Enzymatic synthesis of ATP from RNA and adenine

B I O T E C H N O L O G Y L E T T E R S Volume 17 No .6 (June 1995) pp .599 -602 Received 28th Apri l

ENZYMATIC SYNTHESIS OF ATP FROM RNA AND ADENINE

V.N. Barai*, A.I. Zinchenko, L.M. Zalashko, L.A. Eroshevskaya, and I.A. Mikhailopulo #

Institute of Microbiology and #Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, 220141 Minsk, Republic of Belarus

SUMMARY The title compound was prepared by a three-stage enzymatic procedure consisting of (i) RNA hydrolysis to a mixture of ribonucleosides using intact myceUum of Spicaria violacea, (ii) transribosylation of exogenous adenine employing whole cells of Escherichia coil as a biocatalyst, and (iii) conversion of formed adenosine into ATP by the enzymes of alcohol fermentation and the kinases extracted from baker's yeast.

INTRODUCTION

Since ATP is a precursor of RNA and an energy donor in various biochemical reactions, it has been

widely used as a biochemical and therapeutic agent. Therefore, methods of producing this substance and

their improvement are of great importance (for a review, see Yamada and Shimizu, 1988). One of

approaches to production of ATP, described in literature is hydrolysis of RNA by nuclease P1 to a

mixture of four 5'-nucleoside monophosphates followed by a conversion of AMP to ATP catalyzed by

adenylate kinase and acetate kinase, where acetyl phosphate serves as phosphoryl donor (Leuchs et al.,

1979; Haynie and Whitesides, 1990).

Earlier, we have reported on the synthesis of 2'-deoxyadenosine by an enzymatic

transdeoxyribosylation of adenine (Ade) using the mixture of 2'-deoxynucleosides resulting from

enzymatic hydrolysis of DNA as donors of carbohydrate moiety (Zinchenko et al., 1991). Moreover, it is

well known that enzymes of alcohol fermentation and the kinases extracted from permeabilized yeast

cells catalyze ATP regeneration from adenosine (Ado), using glucose as an energy source (Asada et al..

1978; Zinchenko et al., 1990c). With this background, in the present work we have studied an

analogous approach for the synthesis of Ado from enzymatic RNA hydrolysate and Ade in order to

convert it then to ATP.

MATERIALS AND METHODS

Reagents. RNA (from yeast) and Ade were purchased from Fluka (Buchs, Switzerland). Silufol UV254 plates (Serva, Germany) were used for thin layer chromatography (TLC).

Microorganisms. A fungal strain ~ for RNA hydrolysis to ribonucleosides was Spicaria violacea BM- 105D. The mycelium showing exonuelease and phosphatase activities was prepared as reported previously (Zinchenko et al., 1990a). Strain E. coli BMT-ID/1A was selected according to Munch- Petersen et al. (1972). The bacterial cells possessing high uridine (Urd) phosphorylase (UPasc), purine

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nucleoside phosphorylase (PN-Pas¢), and cytidine (Cyd) deaminase (CDase) activities were grown as described earlier (Zinchenko et al., 1990b). The yeast used for preparing Ado phosphorylating cell-free extract was a pressed commercial baker's yeast.

HFdroiFsis ofRNA. RNA (2 g) was dissolved in 80 mL of distilled water. The pH was adjusted to 5.3 with 1 M KOH. The volume was adjusted to 100 mL and intact mycelium of Spicaria violacea (1 g, calculated as abs. dry wt.) was added. The mixture was then incubated with stirring at 50 °C for 5 h. The pH of the medium was kept between 5.0 and 5.3 by the pH star-controlled addition of 1 M KOH. The reaction was terminated by boiling for 5 min with subsequent withdrawal of mycelium by filtration.

TransribosFlation o f Ade. The reaction mixture contained the filtrate obtained at the preceding stage, 0.9 g of Ade and wet paste ofE. coli cells (0.1 g, dry wt.). The pH value of the mixture was adjusted to 7.0 with 1 M KOH, and then it was incubated with genOe stirring at 60 °C for 2 h. The resultant reaction mixture was further used as a source of Ado in the reaction of ATP synthesis.

Preparation ofphosphorFlating Feast extract. The yeast ceils were dried at 28 °C for 24 h and treated with 0.4 M solution of K-phosphate buffer (pH 7.0; 10 mL/g of dry yeast) containing 4% glucose and 10 mM MgCI 2. The suspension was kept for 3 h at 28 °C and then centrifuged (8000 x g; 5 min). The supematant was used as the source of enzymes.

Transformation o f Ado to ATP. For the ATP synthesis, the phosphorylating yeast extract (100 mL) was added to final reaction mixture obtained at the preceding stage. The suspension was incubated with gentle stirring at 28 °C for 16 h followed by heating for 3 rain in a boiling water bath and removing bacterial ceils by centrifugation. For isolation of ATP, the supernatant obtained (approximately 200 mL)

was diluted with water to 1 L and loaded onto a column with Dowex 1 x 8 (CI- ; 20 x 100 mm) ion- exchange resin. The column was initially washed with 3 mM HCI to remove Ado and then with 10 mM solution of HCi containing 90 mM NaCI which ¢luted AMP and ADP. The ATP was tinted from the column with 10 mM solution of HCI containing 16 mM NaCI. A'[P-containing fractions were pooled and concentrated to a small volume under reduced pressure. The concentrate was brought to pH 2 with HCI followed by the addition of 4 volumes of cold ethanol to precipitate ATP as the disodium salt. The procedure described yielded 2.09 g (3.80 mmol) of ATP-Na 2 which was identical with the reference specimen (UV spectral data and TLC). To recover the unreacted Ade, solution containing Ade was

passed through a column with Dowcx 50 x 8 (H+; 20 x 100 mm) ion-exchange resin. The column was washed with water, and Ade was then eluted with 0.5 M ammonia. The ¢luat¢ was concentrated in vacuo to 20 mL and Ade was crystallized at 4 °C to yield 405.3 mg of Ade.

