Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.     

Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts.

A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.     

rac-Glycerol 1:2-cyclic phosphate 2-phosphodiesterase, a new soluble phosphodiesterase of mammalian tissues.

1. A soluble phosphodiesterase is present in mammalian tissues which rapidly hydrolyses enantiomorphs of rac-glycerol 1:2-cyclic phosphate, producing rac-glycerol 1-phosphate. 2. The enzyme has been purified up to 1700-fold by a combination of acetone precipitation and chromatography on DEAE-Sephadex A-50, Sephadex G-150 and hydroxyapatite. 3. The Km with glycerol cyclic phosphate as substrate is 7.2 mM, and the pH optimum broad (6.9--7.5). The molecular weight (by gel filtration) of the enzyme is approx. 35500. 4. The phosphodiesterase has no requirement for Ca2+ or Mg2+, but is stimulated by reducing agents (cysteine, dithiothreitol) and Fe2+. 5. The purified phosphodiesterase preparation also hydrolysed 3':5'-cyclic AMP, producing 5'-AMP exclusively, and 2':3'-cyclic AMP, forming 3'-AMP and 2'-AMP in the ratio 7:3. Bis-(p-nitrophenyl) phosphate was slowly hydrolysed, but other phosphodiesters tested were not attacked. 6. The phosphodiesterase is inhibited by theophylline and o-phenanthroline. It is inhibited by Pi and by a variety of phosphomonoesters, of which certain aromatic primary phosphates are particularly effective.     

A proton magnetic resonance investigation of the glycosyl torsion angle of uracil nucleosides and nucleotides.

The use of line-shape decomposition techniques permitted the small 5-bond (5-J51') and 4-bond (4-J61') proton-proton coupling constants of a series of uracil nucleosides and nucleotides to be determined accurately. From an analysis of these coupling constants we have determined that the uracil base is in a predominantly anti conformation in aqueous solution and the mean position is not substantially altered by phosphate substitution at the 2', 3', or 5' positions, by changing the furanose stereochemistry from a ribose to a deoxyribose or an arabinose, or by an increase in temperature of 43 degree C.     

D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH.

The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea. In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion. In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes. A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate. Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M. thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.     

Zn2+-dependent deoxyribozymes that form natural and unnatural RNA linkages

We report Zn(2+)-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k(obs) up to 0.5 min(-)(1) at 1 mM Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2',3'-cyclic phosphate on a second RNA substrate. Some of the Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5'-hydroxyl group. Two of these unnatural linkages are the 3'-2' and 2'-2' linear junctions created when the 2'-hydroxyl of the 5'-terminal guanosine of one RNA substrate attacks the 2',3'-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2'-hydroxyl of one RNA substrate at the 2',3'-cyclic phosphate. When compared with the consistent creation of 2'-5' linkages by Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.     

β-d-2'-α-F-2'-β-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: A structure-activity relationship study

The 3',5'-cyclic phosphate prodrug 9-[β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100μm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.     

Carbon-13 nuclear-magnetic-resonance spectra of adenine cyclonucleosides and their phosphates. Effects of neighboring groups for elucidation of fine structure of nucleosides and nucleotides.

Carbon-13 nuclear magnetic resonance spectra of adenine cyclonucleosides, which have a fixed glycosidic conformation in an anti range, and their isopropylidene and phosphate esters are reported; those of 9-beta-D-arabinofuranosyladenine and its 5'-phosphate are also presented. The chemical shifts of the base carbons are affected not only by the bridging atom but also by the position of the bridged sugar carbon which determine the planarity of the third ring formed by cyclization between the base and the sugar. The effects of glycosidic conformation on the sugar-carbon chemical shifts are discussed by comparison of the data for 8:5'-cycloadenosines with the data for adenosine and its 8-substituted derivatives. The effects of a 2'-oxygen on sugar-carbon chemical shifts have been examined by comparing the data for 2'-deoxyadenosine, arabinosyladenine and 8:2'-anhydro-8-oxy-9-beta-D-arabinofuranosyladenine. The effects of phosphomonoester groups on base and sugar carbon resonances have been examined and it is noted that these groups cause downfield shifts for C-8 of all cyclonucleotides. Data for the 3':5'-cyclic monophosphate derivative of 8:2'-anhydro-8-thio-9-beta-D-arabinofuranosyladenine suggest that the previous assignments of C-4' and C-3' for nucleoside 3':5'-cyclic monophosphates must be reversed. According to the reversed assignments, it seems that C-3' and C-5' show moderate downfield shifts and C-4' shows a marked upfield shift.     

Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties

Uracil phosphoribosyltransferase was purified 34-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, binding to DEAE-Sephadex, Sephadex G-200 chromatography, and hydroxylapatite chromatography. The molecular weight of the enzyme by gel filtration was approximately 80,000. The pH optimum for phosphoribosylation was around 7.5, and the optimum MgCl2 concentration was 5 mM. Initial velocity studies were conducted over a wide range of both uracil and 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP) concentrations, and various equations for biomolecular reaction mechanisms were fitted to the data by nonlinear regression. When the equation for an ordered sequential mechanism was fitted to the data, the Kia thus obtained was not statistically different from zero. This is interpreted as evidence for a nonsequential ("ping-pong") reaction. Graphic analysis of the data by the Hanes-Woolf linear transform supported this conclusion. The enzyme has high affinity for uracil (KmUra = 4.2 microM; KmP-Rib-PP = 66 microM), which provides supporting evidence that this activity is responsible for the incorporation of uracil and uridine into nucleotides.     

The phosphorylation of troponin I from cardiac muscle.

1. Troponin I isolated from fresh cardiac muscle by affinity chromatography contains about 1.9 mol of covalently bound phosphate/mol. Similar preparations of white-skeletal-muscle troponin I contain about 0.5 mol of phosphate/mol. 2. A 3':5'-cyclic AMP-dependent protein kinase and a protein phosphatase are associated with troponin isolated from cardiac muscle. 3. Bovine cardiac 3':5'-cyclic AMP-dependent protein kinase catalyses the phosphorylation of cardiac troponin I 30 times faster than white-skeletal-muscle troponin I. 4. Troponin I is the only component of cardiac troponin phosphorylated at a significant rate by the endogenous or a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. 5. Phosphorylase kinase catalyses the phosphorylation of cardiac troponin I at similar or slightly faster rates than white-skeletal-muscle troponin I. 6. Troponin C inhibits the phosphorylation of cardiac and skeletal troponin I catalysed by phosphorylase kinase and the phosphorylation of white skeletal troponin I catalysed by 3':5'-cyclic AMP-dependent protein kinase; the phosphorylation of cardiac troponin I catalysed by the latter enzyme is not inhibited.     

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.     

The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

The RNA ligase RtcB splices broken RNAs with 5'-OH and either 2',3'-cyclic phosphate or 3'-phosphate ends. The 3'-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3')pp(5')G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5'-OH end attacks the 3'-phosphorus of the N(3')pp(5')G end to form a 3',5'-phosphodiester and release GMP. Scheme 2 posits that the N(3')pp(5')G end is converted to a 2',3'-cyclic phosphodiester, which is then attacked directly by the 5'-OH RNA end to form a 3',5'-phosphodiester. Here we show that the sealing of a 2',3'-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB-GMP adduct, and involves a kinetically valid RNA(3')pp(5')G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2',3'-cyclic phosphate to a 3'-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB-GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3'-phosphate ligase steps of the RNA splicing pathway.     

Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.     

Substrate specifity in Amoeba proteus

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.     

Novel mechanism of RNA repair by RtcB via sequential 2',3'-cyclic phosphodiesterase and 3'-Phosphate/5'-hydroxyl ligation reactions

RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.     

RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing.     

Synthesis of poly(ethylene glycol)-bound NADP by selective modification at the 6-amino group of NADP

The N-1 position of the adenine ring of NADP was selectively alkylated by the reaction of 2',3'-cyclic NADP with 3-propiolactone to yield 2',3'-cyclic 1-(2-carboxyethyl)-NADP (I). Derivative I was converted to a mixture of the isomers of N6-(2-carboxyethyl)-NADP with their phosphate groups at the 2' or 3' position (IIa and IIb) by chemical reduction, alkaline rearrangement and chemical reoxidation. Carbodiimide coupling of the mixture of IIa and IIb to alpha, omega-diaminopoly(ethylene glycol) gave the 2', 3'-cyclic derivative of poly(ethylene glycol)-bound NADP (III), which was enzymically hydrolyzed to yield poly(ethylene glycol)-bound NADP (PEG-NADP). PEG-NADP has good cofactor activity (16-100% of that of NADP) for NADP-specific and NAD(P)-specific dehydrogenases except isocitrate and glucose dehydrogenases. For NAD-specific enzymes, PEG-NADP has higher cofactor activity than NADP: for horse liver alcohol dehydrogenase, the cofactor activity of PEG-NADP is 40 times that of NADP and 14% of that of NAD. Kinetic studies show that for most of enzymes tested, Km values for PEG-NADP are larger than those for NADP and V values for PEG-NADP are similar to those for NADP. PEG-NADP proved to be applicable in a continuous enzyme reactor, in which reactions of glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were coupled by the recycling of PEG-NADP.     

RtcB is the RNA ligase component of an Escherichia coli RNA repair operon

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

Formation of ribonucleotide 2'',3''-cyclic carbonates during conversion of ribonucleoside 5''-phosphates to diphosphates and triphosphates by the phosphorimidazolidate procedure.

Ribo- and 2'-deoxyribonucleoside 5'-di- or triphosphates are commonly synthesized by reaction of inorganic phosphate or pyrophosphate with phosphorimidazolidates obtained by reaction of nucleoside 5'-phosphates with 1,1'-carbonyldiimidazole. The latter reaction, however, converted UMP, CMP, IMP, GMP, and AMP in high yield to the 2',3'-cyclic carbonate derivatives of their phosphorimidazolidates. Acidic treatment of the product from AMP gave AMP 2',3'-cyclic carbonate dihydrate; this was characterized by its uv, ir, and pmr spectra and by its conversion to adenosine 2',3'-cyclic carbonate by acid phosphatase and to AMP by basic hydrolysis. ADP or ATP synthesized by the phosphorimidazolidate method contained equal or greater amounts of their respective 2',3'-cyclic carbonates. The latter could be quantitatively converted to ADP and ATP, respectively, by 4-hr hydrolysis at pH 10.5, 22 degrees. ADP or ATP can be synthesized without concomitant 2',3'-cyclic carbonate formation by reaction of AMP with phosphorimidazolidates of inorganic phosphate or pyrophosphate.