The structure of Ap(4)A hydrolase complexed with ATP-MgF(x) reveals the basis of substrate binding

Ap(4)A hydrolases are Nudix enzymes that regulate intracellular dinucleoside polyphosphate concentrations, implicating them in a range of biological events, including heat shock and metabolic stress. We have demonstrated that ATP x MgF(x) can be used to mimic substrates in the binding site of Ap(4)A hydrolase from Lupinus angustifolius and that, unlike previous substrate analogs, it is in slow exchange with the enzyme. The three-dimensional structure of the enzyme complexed with ATP x MgF(x) was solved and shows significant conformational changes. The substrate binding site of L. angustifolius Ap(4)A hydrolase differs markedly from the two previously published Nudix enzymes, ADP-ribose pyrophosphatase and MutT, despite their common fold and the conservation of active site residues. The majority of residues involved in substrate binding are conserved in asymmetrical Ap(4)A hydrolases from pathogenic bacteria, but are absent in their human counterparts, suggesting that it might be possible to generate compounds that target bacterial, but not human, Ap(4)A hydrolases.     

Crystal structure of the 25 kDa subunit of human cleavage factor Im

Cleavage factor I(m) is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor I(m) is an oligomer composed of a small 25 kDa subunit (CF I(m)25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF I(m)25, a hydrolase motif with a characteristic alpha/beta/alpha fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF I(m)25 in its free and diadenosine tetraphosphate (Ap(4)A) bound forms at 1.85 and 1.80 A, respectively. CF I(m)25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF I(m)25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap(4)A. The complex and apo protein structures provide insight into the active oligomeric state of CF I(m) and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.     

Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.     

The g5R (D250) gene of African swine fever virus encodes a Nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates.

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5'-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k(cat)/K(m) ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-beta-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K(m) values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.     

The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms

The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 A and 1.8 A, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.     

The Nudix hydrolase Ndx1 from Thermus thermophilus HB8 is a diadenosine hexaphosphate hydrolase with a novel activity

The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins. Ndx1 was overexpressed in Escherichia coli and purified. Ndx1 was stable up to 95 degrees C and at extreme pH. Size exclusion chromatography indicated that Ndx1 was monomeric in solution. Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always generated ATP as the product. Diadenosine hexaphosphate (Ap(6)A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap(6)A, with a K(m) of 1.4 microm and a k(cat) of 4.1 s(-1). These results indicate that Ndx1 is a (di)adenosine polyphosphate hydrolase. Ndx1 activity required the presence of the divalent cations Mn(2+), Mg(2+), Zn(2+), and Co(2+), whereas Ca(2+), Ni(2+), and Cu(2+) were not able to activate Ndx1. Fluoride ion inhibited Ndx1 activity via a non-competitive mechanism. Optimal activity for Ap(6)A was observed at around pH 8.0 and about 70 degrees C. We found two important residues with pK(a) values of 6.1 and 9.6 in the free enzyme and pK(a) values of 7.9 and 10.0 in the substrate-enzyme complex. Kinetic studies of proteins with amino acid substitutions suggested that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis. Trp-26 was likely involved in enzyme-substrate interactions based on fluorescence measurements. Based on these results, the mechanism of substrate recognition and catalysis are discussed.     

Solution structure of hypothetical Nudix hydrolase DR0079 from extremely radiation-resistant Deinococcus radiodurans bacterium

Using nuclear magnetic resonance (NMR) based methods, including residual dipolar coupling restraints, we have determined the solution structure of the hypothetical Deinococcus radiodurans Nudix protein DR0079 (171 residues, MW = 19.3 kDa). The protein contains eight beta-strands and three alpha-helices organized into three subdomains: an N-terminal beta-sheet (1-34), a central Nudix core (35-140), and a C-terminal helix-turn-helix (141-171). The Nudix core and the C-terminal helix-turn-helix form the fundamental fold common to the Nudix family, a large mixed beta-sheet sandwiched between alpha-helices. The residues that compose the signature Nudix sequence, GX5EX7REUXEEXGU (where U = I, L, or V and X = any amino acid), are contained in a turn-helix-turn motif on the face of the mixed beta-sheet. Chemical shift mapping experiments suggest that DR0079 binds Mg2+. Experiments designed to determine the biological function of the protein indicate that it is not a type I isopentenyl-diphosphate delta-isomerase and that it does not bind alpha,beta-methyleneadenosine 5'-triphosphate (AMPCPP) or guanosine 5'-[beta,gamma-imido]triphosphate (GMPPNP). In this article, the structure of DR0079 is compared to other known Nudix protein structures, a potential substrate-binding surface is proposed, and its possible biological function is discussed.     

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.     

Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates

DNA ligase from the hyperthermophilic marine archaeon Pyrococcus furiosus (Pfu DNA ligase) synthesizes adenosine 5'-tetraphosphate (p4A) and dinucleoside polyphosphates by displacement of the adenosine 5'-monophosphate (AMP) from the Pfu DNA ligase-AMP (E-AMP) complex with tripolyphosphate (P3), nucleoside triphosphates (NTP), or nucleoside diphosphates (NDP). The experiments were performed in the presence of 1-2 microM [alpha-32P]ATP and millimolar concentrations of NTP or NDP. Relative rates of synthesis (%) of the following adenosine(5')tetraphospho(5')nucleosides (Ap4N) were observed: Ap4guanosine (Ap4G) (from GTP, 100); Ap4deoxythymidine (Ap4dT) (from dTTP, 95); Ap4xanthosine (Ap4X) (from XTP, 94); Ap4deoxycytidine (Ap4dC) (from dCTP, 64); Ap4cytidine (Ap4C) (from CTP, 60); Ap4deoxyguanosine (Ap4dG) (from dGTP, 58); Ap4uridine (Ap4U) (from UTP, <3). The relative rate of synthesis (%) of adenosine(5')triphospho(5')nucleosides (Ap3N) were: Ap3guanosine (Ap3G) (from GDP, 100); Ap3xanthosine (Ap3X) (from XDP, 110); Ap3cytidine (Ap3C) (from CDP, 42); Ap3adenosine (Ap3A) (from ADP, <1). In general, the rate of synthesis of Ap4N was double that of the corresponding Ap3N. The enzyme presented optimum activity at a pH value of 7.2-7.5, in the presence of 4 mM Mg2+, and at 70 degrees C. The apparent Km values for ATP and GTP in the synthesis of Ap4G were about 0.001 and 0.4mM, respectively, lower values than those described for other DNA or RNA ligases. Pfu DNA ligase is used in the ligase chain reaction (LCR) and some of the reactions here reported [in particular the synthesis of Ap4adenosine (Ap4A)] could take place during the course of that reaction.     

The binding of adenosine(5'')tetraphospho(5'')adenosine to calf thymus histones measured by non-equilibrium dialysis.

Binding of adenosine(5')tetraphospho(5')adenosine (Ap4A) to histones of calf thymus was investigated by non-equilibrium dialysis. Histone H1 interacts with the dinucleotide via two strong sites and competes with Mg2+ ions. Intrinsic dissociation constants were 1.6 +/- 0.1 microM and 11 +/- 1 microM for zero and 0.4 mm-Mg2+ concentration respectively. Binding of poly(dT) and of other nucleotides to histone H1 was measured in an [3H]Ap4A-competition assay. The tendency to form complexes among nucleotides was highest for bisnucleoside tetraphosphates and decreased in the order poly(dT) greater than or equal to Ap4A approximately Gp4G greater than Ap4 much greater than Ap3A approximately Ap5A greater than or equal to ATP, GTP and dTTP. The co-ordination complex derived from Ap4A and cis-diammine-dichloroplatinum(II) was not reactive. The other histones of calf thymus also bound Ap4A with affinities decreasing in the order H4 approximately H3 greater than H1 greater than H2b greater than H2a. Ap4A stimulated the exchange of histone H1 between nucleosomes, but this effect was referred to ionic strength. It did not bind to assembled nucleosomes. Binding of Ap4A to histone H1 was decreased by salt (NaCl). At physiological saline concentration the value of the dissociation constant is commensurable with the value of the Ap4A concentration in the nucleus and thus indicative of complex-formation in vivo.     

T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates.

T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [alpha-32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, microm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [alpha-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2'p.     

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.     

Investigation into the interactions between diadenosine 5',5'-P1,P4-tetraphosphate and two proteins: molecular chaperone GroEL and cAMP receptor protein

Diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A) is a dinucleoside polyphosphate found ubiquitously in eukaryotic and prokaryotic cells. Despite Ap(4)A being universal, its functions have proved to be difficult to define, although they appear to have a strong presence during cellular stress. Here we report on our investigations into the nature and properties of putative Ap(4)A interactions with Escherichia coli molecular chaperone GroEL and cAMP receptor protein (CRP). We confirm previous literature observations that GroEL is an Ap(4)A binding protein and go on to prove that binding of Ap(4)A to GroEL involves a set of binding sites (one per monomer) distinct from the well-known GroEL ATP/ADP sites. Binding of Ap(4)A to GroEL appears to enhance ATPase rates at higher temperatures, encourages the release of bound ADP, and may promote substrate protein release through differential destabilization of the substrate protein-GroEL complex. We suggest that such effects should result in enhanced GroEL/GroES chaperoning activities that could be a primary reason for the improved yields of the refolded substrate protein observed during GroEL/GroES-assisted folding and refolding at >or=30 degrees C in the presence of Ap(4)A. In contrast, we were unable to obtain any data to support a direct role for Ap(4)A interactions with CRP.     

Diadenosine tetraphosphate hydrolase from mouse liver. Purification to homogeneity and partial characterization

An enzyme hydrolyzing diadenosine 5',5"'P1, P4-tetraphosphate (Ap4A) to AMP and ATP has been purified to apparent homogeneity from mouse liver cell extracts. The isolation procedure comprised ammonium sulfate precipitation, chromatography on Sephadex G-75. DEAE-cellulose, blue Sepharose and AMP-Sepharose. The enzyme is a single polypeptide chain with a native Mr = 64,000 with a Km of 1.66 microM and Vmax of 1.25 mumol/min. AMP, ADP, Ap4, GTP, Gp4, Ap3A, Ap5A, Gp3G, and Gp5G are noncompetitive inhibitors of the Ap4A hydrolase activity, whereas Gp4G inhibits Ap4A hydrolysis competitively with a Ki of 6 microM. Theophylline, caffeine, and isobutylmethylxanthine do not or only slightly inhibit Ap4A hydrolysis. Mitogenic factors have no effect on the enzymatic activity of Ap4A hydrolase, excluding that a direct influence of internalized mitogens on Ap4A degradation could be responsible for mitogen-dependent fluctuation of intracellular Ap4A pool sizes.     

Hydrolytic activity of bovine tryptophanyl-tRNA-synthetase cause by removal of Zn2+

Bovine tryptophanyl-tRNA synthetase (E.C.6.1.1.2) lacking Zn2+ ions removed by chelation with phosphonate analog of P1,P4-bis-(5'-adenosyl)tetraphosphate (Ap4A) was obtained (E-Zn). E-Zn lost the ability to form tryptophanyl adenylate, however it hydrolyses ATP to ADP and further on to AMP and Pi. GTP serves as a substrate with Km approximately 0.6 mM. It is proposed that the hydrolysable nucleotides bind to a nucleotide binding site(s) distinguishable from the substrate (catalytic) ones. After incubation of E-Zn with Zn2+ and Mg2+ the initial catalytic activity (ATP-PPi exchange and amino-acylation reactions) is restored whereas the hydrolytic activity becomes fully suppressed.     

Diadenosine Tetraphosphate Is Co-Released with ATP and Catecholamines from Bovine Adrenal Medulla

Secretion of adenosine(5')tetraphospho(5')adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co-release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1-2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.     

Synthesis of a fluorescent 7-methylguanosine analog and a fluorescence spectroscopic study of its reaction with wheatgerm cap binding proteins.

In the initiation of protein synthesis, the mRNA 5'-terminal 7-methylguanosine cap structure and several recognition proteins play a pivotal role. For the study of this cap binding reaction, one approach is to use fluorescence spectroscopy. A ribose diol-modified fluorescent cap analog, anthraniloyl-m7GTP (Ant-m7GTP), was designed and synthesized for this purpose. This fluorescent cap analog was found to have a high quantum yield, resistance to photobleaching and avoided overlap of excitation and emission wavelengths with those of proteins. The binding of Ant-m7GTP with wheatgerm initiation factors elF-4F and elF-(iso)4F was determined. The fluorescent cap analog and m7GTP had similar interactions with both cap binding proteins. Fluorescence quenching experiments showed that the microenvironment of Ant-m7GTP when bound to protein was hydrophobic.     

P alpha-chiral phosphorothioate analogues of bis(5''-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.

Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae. The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S. No dithiophosphate analogue was formed. Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively. The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate. The adenylate was accepted by corresponding thioanalogues of ATP. In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60. The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E. coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S. cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S. For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively. Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.     

The gene e.1 (nudE.1) of T4 bacteriophage designates a new member of the Nudix hydrolase superfamily active on flavin adenine dinucleotide, adenosine 5'-triphospho-5'-adenosine, and ADP-ribose

The T4 bacteriophage gene e.1 was cloned into an expression vector and expressed in Escherichia coli, and the purified protein was identified as a Nudix hydrolase active on FAD, adenosine 5'-triphospho-5'-adenosine (Ap(3)A), and ADP-ribose. Typical of members of the Nudix hydrolases, the enzyme has an alkaline pH optimum (pH 8) and requires a divalent cation for activity that can be satisfied by Mg(2+) or Mn(2+). For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap(3)A, the T4 enzyme hydrolyzes higher homologues including Ap(4-6)A. This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4. Although the protein was predicted to be orthologous to E. coli MutT on the basis of a sequence homology search, the properties of the gene and of the purified protein do not support this notion because of the following. (a) The purified enzyme hydrolyzes substrates not acted upon by MutT, and it does not hydrolyze canonical MutT substrates. (b) The e.1 gene does not complement mutT1 in vivo. (c) The deletion of e.1 does not increase the spontaneous mutation frequency of T4 phage. The properties of the enzyme most closely resemble those of Orf186 of E. coli, the product of the nudE gene, and we therefore propose the mnemonic nudE.1 for the T4 phage orthologue.