AnalFses. Nucleoside accumulation in the course of RNA cleavage as well as Ado formation in the reaction of Ade transribosylation were monitored by HPLC according to procedure described earlier (Zinchenko et al., 1991). The conversion of Ado to ATP was examined by TLC (Silufol UV254; dioxane/isopropanol/water/25% aq. ammonia, 4:2:4:1).

RESULTS AND DISCUSSION

The general scheme for enzymatic synthesis of ATP from RNA and Ade is illustrated ill Fig. 1.

First, exonuclease in combination with phosphatase of Spicaria violacea catalyze the decomposition of

RNA yielding four ribonucleosides (stage 1). At the second stage, Cyd present in the hydrolysate is

deaminated to Urd by CDase ofE. coli. Urd and guanosine (Guo) in the presence of inorganic phosphate

are subjected to phosphorolysis to give ribofuranose-l-<x-phosphate (not shown in Fig. 1) and free

guanine (Gua) and uracil (Ura), under the action of E. coli UPase and PNPase. Then, PNPas¢

transforms ribofuranose-l-<x-phosphate and exogenous Ade into Ado with the release of phosphate.

Finally, Ado is converted to ATP using enzymes of alcohol fermentation and the kinases extracted from

cells of baker's yeast (stage 3).

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The main peculiarity of the offered approach consists in rather effective utilization of a mixture of

nucleosides produced at RNA hydrolysis stage for the synthesis of Ado that is then used for ATP

production "without isolation in individual form.

RNA ---

2

Cyd + Urd

AlP

+ Guo + Ado

Ade Gua + Um

Fig. 1. Pathways of ATP synthesis from RNA and Ade using whole cells ofSpicaria violacea (stage 1), E. coli (stage 2) and cell-free extract of baker's yeast (stage 3).

Ado contents in the reaction mixtures at intermediate stages of ATP synthesis as well as the

amount of ATP in the resultant solution are given in Table 1.

It should be noted that several important factors affecting Ado and ATP production (pH,

temperature, concentration of substrates and biocatalyst, etc.) were pre-optimized. For example, the

reactions of RNA hydrolysis and Ade transribosylation were performed at high temperature (50 and 60 °

C, respectively), when such enzymes as nucleosidases and Ado deaminases are inactivated.

Table 1. Amounts of Ado and ATP formed at different stages of ATP synthesis.

Stage

1. Hydrolysis of RNA

Products (mmol) Ado I 1.49

ATP

2. Transribosylation of 4.19 Ade

3. Phosphorylation of traces 4.11(3.82)* Ado

*Value obtained while Ado phosphorylation was carried out in the absence ofE. coli cells.

The resultant solution obtained in the typical experiment under the optimum conditions contained

(mmol) ATP (4.11), ADP (0.19), AMP (0.07), Ado (traces), inosine (0.14), Ade (3.60), Ura (2.47), and

Gua (traces, due to very low solubility). Such figures correspond to a 91% selectivity for conversion of

Ado into ATP.

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Noteworthy, ATP yield is increased if the transformation of Ado into ATP proceeds in the presence

orE. coil cells which are not removed from reaction mixture upon completion of the second stage of the

process. This effect can apparently be explained as follows. The reaction of Ade transribosylation

catalyzed by E. coli PNPase is known to be reversible, therefore, at the end of the second stage a

dynamic equilibrium is established between Ado and its precursors. The reduction in the Ado level as a

result of its phosphorylation by yeast kinase, is counterbalanced by formation of additional quantities of

this nucleoside. This, in turn, favours the accumulations of ATP.

Our experiments indicated that dried cells of baker's yeast could also phosphorylate Ado to ATP,

but in accordance with the data reported by Asada et al. (1978) cell-free extract exhibited higher

efficiency as a source of biocatalysts in comparison with whole cells. Moreover, in the reaction with cell-

free extract, the level of ATP after its maximum accumulation was kept constant for a long time. On the

contrary, in the case of dried cells the net conversion of Ado to ATP was very sensitive to the reaction

time. This effect might be due to the influence of ATPase not released from the cells when preparing the

cell-free extract.

The resultant mixture generated by the aforementioned procedure contains nearly 20 mM ATP. We

suppose that purification step is not essential prior to its use in some enzymatic reactions involving ATP

recycling. However, pure ATP can be readily isolated from this mixture if required (see Materials and

Methods).

In conclusion, the method described in this communication supplements the methods available in

the literature for the synthesis of ATP from RNA as a starting material (Leuchs et al., 1979; Ping and

Lu, 1989; Haynie and Whitesides, 1990).

Acknowledgement I.A.M, is deeply grateful to the Alexander von Humboldt-Stifiung (Bonn - Bad-Godesberg,

Germany) for the partial financial support of this work.

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