Streptomyces nucleotide 3′-pyrophosphokinase. Synthesis of deoxynucleotide-3′-pyrophosphates and 3′-pyrophosphoryl→3′-OH transfer activity

Special Publication No. 5 N u c l e i c A c i d s Research Streptomyces nucleotide 3'-pyrophosphokinase. Synthesis of deoxynucIeotide-3'-pyrophosphates and S'-pyrophosphoryl-^'-OH transfer activity Jun-Ichiro Mukai*, Abdur Razzaque*, Toshio Kukita*, Sawao Murao+ and Toyokazu Nishino+ 'Department of Agricultural Chemistry, Kyushu University, Fukuoka, and +Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Japan Nucleotide-3•-pyrophosphoklnase(ATP:nucleoside-5'-phosphate pyrophosphotransferase, E.C.2.7.6.4) of Streptomyces adephospholytlcus has been shown to transfer 5'-P,y-pyrophosphoryl group of either ATP, dATP or adenosine-5 1 tri-3'-diphosphate(pppApp) to 3'-slte of a variety of nucleotidlc substances Including both purine and pyrimidine rlboraononucleoside-5'-phosphates, short oligoribonucleotides, tRNA and 5'-di- or trlphosphonucleosidle sugars and their derivatives such as GDP-glucose, UDP-glucose, COP-chollne, NAD, FAD and rbG-5'-ppp-5'-Am, a cap nucleotide at the 5'-terminus of eucaryote mRNAs. The present paper reports two newly found facts of this enzyme: 1) deoxyATP(pppdA) is active not only as a donor but also as an acceptor of the pyrophosphoryl group giving rise to Its 3'-pyrophosphoryl derivative, pppdApp. pppdCpp could also be prepared using dGTP as the acceptor. 2 ) the enzyme catalyses both 5'P.y-pyrophosphoryl—>3'-0H kinase reaction(irreversible) and 3'-a,P-pyrophosphoryl^-^3'-0H transfer reaction(reverslble). INTRODUCTION ATP:nucleotide 3'-pyrophosphokinase was first discovered by Murao and Nishino(l,2) and its node of action was elucidated to be a transfer of 5'-P,ypyrophosphoryl group(-PP) from ATP, pppApp or dATP to 3'-0H site of acceptor nucleotldes. The acceptor specificity of the enzyme was recently found(3,4) to be unusually broad as mentioned above in comparison with the so-called stringent factor of £ . coll(5) and related polyphosphate synthetases ever known(6,7). Thus, its resemblance In principle in the mode of catalysis to and much broader acceptor specificity than those of the stringent factor are strongly indicative of some important physiological role(s) of the enzyme- whether It be similar to or different from the stringent control. It might be more than that. Under these circumstances, the present findings, namely the synthesis of deoxynucleoslde-5'-tri-3'-diphosphates and dual catalytic actlvi t y ( 5 ' ~ > 3 ' kinase and 3'<->3' transferase actions) make this enzyme more puczllng in search of its biological functions and more challenging In getting more insight into the cellular regulatory mechanisms in general. © Information Retrieval Limited 1 FalconbergCourt London W 1 V 5 F G England s451 ABSTRACT Nucleic Acids Research MATERIALS AND METHODS Streptomyces pyrophosphokinase was purified as reported(2). Reaction products were analysed by avicel thinlayer chromatography with isobutyric acid -NH.OH, pH 3.7 and Shimadzu dual wave CS 900 scanner at 270/310 nm(4). PI and PP1 were visualysed by spray on the plates(8) and separately determined colorimetrlcally(9). 