the possibility of using Ca-RNA as a starting material for ribonucleoside production. We assumed that this would provide conditions for the most effective removal of orthophosphatefrom the reaction medium. The data obtained are listed in Table 1. To evaluate the efficiency of RNA hydrolysis we have chosen yield of ribonucleosides and intermediates as well as the time required to complete enzymatic and chemical steps. Table 1. Yield (mol. %) of reaction products under different variants of RNA hydrolysis

Products

Mononucleotides

Time of chemical

-

0.75

0.75

reaction (h) Time of enzymatic

9

9

6

15

1.5

5

reaction (h) * SeeChemo-enzvmatichydrolysis of RNA in MATERIALS

AND METHODS.

From Table 1, phosphohydrolasesof Spicaria violacea BM-105D are able to hydrolyze Ca-RNA and dephosphorylate calcium salts of formed mononucleotides, both in acidic and alkaline media. Further examination of the results shows that the replacement of Na-RNA by Ca-RNA reduces the amount of non-transformed mononucleotides in the final reaction mixture. This phenomenon may apparently indicate the lack of acid and alkaline phosphataseinhibition by inorganic phosphate under specified set of conditions. It should be noted that Ca-RNA digeststo a lesserdegreeas comparedwith Na-RNA, especially in acid media. This effect may be accountedfor by the formation of strong complexes between Ca2+ ions and certain RNA sites, appearing as poor substratesfor the exonucleases.

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RNA is known to be readily hydrolyzed into mononucleotidesunder basic conditions by Ca2+ ions. Taking this into account together with our finding that phosphatasesof Spicaria violacea are able to dephosphorylate calcium salts of mononucleotides,we presumedthat coupling of these chemica1and enzymatic reactions would result in elevatednucleosideyields. Indeed, as can be seenfrom Table 1, this approach (variants III and Vr) in both casesbrings about essential efficiency improvements, manifested as the highest yield and the shortestduration of the process. It is noteworthy that the efficiencies of III and U variants do not differ markedly, though the mycelium acid phosphataseactivity exceedsthat of alkaline phosphatasefour times (see Materials and Methods). This discrepancy is attributable to the fact that orthophosphateremoval by Ca2+ ions from the reaction medium proceedsmore effectively at pH 9.0 than at pH 5.0. The resultant solution obtained in the variants 111and VI contained (mg) adenosine (89.6; 89.3) guanosine (91.3; 90.1), uridine (72.1; 65.5) and cytidine (53.1; 59.0). A cytidine deficiency in the alkaline media is due to its partial deamination by cytidine deaminase. It should be noted that the final mixture of nucleosidesgeneratedby the chemo-enzymatic method, is lacking in inorganic phosphateand contains minute amountsof oligonucleotides and mononucleotides that appreciably simplifies the isolation of individual nucleosidesthrough crystallization of guanosine followed by ion-exchange chromatographyof remaining nucleosides. Furthermore, according to our preliminary data, Ca-RNA (unlike Na-RNA) ensures drastic increase (up to 200 mg/mL) of RNA concentration in the initial reaction mixture. In conclusion, this study demonstratesfor the first time a considerable potential of Ca-RNA as a starting material for chemo-enzymatichydrolysis to nucleosides. REFERENCES Arnold, W.N., Mann, L.C., Sakai, K.H., Garrison, R.G. and Coleman,P.D. (1986). .I. Gen. Microbial. 132, 3421-3432. Barai, V.N., Kukharskaya, T.A. and Zinchenko, A.I. (1995). Prikl. Biokhim. Microbial. [in Russian] 31, 494-497. Brown, R.S., Dewan, J.C. and Klug, A. (1985). Biochemistry 24,4785-4801. Dimroth, K., Witzel, H., H&en, W. and Mirbach, H. (1959). Liebig’sAnn. Chem. 620, 94-108. Dom, G.L. and Harsanyi, Z. (1972). J. Bacterial. 110,246-255. Hayes,D.H. (196O).J. C/rem.Sot. 1184-1187. Kliment, J., Gaspar,R., Kasar, J., Zelinkova, E. and Zelinka, J. (1987). Biologia (Bratislava) 42, 753764. Nakao, Y. (1976). In: Microbial Production of Nucleic Acid-Related Substances,K. Ogata, S. Kinoshita, T. Tsunoda and K. Aida, eds.pp. 87-100, Tokyo: Kodansha. Sowa, T. (1976). In: Microbial Production of Nucleic Acid-Related Substances,K. Ogata, S. Kinoshita, T. Tsunoda and K. Aida, eds.pp. 112-121,Tokyo: Kodansha. Teshiba, S. and Furuya, A. (1989). Production of Nucleotides and Nucleosides by Fermentation, New York etc: Gordon and Breach SciencePublishers. Shakhova,T.V., Mayorova, G.I., Nesterenko,E.A. and Orlova, T.V. (1988). Biotechnofogiya [in Russian]. 4, 501-505. Wiegand, R.C., Godson, G.N. and Radding, C.M. (1975).J. Biol. Chem. 250,8848-8855. Wang, C.H. and Whitesides, G.M. (1995). Enzymesin Synthetic Organic Chemistry, Oxford: Pergamon Press. Zinchenko, AI., Bar%, V.N., Bokut, S.B., Dudchik, N.V., Belyaeva,Yu.V. and Mikhailopulo, I.A. (1990). BiotechnoE.Lett. 12,341-346.

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