Kinetic analysis of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase

Properties of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase were characterized using a modified cyanide addition method by which initial velocities of the transglycosidation (vT) and hydrolysis (vH) of pyridine nucleotides could be monitored simultaneously. (1) The vT was routinely determined with NMN and nicotinic acid used as substrates and was observed to be maximal at pH 6. Arrhenius plots of vT and vH indicated that the activation energies for transglycosidation and hydrolysis were 8.7 and 10.7 kcal/mol, respectively. (2) The enzyme showed a broad spectrum of substrate specificity with respect to both pyridine nucleotides and bases. Of the compounds tested, NMN and nicotinic acid were shown to be the best substrates when compared on the basis of Vmax/Km values. Kinetic constants for the enzyme-catalyzed transglycosidation reaction were as follows; Km(NMN) = 0.53 mM, Km(nicotinic acid), as acid form = 15 mM, apparent Vmax = 7.8 mumol/min/mg protein, in the presence of 0.2 M nicotinic acid. (3) The ratio of vT/vH was shown to be dependent on both pH and nicotinic acid concentration. However, transglycosidation versus hydrolysis partition at a fixed pH was constant regardless of the nicotinic acid concentration employed and approximated to be 1.2 x 10(4) at the maximal pH. (4) Nicotinamide, one of the most potent inhibitors for the enzyme-catalyzed hydrolysis, was shown to function as an antagonist for the transglycosidation reaction with NMN and nicotinic acid used as substrates. The inhibition mechanism with nicotinamide was purely noncompetitive with respect to nicotinic acid; on the other hand, the double reciprocal plot of the transglycosidation velocity against NMN concentration at a fixed concentration of nicotinamide was concave downwards. (5) The equilibrium constant of the reaction, NMN + 3-acetylpyridine----3-acetylpyridine mononucleotide + nicotinamide, was 0.61, whereas the conversion of NMN with nicotinic acid to nicotinic acid mononucleotide was essentially irreversible. These enzymatic properties of rabbit spleen pyridine nucleotide glycohydrolase suggested that the enzyme should not function as a glycohydrolase but as a transglycosidase and could serve in an important mechanism for an alternative biosynthetic pathway of nicotinic acid mononucleotide, one of the precursors for NAD synthesis, when nicotinic acid is supplied.     

Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium

A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.     

Purification and characterization of a pyridine nucleotide glycohydrolase from rabbit spleen

A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a Km of 0.25 mM, a Vmax of 5.3 mumol/min/mg protein, an activation energy of 7.9 kcal/mol, and a molecular weight of approximately 400,000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN:NAD:NADP = 1.00:1.45:0.44 and Vmax/Km ratios of 1.00:1.7:2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with Ki values similar to Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme in vivo.     

Pyridine nucleotide biosynthesis in Claviceps

Claviceps purpurea grown on synthetic medium incorporated labeled [7-¹⁴]nicotinic acid and [7-¹⁴C]nicotinamide into NaMN, des-NAD, NAD, and NADP. Label also appeared in NMN and N-methyl nicotinamide. The specific activities of NAD, NADP, and NMN are compatible with the operation of the Preiss-Handler pathway of NAD biosynthesis (nicotinic acid → NaMN → des-NAD → NAD → NADP). The relative amounts of NaMN:des-NAD:NAD and NADP were about 8:1:36:10 on incubation of Claviceps with nicotinic acid for 6 hr. The incorporation of nicotinamide into NAD proceeds mainly by conversion to nicotinic acid catalyzed by nicotinamide deamidase.Tryptophan ([U-¹⁴C]benzene ring) was incorporated into NAD demonstrating the presence of the tryptophan-nicotinic acid pathway. No qualitative difference in pyridine nucleotide intermediates was noted in C. purpurea CPM, which does not produce clavine alkaloids, and Claviceps 47A which does produce clavine alkaloids.     

Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide adenine dinucleotide glycohydrolase, nicotinamide mononucleotide glycohydrolase, and nicotinamide adenine dinucleotide pyrophosphatase activities.