0- P ATP was prepared by the method of Yamazakl(lO). RESULTS AND DISCUSSION Synthesis of deoxynucleoslde-5'-trl-3'-dlphosphates Ten ml reaction mixture containing 7 mM dATP, 0.1 M Tris-HCl, pH 9.0, 3 mM CoCl, and 600 units hrs as checked by TLC analysis. Presence of Co ions was imperative while Mg ions were inactive. The mixture was chromatographed on a DEAE-Sephadex A 25 column(2 x 35 cm) by 1 liter linear gradient from 0,1 to 0.3 M LiCl buffered with 0.05 M Tris-HCl, pH 7.5. dAMP, dATP and a new peak emerged at 34, 60 and 99 % gradient respectively. The last peak was recovered by an extended elutlon with the same gradient. The combined peak was flash-concentrated under reduced pressure, and precipitated and dried with ethanol, 18 mg. For structural analysis, 40 ug product was digested with 4 yg Penlcillium nuclease P.(4,11) in 10 ul 0.1 M ammonium acetate, pH 5.0 for 4 hrs at 37 C. The product(Rf 0.13) was completely hydrolysed to dATP(0.34) and PPi(0.20) in equimolar amounts as checked by TLC analysis and PPi colorimetry. No Pi was detected. In accordance with the previous results(4,12), the product was concluded to be deoxyadenosine-5'-tri-3'-diphosphate. dGTP was also found to accept -PP from dATP at a little lower rate. 3'-a.,P-PP<->3'-OH transfer reaction Knowledge on the metabolic fates of magic spots and related 3'-PP compounds is essential in understanding the molecular mechanisms of stringent and other related cellular controls in view of their well-known, very rapid synthesis and disappearance. Sy reported on the reversibility of the stringent reaction ATP + GD(T)P = AMP + GD(T)Ppp in E_. coll system(13) and also on an enzyme which specifically pyrophosphohydrolyses p(p)Gpp to p(p)G + PPi(14). On the contrary, using Streptomyces enzyme, Murao and Nishlno(15) found the reaction 2 ATP = pppApp + pA was not reversible and the reaction pppApp + pA yielded 2 pApp. They further claimed the reactions pp(P)Gpp + pA—>pApp + guanoslne di(tri)phosphate, leaving unambiguous structural Identification of the latters undone. In the present study we employed ppGpp and ADP to see which reaction (—>ppG + ppApp) or (—^Gpp + ppApp) occurres. Each 5 mM ppGpp and ppA were incubated with 0.6 units of the s452 enzyme was incubated at 45 C. The reaction was found 70 % complete after 22 2+ 2+ Nucleic Acids Research enzyme in 10 pi 0.05 M Tris-HCl, pH 8.2-10 mM MgCl 2 for 8 hrs at 37°C. TLC analysis revealed two closely overlapping new bands corresponding to authentic ppG(Rf 0.19) and ppApp(0.21) besides ppGpp(O.lO) and ppA(0.40). The extent of the reaction was 37 X. The two new bands were eluted together and rechromatographed by the same solvent system except that pH was adjusted to 4.3. Here the two bands were distinctly separated, corresponding to ppG(0.60) and ppApp (0.70). Each band was eluted and chromatographed by isopropanol-(NH,)«S0,0.1 M NH OAc, pH 6.0(2:79:18). Further confirmation of ppApp(0.37) and ppG (0.45) was made. 3'-GDP(Gpp), prepared by RNase T. digestion of ApGpp which was synthesised from ApG and dATP using Streptomyces enzyme(4) migrated to Rf 10 mM GDP were reacted with 0.6 units Streptomyces enzyme in 10 ul 0.05 M Tris HC1, pH 8.2-10 mM MgCl, for 3 hrs at 37°C. TLC separation followed by radioautography revealed isolation of 3'-a- P ppGpp from faint radioactive bands corresponding to pppApp and pApp and from any UV-absorbing components. The band corresponding to ppGpp was eluted from the plate with 1 mM cold ppGpp 0.1 mM EDTANa. and concentrated under vacuum. Using this as the labeled donor, 3'-a,fi-PP—^3'-OH transfer specificity of Streptomyces enzyme was studied under the identical conditions as above. Besides ppA used above, pA, ppU and even NAD reacted with ppGpp to give the radioactive products corresponding to the respective cold 3'-pyrophosphoryl derivatives(4). The reaction between ppGpp and ppA was found reve (...truncated)