Extracts of Salmonella typhimurium were chromatographed by using Sephadex G-150 to separate the various enzymes involved with pyridine nucleotide cycle metabolism. This procedure revealed a previously unsuspected nicotinamide adenine dinucleotide (NAD) glycohydrolase (EC 3.2.2.5) activity, which was not observed in crude extracts. In contrast to NAd glycohydrolase, NAD pyrophosphatase (EC 3.6.1.22) was readily measured in crude extracts. This enzyme possessed a native molecular weight of 120,000. Other enzymes examined included nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.00), molecular weight of 43,000; NMN glycohydrolase (EC 3.2.2.14), molecular weight of 67,000; nicotinic acid phosphoribosyl transferase (EC 2.4.2.11), molecular weight of 47,000; and nicotinamide deamidase (EC 3.5.1.19), molecular weight of 35,000. NMN deamidase and NMN glycohydrolase activities were both examined for end product repression by measuring their activities in crude extracts prepared from cells grown with and without 10(-5) M nicotinic acid. No repression was observed with either activity. Both activities were also examined for feedback inhibition by NAD, reduced NAD, and NADP. NMN deamidase was unaffected by any of the compounds tested. NMN glycohydrolase was greatly inhibited by NAD and reduced NAD, whereas NADP was much less effective. Inhibition of NMN glycohydrolase was found to level off at an NAD concentration of ca. 1 mN, the approximate intracellular concentration of NAD.     

Utilization and metabolism of NAD by Haemophilus parainfluenzae

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the beta-configuration of the glycopyridine linkage. (4) An intact carboxamide groups is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.     

The pathway of biosynthesis of nicotinamide–adenine dinucleotide in rat mammary gland

1. The pathway of NAD synthesis in mammary gland was examined by measuring the activities of some of the key enzymes in each of the tryptophan, nicotinic acid and nicotinamide pathways. 2. In the tryptophan pathway, 3-hydroxyanthranilate oxidase and quinolinate transphosphoribosylase activities were investigated. Neither of these enzymes was found in mammary gland. 3. In the nicotinic acid pathway, nicotinate mononucleotide pyrophosphorylase, NAD synthetase, nicotinamide deamidase and NMN deamidase were investigated. Both NAD synthetase and nicotinate mononucleotide pyrophosphorylase were present but were very inactive. Nicotinamide deamidase, if present, had a very low activity and NMN deamidase was absent. 4. In the nicotinamide pathway both enzymes, NMN pyrophosphorylase and NMN adenylyltransferase, were present and showed very high activity. The activity of the pyrophosphorylase in mammary gland is by far the highest yet found in any tissue. 5. The apparent K(m) values for the substrates of these enzymes in mammary gland were determined. 6. On the basis of these investigations it is proposed that the main, and probably only, pathway of synthesis of NAD in mammary tissue is from nicotinamide via NMN.     

The pyridine nucleotide cycle: presence of a nicotinamide mononucleotide-specific amidohydrolase in Propionibacterium shermanii

A specific nicotinamide mononucleotide amidohydrolase which catalyzes the stoichiometric conversion of NMN to nicotinate mononucleotide and ammonia has been partially purified from an extract of $\text{Propionibacterium}\text{shermanii}$. The reaction has optimum activity at pH 5.6, a K_m of 70 μM, and an experimental activation energy of 14.5 Kcal/mole. The enzyme appears to be highly specific for NMN. Neither free nicotinamide nor NAD, NADH, NADP, NADPH compete with NMN. Numerous substances such as isonicotinic acid hydrazide and quinolinic acid are also without effect. It can be stored at ?15° in 12% glycerol, but is somewhat unstable in the absence of this solvent. The enzyme is composed of a heatstable and a heat-sensitive subunit. This enzyme considerably simplifies the pyridine nucleotide cycle, and may, besides this salvage function for NAD, play a role in B₁₂ biosynthesis and in the bacterial DNA ligase reaction.     

Metabolism of the pyridine nucleotides involved in nicotinamide adenine dinucleotide biosynthesis by Clostridium butylicum

In order to elucidate the mechanism of the accumulation of considerable amounts of free nicotinic acid (NA) in the culture medium of Clostridium butylicum, this organism was investigated with regard to its ability to metabolize nicotinamide adenine dinucleotide (NAD) and its immediate biosynthetic precursors, nicotinic acid mononucleotide (NAMN) and nicotinic acid adenine dinucleotide (deamido-NAD). Cell-free extracts of C. butylicum were found to degrade NAMN and deamido-NAD to NA. NAMN, in the presence of adenosine triphosphate (ATP), was converted to deamido-NAD, but only at high concentrations of ATP (20 mM) was significant synthetic activity observed in competition with NAMN degradation. Degradation of both NAMN and deamido-NAD was activated by ATP at concentrations of 5 and 10 mm. Anaerobiosis markedly enhanced the degradation of the nucleotides. The data indicate that the synthesis of NAMN and deamido-NAD prevails over their degradation only in the presence of high concentrations of ATP. NAD was degraded to nicotinamide mononucleotide (NMN) by a pyrophosphatase. Phosphate markedly inhibited both the deamido-NAD and NAD pyrophosphatases. Under anaerobic conditions there was practically no further degradation of NMN to NA, whereas barely measurable amounts of NA were formed under aerobic conditions. All of these observations suggest that, under the given conditions of anaerobiosis and physiological phosphate concentrations, there is very little degradation of NAD to NMN and practically no degradation to NA by C. butylicum. Thus, NAD represents an insignificant source of the NA accumulated in the culture medium. The intermediates in the biosynthetic pathway (NAMN and deamido-NAD) have been shown to be the major source of the NA which is accumulated by C. butylicum.     

NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by ADP-ribosylation of a specific arginine residue of dinitrogenase reductase based on the cellular nitrogen or energy status. In this paper, we have investigated the ability of nicotinamide adenine dinucleotide, NAD (the physiological ADP-ribose donor), and its analogs to support covalent modification of dinitrogenase reductase in vitro. R. rubrum dinitrogenase reductase can be modified by DRAT in the presence of 2 mM NAD, but not with 2 mM nicotinamide mononucleotide (NMN) or nicotinamide adenine dinucleotide phosphate (NADP). We also found that the apo- and the all-ferrous forms of R. rubrum dinitrogenase reductase are not substrates for covalent modification. In contrast, Azotobacter vinelandii dinitrogenase reductase can be modified by the dinitrogenase reductase ADP-ribosyl transferase (DRAT) in vitro in the presence of either 2 mM NAD, NMN or NADP as nucleotide donors. We found that: (1) a simple ribose sugar in the modification site of the A. vinelandii dinitrogenase reductase is sufficient to inactivate the enzyme, (2) phosphoADP-ribose is the modifying unit in the NADP-modified enzyme, and (3) the NMN-modified enzyme carries two ribose-phosphate units in one modification site. This is the first report of NADP- or NMN-dependent modification of a target protein by an ADP-ribosyl transferase.     

The pyridine-nucleotide cycle in tobacco Enzyme activities for the de-novo synthesis of NAD

The enzyme activities of the pyridine-nucleotide cycle, which transform nicotinic acid mononucleotide (NaMN) into NAD, have been characterized. The investigations were based on the extraction of protein, its purification on disposable gel-filtration columns, and determination of the enzymatic activities by high-performance liquid chromatography techniques. The latter technique avoided the synthesis and use of radioactive precursors. The NaMN-adenylyltransferase which converts NaMN into NaAD (nicotinic acid adenine dinucleotide) and NAD-synthetase which converts NaAD into NAD were characterized by their kinetic parameters and their specific activities in different tobacco tissues. This is the first report on NAD-synthetase from tissue of a higher plant. It was found that NAD-synthetase accepted both glutamine and asparagine for the amide transfer. Adenylyltransfer also occured with nicotinamide mononucleotide (NMN) which was transformed to NAD, whereas the glutamine-dependent amidation was only observed with NaAD. Thus, an additional route for the synthesis of NAD (NaMNNMNNAD) obviously does not exist. A comparison of the enzyme activities in tobacco tissues with different capacities for the synthesis of nicotine showed that, in contrast to quinolinic acid phosphoribosyltransferase whose activity was strictly correlated with the nicotine content, only NaMN-adenylyltransferase showed a smooth correlation, whereas NAD-synthetase was not affected at all.Abbreviations HPLC high-performance liquid chromatography - QA quinolinic acid - NaMN nicotinic acid mononucleotide - NaAD nicotinic acid adenine dinucleotide - NMN nicotinamide mononucleotide     

Properties and Structure of a Novel Peptide Antibiotic No. 1907

There are three NAD biosynthetic pathways: the nicotinic acid-NAD, nicotinamide-NAD, and quinolinic acid (derived from tryptophan)-NAD pathways. To discover the main pathways of NAD biosyntheses in various tissues of the rat, the tissue distribution of nicotinamidase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase, nicotinamide phosphoribosyl-transferase, nicotinamide mononucleotide adenylyltransferase, and NAD⁺ synthetase were investigated. All of the tissues could synthesize NAD from nicotinamide, judging from that the activities of nicotinamide phosphoribosyltransferase and NMN adenylyltransferase detected in all of the tissues. From nicotinic acid, only liver, kidneys, and heart could. Liver and kidney can also synthesize NAD de novo from quinolinic acid.     

Interaction of muscle glycogen phosphorylase b with nicotinic acid, nicotinamide, N-nicotinyl-gamma-aminobutyric acid and nicotinamide coenzymes

The inhibitory action of nicotinic acid, nicotinamide, N-nicotinoyl-gamma-aminobutyric acid, NAD, NADH, NADP, and NADPH on the rabbit skeletal muscle glycogen phosphorylase b has been studied. The inhibition is reversible and positively cooperative (the value of Hill coefficients were determined for the following compounds: nicotinic acid (28 mM; 1.4), nicotinamide (4.4 mM; 1.2), N-nicotinoyl-gamma-aminobutyric acid (9.5 mM; 1.4), NAD (4.4 mM; 1.2), NADH (0.93 mM; 1.2). NADH-binding site of glycogen phosphorylase b subunit was characterized by the sedimentation velocity method. Microscopic dissociation constant was found to be 86 +/- 9 microM (pH 6.8; 20 degrees C). AMP-induced association of glycogen phosphorylase b is hindered by NADH.     

Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica

Salmonella enterica can obtain pyridine from exogenous nicotinamide mononucleotide (NMN) by three routes. In route 1, nicotinamide is removed from NMN in the periplasm and enters the cell as the free base. In route 2, described here, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and the produced nicotinamide ribonucleoside (NmR) enters the cell via the PnuC transporter. Internal NmR is then converted back to NMN by the NmR kinase activity of NadR. Route 3 is seen only in pnuC* transporter mutants, which import NMN intact and can therefore grow on lower levels of NMN. Internal NMN produced by either route 2 or route 3 is deamidated to nicotinic acid mononucleotide and converted to NAD by the biosynthetic enzymes NadD and NadE.     

Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid are mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphate ribose. NAD inhibits the IMPDH reaction at concentrations greater than 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (alpha) between 0 and 1. However, alpha was greater than 1 in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

An enzymatic method for the measurement of nicotinamide mononucleotide pyrophosphorylase in cells and tissues

A simple enzymatic method is described for the measurement of NMN pyrophosphorylase in tissue homogenates at levels as low as 10(-12) to 10(-9) mol. The product, nicotinamide mononucleotide, is converted to NAD using NAD pyrophosphorylase and the NAD is quantified in an enzymatic cycling assay. The enzyme described here is stimulated more at low concentrations of Mn2+ than Mg2+. ATP is not required for NMN pyrophosphorylase activity; the reaction is neither stimulated nor inhibited by ATP concentrations as high as 3 mM. The enzyme is totally dependent on phosphoribosylpyrophosphate. The method is highly reproducible in all tissues examined. Various cell lines and tissues from mouse were analyzed for NMN pyrophosphorylase.     

Identification of a novel human nicotinamide mononucleotide adenylyltransferase

The enzyme nicotinamide mononucleotide adenylyltransferase is an ubiquitous enzyme catalyzing an essential step in NAD (NADP) biosynthetic pathway. In human cells, the nuclear enzyme, which we will now call NMNAT-1, has been the only known enzyme of this type for over 10 years. Here we describe the cloning and expression of a human cDNA encoding a novel 34.4kDa protein, that shares significant homology with the 31.9kDa NMNAT-1. We propose to call this enzyme NMNAT-2. Purified recombinant NMNAT-2 is endowed with NMN and nicotinic acid mononucleotide adenylyltransferase activities, but differs from NMNAT-1 with regard to chromosomal and cellular localization, tissue-specificity of expression, and molecular properties, supporting the idea that the two enzymes might play distinct physiological roles in NAD homeostasis.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.     

Structure of nicotinamide mononucleotide adenylyltransferase: a key enzyme in NAD(+) biosynthesis

BACKGROUND: Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in fundamental processes in cell metabolism. The enzyme nicotinamide mononucleotide adenylyltransferase (NMN AT) plays a key role in NAD(+) biosynthesis, catalysing the condensation of nicotinamide mononucleotide and ATP, and yielding NAD(+) and pyrophosphate. Given its vital role in cell life, the enzyme represents a possible target for the development of new antibacterial agents. RESULTS: The structure of NMN AT from Methanococcus jannaschii in complex with ATP has been solved by X-ray crystallography at 2.0 A resolution, using a combination of single isomorphous replacement and density modification techniques. The structure reveals a hexamer with 32 point group symmetry composed of alpha/beta topology subunits. The catalytic site is located in a deep cleft on the surface of each subunit, where one ATP molecule and one Mg(2+) are observed. A strictly conserved HXGH motif (in single-letter amino acid code) is involved in ATP binding and recognition. CONCLUSIONS: The structure of NMN AT closely resembles that of phosphopantetheine adenylyltransferase. Remarkably, in spite of the fact that the two enzymes share the same fold and hexameric assembly, a striking difference in their quaternary structure is observed. Moreover, on the basis of structural similarity including the HXGH motif, we identify NMN AT as a novel member of the newly proposed superfamily of nucleotidyltransferase alpha/beta phosphodiesterases. Our structural data suggest that the catalytic mechanism does not rely on the direct involvement of any protein residues and is likely to be carried out through optimal positioning of substrates and transition-state stabilisation, as is proposed for other members of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily.