Joonas Jämsen

Heidi Tuominen

Anu Salminen

Georgiy A Belogurov

Natalia N Magretova

Alexander A Baykov
email: baykov@genebee.msu.su

Reijo Lahti
email: reijo.lahti@utu.fi

A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides

Keywords: inorganic pyrophosphatase, CBS domain, Moorella thermoacetica, bidirectional regulation, phylogenetic analysis, quaternary structure

Cystathionine--synthase (CBS) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. Here, we cloned, expressed in Escherichia coli, and characterized a family II inorganic pyrophosphatase (PPase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co 2+ or Mn 2+ ) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6fold by ATP. Inhibition by AMP is competitive whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide binding affinities are thus 100-to 10,000-fold higher than for other CBS domain-containing proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H + -translocating PPase with a comparable PP i -hydrolyzing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in M. thermoacetica by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP] + [ADP]) ratios, respectively.

INTRODUCTION

Inorganic pyrophosphate (PP i ) is produced in vast amounts by biosynthetic reactions, such as protein, RNA, and DNA synthesis, and its concentration affects the equilibria of these reactions [START_REF] Kornberg | On the metabolic significance of phosphorolytic and pyrophosphorolytic reactions[END_REF]. In addition, PP i regulates many other cellular processes, including calcification, cell proliferation, and iron transport [START_REF] Heinonen | Biological Role of Inorganic Pyrophosphate[END_REF]. Consequently, disruption of PP i metabolism can lead to a variety of pathological conditions [START_REF] Heinonen | Biological Role of Inorganic Pyrophosphate[END_REF].

PP i is mainly hydrolyzed to orthophosphate by inorganic pyrophosphatase (PPase) (EC 3.6.1.1), an enzyme that is essential for life [START_REF] Chen | Pyrophosphatase is essential for growth of Escherichia coli[END_REF][START_REF] Lundin | Yeast PPA2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function[END_REF][START_REF] Sonnewald | Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partioning[END_REF][START_REF] Ogasawara | Systematic function analysis of Bacillus subtilis genes[END_REF]. There are two known types of PPase: soluble and integral membrane-bound. Soluble PPases are further subdivided into families I and II, which are not homologous [START_REF] Shintani | Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis. Evidence for a new family of soluble inorganic pyrophosphatases[END_REF][START_REF] Young | Bacillus subtilis ORF yybQ encodes a manganese-dependent inorganic pyrophosphatase with distinctive properties: the first of a new class of soluble pyrophosphatase?[END_REF]. Family I PPases are found in all kingdoms of life and are among the best-characterized phosphoryl transfer enzymes [START_REF] Cooperman | Evolutionary conservation of the active site of soluble inorganic pyrophosphatase[END_REF][START_REF] Baykov | Cytoplasmic inorganic pyrophosphatases[END_REF]. Family II PPases, which were discovered more recently [START_REF] Shintani | Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis. Evidence for a new family of soluble inorganic pyrophosphatases[END_REF][START_REF] Young | Bacillus subtilis ORF yybQ encodes a manganese-dependent inorganic pyrophosphatase with distinctive properties: the first of a new class of soluble pyrophosphatase?[END_REF], are found in Bacilli and Clostridia and in some other bacterial lineages, including several human pathogens, and belong to the DHH family of phosphohydrolases [START_REF] Aravind | A novel family of predicted phoshoesterases includes Drosophila prune protein and bacterial RecJ exonuclease[END_REF]. Family II PPases are homodimers of subunits formed by two welldefined domains, whereas family I PPases have two or six compact one-domain subunits. The N-and C-terminal domains of family II PPases are connected by a flexible linker, and the active site is located at the domain interface [12,[START_REF] Ahn | The "open" and "closed" structures of the type-C inorganic pyrophosphatases from Bacillus subtilis and Streptococcus gordonii[END_REF]. Also, unlike family I PPases, family II PPases contain a tightly bound transition metal ion, usually Mn 2+ or Co 2+ , although they also require Mg 2+ for maximal activity. In addition, divalent cations, especially Mn 2+ , promote dimerization of family II PPases [START_REF] Parfenyev | Quaternary structure and metal-ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii and Streptococcus mutans[END_REF].

Interestingly, the N-terminal domain of approximately a quarter of the known family II PPase sequences contains a large (~250-amino acid) insert, comprising two CBS domains.

None of these CBS-PPases has been isolated and characterized. CBS domains, originally found in cystathionine--synthase [START_REF] Bateman | The structure of a domain common to archaebacteria and the homocystinuria disease protein[END_REF], are widely distributed among proteins in all three kingdoms of life, but their roles are not well understood [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF]. In some cases, CBS domains are potential targets for regulation by adenosine derivatives [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF][START_REF] Kemp | Bateman domains and adenosine derivatives form a binding contract[END_REF][START_REF] Niemeyer | Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies[END_REF][20][START_REF] Wellhauser | Nucleotides bind to the C-terminus of ClC-5[END_REF]. Importantly, point mutations in CBS domains cause several hereditary diseases in humans [START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF].

To elucidate the role of CBS domains in family II PPases, we cloned, expressed, and purified a CBS-PPase from Moorella thermoacetica (former name Clostridium thermoaceticum), a low G + C Gram-positive thermophilic acetogen with an optimum growth temperature at 55-60°C [START_REF] Drake | Physiology of the thermophilic acetogen Moorella thermoacetica[END_REF]. We found that, unlike all other known PPases, this enzyme is subject to a strong bidirectional regulation by adenine nucleotides.

EXPERIMENTAL Cloning and mutagenesis

Genomic DNA extracted from M. thermoacetica strain ATCC 35608 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). The mtCBS-PPase open reading frame (GenBank accession number NC_007644) was amplified by PCR using primers 5'-TTATCATATGGGTAAAGAGAT TCTGGTTATCG-3' (forward) and 5'-TTATC TCGAGTTATCCCTGCAGCAACCGCCG-3' (reverse). The amplified gene was sequenced in both directions and one difference compared to the sequence shown in GenBank was observed (TTG encoding Leu190 was replaced by CTG), but this did not affect the protein sequence. The PCR fragment was inserted into pBLUESCRIPT SK (Stratagene) and site-directed mutagenesis was performed using an overlapping PCR technique (QuikChange, Stratagene). The mutation was verified by DNA sequencing.

Protein expression and purification

To produce wild-type and variant mtCBS-PPases, the PCR products were cloned into the pET36b vector (Novagen) using NdeI and XhoI. The plasmid construct was transformed into Escherichia coli BL21(DE3) RIL cells (Stratagene) and the transformants were grown in Terrific Broth [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] containing 30 g/mL kanamycin and 30 g/mL chloramphenicol. mtCBS-PPase expression was induced for 3 h by 0.4 mM isopropyl--D-thiogalactopyranoside.

Cell paste (10 g wet weight) obtained by centrifugation was resuspended in 30 ml of icecold 25 mM Tris-HCl buffer, pH 7.3, containing 24 mM MgCl 2 , 1 mM CoCl 2 , 20 µM diethylenetriaminetetraacetic acid (DTPA) and homogenized twice with a French press (SLM Instruments) at 900 psi. The crude extract obtained was loaded onto a 150-ml column containing Fast Flow DEAE-Sepharose (GE Healthcare). The column was washed with the same buffer, and protein was eluted with a 450-ml linear gradient of 0.1-0.3 M NaCl.

Fractions containing mtCBS-PPase (150-190 mM NaCl) were pooled, concentrated to 12 ml with Centriprep10 (Amicon), and further purified by gel filtration on a Superdex 200 26/60 column (GE Healthcare) equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 50 mM KCl, 2 mM MgCl 2 , 0.1 mM CoCl 2 and 20 µM DTPA. The fractions containing mtCBS-PPase were pooled, concentrated to 20-40 mg/mL and stored frozen at 70°C.

Because the enzyme was most stable in the presence of 0.1 mM Co 2+ and 2 mM Mg 2+ , these cations were routinely added to the solutions used to purify, store, and dilute the enzyme. When needed, these metal ions were removed from the enzyme stocks by incubation with 5 mM DTPA for 1 day at 4°C. The bulk of the chelator was removed by dialysis against three 1-l changes of 100 mM Mops-KOH buffer (pH 7.5) containing 50 µM DTPA.

The purity of enzyme samples was assessed by electrophoresis on 8-25% gradient polyacrylamide gels in the presence of 0.55% sodium dodecyl sulfate using the Phast System (GE Healthcare). Concentrations of mtCBS-PPase solutions were determined on the basis of a subunit molecular mass of 48.1 kDa and an extinction coefficient 1% 280 of 4.5, as estimated from the amino acid composition using the ProtParam program [START_REF] Gasteiger | Protein identification and analysis tools on the ExPASy server[END_REF].

Activity measurements

Except where noted, the activity was measured at 25°C in 25 ml of 100 mM Mops-KOH buffer (pH 7.2) containing 0.1 mM CoCl 2 , 5 mM MgCl 2 , and the nucleotide tested. An aliquot (5-150 µl) of diluted enzyme solution was added to the mixture (0.2-2 g/mL final enzyme concentration), followed 1 min later by 0.16 mM PP i . The formation of P i was then monitored for 3-10 min using an automatic P i analyzer [START_REF] Baykov | A simple and sensitive apparatus for continuous monitoring of orthophosphate in the presence of acid-labile compounds[END_REF], and initial reaction rates were estimated from the recorder tracing. AMP, ADP, and ATP were from Fluka. All other nucleotides were from Sigma. AMP was the free acid, ADP was the potassium salt, and all other nucleotides were sodium salts.

Immediately prior to the use of ATP, any contaminating ADP was converted to ATP by treating 5 mM stock solutions of ATP for 1 h at room temperature with 10 U/mL rabbit muscle creatine kinase (Roche) in 100 mM Mops-KOH buffer (pH 7.2) containing 10 mM creatine phosphate (Fluka) and 5 mM MgCl 2 . The AMP preparation was judged to be reasonably pure because a similar treatment with creatine kinase and creatine phosphate had no effect on the PPase activity measured in the presence of AMP. In addition, treating stock ADP solutions with 10 U/mL of hexokinase and 10 mM glucose to convert any contaminating ATP into ADP also had no effect on the PPase activity. For competition measurements, in which ADP and ATP were added simultaneously, creatine kinase in stock solutions of ATP was removed by ultrafiltration using Vivaspin 4 ml concentrators (Sartorius AG) with a 10 kDa MWCO PES membrane.

Sedimentation

Analytical ultracentrifugation was carried out at 20ºC in a Spinco E instrument (Beckman Instruments) with scanning at 280 nm. The samples contained 10 µM enzyme and appropriate ligands. Before each run, the samples were incubated for 2 to 3 h at 20ºC. The sedimentation velocity was measured at 48,000 rpm, and the sedimentation coefficient (s 20,w ) was calculated as described by Chervenka [START_REF] Chervenka | Methods for the Analytical Ultracentrifuge[END_REF].

Crosslinking and electrophoresis

Enzyme was diluted to 8.3 µM with 25 mM HEPES-KOH (pH 7.5) and then incubated with 26 mM glutaraldehyde for 15 or 30 min at room temperature [27]. The reaction was stopped by addition of 1/10 volume of 1.0 M Tris-HCl (pH 7.3). The samples were separated by electrophoresis on Phast System 8-25% SDS-PAGE gradient gels (GE Healthcare), and the gels were stained with PhastGel Blue R (GE Healthcare). A Perfect Protein Marker kit (Novagen) was used as molecular weight standards.

Kinetic data analysis

Nonlinear least squares fitting of the data was performed using SCIENTIST, version 2.01 (Micromath). The dependence of activity on the concentration of the ligand (L) was fit to eqn 1, where A +L and A -L are activities with and without bound ligand, respectively, and K d is the apparent dissociation constant of the enzyme-ligand complex, all measured at a fixed substrate concentration:

A = A +L + (A -L -A +L )/(1 + [L]/K d ) (1)
In competition experiments, the dependence of the apparent K d (K d,app ) for ligand L 1 on the concentration of the second ligand, L 2 , was fit to eqn 2, where K d1 and K d2 are the dissociation constants for ligands L 1 and L 2 , respectively:

K d,app = K d1 ( 1 + [L 2 ]/K d2 ) (2)

RESULTS

Expression, purification and catalytic activity of mtCBS-PPase

SDS-PAGE analysis of crude extracts obtained from recombinant E. coli cells revealed an intense ~50 kDa band that was absent in cells transformed with empty pET36b vector (data not shown). The SDS-PAGE also indicated that mtCBS-PPase represented ~10% of the total protein. The recombinant protein was easily purified to homogeneity by ion exchange chromatography and gel filtration. This procedure yielded 30-40 mg of pure mtCBS-PPase per liter of cell culture (~6 g of cell paste). E. coli PPase, which belongs to family I of soluble PPases, was absent from the purified mtCBS-PPase because the former eluted at a 20 mM lower NaCl concentration during DEAE-Sepharose chromatography.

The purified mtCBS-PPase exhibited a maximal k cat value (1.7 s -1 ) when assayed in the presence of both 1 mM Mg 2+ and 0.1 mM Co 2+ . Replacement of Co 2+ by Mn 2+ reduced k cat to 0.65 s -1 . The k cat values observed in the presence of Co 2+ , Mn 2+ or Mg 2+ alone were 1.15, 0.36 and 0.01 s -1 , respectively. According to its cofactor specificity, mtCBS-PPase resembles the common-type family II PPase of Methanococcus jannaschii [START_REF] Kuhn | Methanococcus jannaschii ORF mj0608 codes for a class C inorganic pyrophosphatase protected by Co 2+ or Mn 2+ ions against fluoride inhibition[END_REF]. However, the maximal observed k cat value for mtCBS-PPase at 25°C was three orders of magnitude lower than that for common family II PPases [START_REF] Parfenyev | Quaternary structure and metal-ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii and Streptococcus mutans[END_REF] and was only two-fold higher at 55°C, the growth temperature of M. thermoacetica.

Effects of nucleotides and nucleosides

Table 1 shows that mtCBS-PPase is strongly inhibited by 100 µM AMP and ADP, whereas ATP activates it 1.6-fold. Some nucleotides, including cAMP, CDP, and UDP were less effective at inhibiting mtCBS-PPase, and most other nucleotides and nucleosides did not significantly affect the activity. Thus, adenine nucleotides are effective modulators of mtCBS-PPase activity. This is strikingly different than the common type family II PPase from Bacillus subtilis and family I PPases from E. coli and other sources, which are not modulated by ATP, ADP, or AMP and lack CBS domains.

Kinetic analysis of adenine nucleotide effects

Figure 1 shows the dose dependencies of AMP, ADP and ATP effects measured at a fixed substrate concentration (160 M total PP i ). Each curve obeyed Eq. 1 for activation or nonlinear inhibition with a finite activity of the enzyme-inhibitor complex. The relative values of these activities (A +L /A -L ) for AMP and ADP were low but significantly different from zero (Table 2). The apparent binding constants (K d ) derived for each nucleotide from these profiles are also summarized in Table 2. As expected from the appearance of the profiles in Figure 1, the K d values were similar for AMP and ATP and an order of magnitude lower for ADP.

The nucleotide effects on activity were further analyzed using a Lineweaver-Burk plot. estimated parameter values. For ATP, true binding constants governing binding to substratefree enzyme (K a ) and to enzyme-substrate complex (K a ´) were not determined separately, but their ratio, K a ´/K a , should be close to the ratio of the Michaelis constants measured at ATP concentrations of zero and near-saturating (10 M), i.e., 1.5 ± 0.2. That K a ´/K a exceeds unity is also evidenced by the fact that the lines measured in the absence and in the presence of ATP intersected above the ordinate axis in Figure 2, top.

To investigate interactions of inhibiting and activating nucleotides on mtCBS-PPase activity, the profiles of ADP modulation were measured at several fixed ATP concentrations and vice versa (Figure 3). for the pair AMP/ATP (data not shown). The effective concentrations of AMP were, however, higher than those of ADP by an order of magnitude. The binding constants derived for each nucleotide, from data in the insets to Figure 3, are summarized in Table 2.

The effects of the adenine nucleotides on Mn 2+ /Mg 2+ -activated mtCBS-PPase were qualitatively similar to those observed for the Co 2+ /Mg 2+ -activated form (Figure 1). The K d values for AMP and ADP were also similar, but the K d value for ATP was 10-fold higher (Table 2). Furthermore, the relative residual activities at infinite AMP or ADP concentrations (A +L /A -L ) were markedly higher in the presence of Mn 2+ . Finally, AMP, ADP, and ATP had similar effects on Co 2+ /Mg 2+ -activated mtCBS-PPase at 55°C, which is the physiological temperature for M. thermoacetica (data not shown).

Effect of Y169A substitution on adenine nucleotide binding

Tyr-169 is located in the CBS domain of mtCBS-PPase, in the region corresponding to the nucleotide binding pocket of the chloride transporter ClC-5, another CBS domain protein whose three-dimensional structure has been recently determined [START_REF] Meyer | Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5[END_REF]. Consistent with this localization, the replacement of Tyr-169 with Ala markedly decreased the affinity of mtCBS-PPase for the adenine nucleotides (Table 2), while having only a small effect (1.8-fold activation) on enzyme hydrolytic activity measured in their absence. Qualitatively, the Y169A variant behaved similarly to wild type mtCBS-PPase and exhibited activation by ATP and inhibition by AMP and ADP (Table 2).

Quaternary structure of mtCBS-PPase

Common family II PPases are dimeric proteins in the presence of metal cofactors but dissociate into nearly inactive monomers in the absence of such cofactors [START_REF] Parfenyev | Quaternary structure and metal-ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii and Streptococcus mutans[END_REF]30]. As CBS domains are reported to control oligomerization of cystathionine--synthase [START_REF] Kery | Trypsin cleavage of cystathionine-synthase into an evolutionarily conserved active core: structural and functional consequenses[END_REF][START_REF] Jhee | Domain architecture of the hemeindependent yeast cystathionine--synthase provides insights into mechanisms of catalysis and regulation[END_REF], we determined the oligomeric structure of mtCBS-PPase in the absence and presence of adenine nucleotides and compared it with that of common family II PPases. The sedimentation coefficient (s 20,w ) at pH 7.2 was 6.4 ± 0.1 S for the metal-depleted mtCBS-PPase and increased to 7.7 ± 0.1, 7.9 ± 0. The values of s 20,w for mtCBS-PPase measured in the presence of the metal ions were somewhat higher than that predicted assuming a spherical protein with a molecular weight of 96 kDa (dimer of 48 kDa monomers). Therefore, we performed crosslinking experiments to confirm that this enzyme is a dimer. Specifically, we reacted mtCBS-PPase with glutaraldehyde and separated the products by SDS-PAGE. Almost all of the protein migrated as the crosslinked dimer, although a small amount of unreacted monomer was observed (Figure 4). Similar electrophoresis patterns were obtained for mtCBS-PPase treated with glutaraldehyde in the presence of different metal ions or at various concentrations of mtCBS-PPase. The relative amount of the monomer, however, slightly increased in the absence of the metal ions or at low enzyme concentrations. Collectively, our data indicate that the oligomeric structure of mtCBS-PPase is similar to that of common family II PPases, which lack CBS domains, and is not affected by nucleotide binding.

DISCUSSION

CBS domains are found both in cytosolic enzymes and membrane-associated enzymes and channels from all species. They usually occur as tandem repeats, which are independent units that retain their structure and binding properties even when separated from the bulk protein [START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF]. The importance of CBS domains is emphasized by the observations that mutations of their conserved residues impair AMP, ATP, and S-adenosyl methionine binding to the CBS domains and cause a variety of human hereditary diseases [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF]. Although they are thought to be regulatory domains, the experimental evidence for this is scarce. Because CBS domains bind adenine nucleotides with varying affinities, they may act as sensors of cellular energy status [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF]. More recently, CBS domains of the chloride channel CLC-5 have been implicated in the control of intracellular trafficking [START_REF] Carr | A role for CBS domain 2 in trafficking of chloride channel CLC-5[END_REF] and the gating of the osmoregulatory transporter OpuA by osmotic strength [START_REF] Bieman-Oldehinkel | A sensor for intracellular ionic strength[END_REF].

CBS domains as regulators of mtCBS-PPase

Several lines of evidence suggest that the modulation of mtCBS-PPase by nucleotides is also mediated by CBS domains. First, isolated CBS domains from several proteins can bind adenine nucleotides [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF]20], whereas common family II PPases that lack CBS domains do not bind adenine nucleotides [START_REF] Zyryanov | Mechanism by which metal cofactors control substrate specificity in pyrophosphatase[END_REF]. Second, a single Y169A replacement in the CBS domain suppressed nucleotide binding to mtCBS-PPase, but increased enzyme catalytic activity.

Finally, the mixed type inhibition of mtCBS-PPase by ADP, and the activation by ATP, indicate formation of a ternary enzyme•substrate•nucleotide complex and are therefore inconsistent with nucleotide binding at the active site.

The observed competition between ATP binding, and the binding of both AMP and ADP, as well as the structural similarities between these compounds, imply a common binding site for all nucleotides on mtCBS-PPase. This conclusion is supported by structural data reported for other proteins. To date, a high-resolution three-dimensional structure is available for only a single intact CBS domain-containing protein, namely IMP dehydrogenase, but it does not contain bound nucleotides in the CBS domain [START_REF] Zhang | Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase[END_REF]. However, three-dimensional structures have been determined for several truncated CBS domain-containing proteins [START_REF] Meyer | Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5[END_REF][START_REF] Miller | Crystal structure of a tandem cystathioninebeta-synthase (CBS) domain protein (TM0935) from Thermotoga maritima at 1.87 Å resolution[END_REF][START_REF] Dutzler | Crystal structure of the cytoplasmic domain of the chloride channel ClC-0[END_REF] Importantly, such an "internally inhibited" system should be very sensitive to structural changes caused by nucleotide binding to the CBS domains and could easily be inhibited or activated. In this context, ATP partially compensates for the effect of the CBS insertion, whereas AMP and ADP increase the effect.

Nucleotide specificity of mtCBS-PPase

Nucleotide binding to mtCBS-PPase was highly specific with respect to both the base and the phosphate moieties. At a concentration of 100 M, adenine nucleotides were the most potent regulators of the enzyme. When guanine and uridine nucleotides were examined, only the diphosphates had significant effects on the activity of mtCBS-PPase; this may be because these nucleotides bound with weaker affinities.

The phosphate moiety of the nucleotide is also important, and determines both binding affinity (adenosine does not bind), and whether the nucleotide acts as an activator or inhibitor.

A diphosphate seems to result in the highest binding affinity, but a triphosphate is needed for activation. Moreover, the phosphate moiety determines the relative affinities of the nucleotide for free enzyme and enzyme•substrate complex. AMP, ADP and ATP can all combine with substrate-free enzyme whereas only ADP and ATP can bind, though with lower affinity, to the enzyme•substrate complex. Accordingly, the inhibition type is competitive with AMP and mixed with ADP, and ATP is a mixed-type activator. Substrate thus interferes with adenine nucleotide binding and vice versa, and the effect decreases with increases in phosphate chain length, especially when comparing AMP effects with ADP effects. A likely structural explanation of these data is that both the -phosphate and the -phosphate are involved in strong, thermodynamically favorable, interactions, that stabilize inactive conformations of mtCBS-PPase, whereas the interaction with the -phosphate is thermodynamically unfavorable but gives rise to an active enzyme conformation. The conformations elicited by AMP and ADP are different because their complexes bind substrate differently. Although a direct contact of the nucleotide phosphate chain with the active site cannot presently be excluded, it seems unlikely, because ATP, which has the longest phosphate chain, facilitated substrate binding by lowering K m (Figure 2). The assumed conformational flexibility of mtCBS-PPase is consistent with the known structures of common family II PPases, in which active sites lie between two domains connected by a highly flexible linker [12,[START_REF] Ahn | The "open" and "closed" structures of the type-C inorganic pyrophosphatases from Bacillus subtilis and Streptococcus gordonii[END_REF]. The dependence of the nucleotide effect on the length of the phosphate moiety may be due, but only in part, to Mg 2+ binding; in the presence of 5 mM Mg 2+ , ATP and ADP predominantly exist as complexes with Mg 2+ , whereas AMP exists as a mixture of free and Mg 2+ -bound forms [START_REF] Phillips | Adenosine and the adenine nucleotides. Ionization, metal complex formation and conformation in solution[END_REF].

The effects of the adenine nucleotides vary in different CBS domain proteins. With the exception of the chloride channel ClC-1, which is inhibited by both AMP and ATP, generally AMP and ATP have opposite effects on these proteins [20]. For example, AMP activates and ATP inhibits AMP-activated protein kinase and the chloride channel ClC-2 [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Niemeyer | Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies[END_REF], and, as in the case of mtCBS-PPase, ATP activates and AMP inhibits IMP dehydrogenase-2 and the chloride channel ClC-5. These findings have led to the hypothesis that CBS domains act as energy sensing modules in proteins [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF]. Generally, AMP, ADP and ATP bind with similar affinities to these proteins, in contrast to adenine nucleotide interactions with mtCBS-PPase, where ADP binding is strongly preferred. Furthermore, AMP and ATP bind at least 100-fold, and ADP 10,000-fold, more tightly (in terms of K d ) to mtCBS-PPase than to ClC-5 [START_REF] Meyer | Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5[END_REF], or to other CBS domain proteins [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF].

Distribution, phylogeny, and possible role of CBS-PPases

Common type family II PPases predominate in Bacilli, whereas CBS-PPases predominate in Clostridia and sulfate-reducing delta-proteobacteria (supplementary material online, Figure S1). Both types of family II PPases occur sporadically in other bacterial lineages. In contrast, structurally distinct family I PPases are more widespread and occur in most of the eubacterial phyla as well as in archaea and eukaryotes. Interestingly, a majority of the CBS-PPases contain an ~120-amino acid DRTGG domain [START_REF] Bateman | The Pfam protein families database[END_REF] between two CBS domains. The function of DRTGG domains is unknown, and they are found exclusively in bacterial and archael proteins. CBS-PPases from M. thermoacetica and Syntrophomonas wolfei are unique in that they possess CBS domains but lack a DRTGG domain.

Phylogenetic analysis of family II PPases suggests that all CBS-PPases are closely related but do not form a monophyletic group (supplementary material online, Figure S1). Some groups of common family II PPases, notably Bacilli PPases, have probably arisen due to the secondary loss of the CBS and DRTGG domain insertion. In addition, M. thermoacetica and S. wolfei are two independent cases where the DRTGG domain appears to have been lost. Biosynthetic reactions are the major source of PP i . Accordingly, in M. thermoacetica, the intracellular level of PP i peaks sharply during the mid-log phase of growth [START_REF] Heinonen | Comparative assessment of inorganic pyrophosphate and pyrophosphatase levels in Escherichia coli, Clostridium pasterianum and Clostridium thermoaceticum[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Surprisingly, the inhibition by AMP and ADP was of different types. AMP increased only the Michaelis constant (Figure2, top) and was thus a competitive inhibitor, whereas ADP additionally decreased the maximal velocity (Figure2, bottom) and was thus a mixed-type inhibitor. The effect of ATP on the kinetic parameters was the opposite of that of ADP (Figure2, top). At 10 M, ATP decreased the Michaelis constant (from 10.6 ± 0.5 M to 7.0 ± 0.6 M) and increased the maximal velocity (from 1.69 ± 0.05 s -1 to 2.3 ± 3 s -1 ). True inhibition constants governing AMP binding to substrate-free enzyme (K i = 19 ± 2 nM) and ADP binding to substrate-free enzyme (K i = 1.3 ± 0.1 nM), and to the enzyme-substrate complex (K i ´ = 0.35 ± 0.03 M) were estimated from the secondary dependencies shown in the insets of Figure 2. The substrate thus afforded partial protection against inhibition by ADP and completely prevented inhibition by AMP. The residual activity of the nucleotide-bound enzyme was ignored in this analysis as this activity would have only a minor effect on 7 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20071017 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071017

  ATP protected mtCBS-PPase from inhibition by ADP by decreasing the sensitivity to ADP (i.e., by shifting the dose-response inhibition curve to the right) and by increasing the basal activity in the absence of ADP. The K d,app for ADP changed in proportion to the ATP concentration (Figure 3, top inset) up to 100 µM ATP, which is 500 times the K d for ATP. Similarly, the K d,app for ATP was a linear function of the ADP concentration up to 0.2 µM ADP (Figure 3, bottom inset), which is 80 times the K d for ADP. These results indicated that the binding of ADP and ATP was mutually exclusive in the concentration ranges tested. If a mixed E•ADP•ATP and/or E•S•ADP•ATP complex were formed, K d,app for ADP would have attained a constant level, intermediate between the K d values for ADP binding to E•ATP and E•S•ATP. These considerations also apply to the bottom panel of Figure 3. Qualitatively similar inhibition/activation patterns were observed

  . The most recent structures of the cytoplasmic part of the chloride transporter ClC-5 [29] contain bound ADP or ATP, and those of the C-terminal domain of AMP-activated protein kinase (Protein Data Bank entries 2OOX and 2OOY) contain bound AMP or ATP, at the same site, formed between two CBS domains. Importantly, the new structures suggest that the nucleotides cause a marked conformational change in the CBS domains, thus explaining nucleotide regulatory action.Although all polar active site residues found in common family II PPases[12,[START_REF] Ahn | The "open" and "closed" structures of the type-C inorganic pyrophosphatases from Bacillus subtilis and Streptococcus gordonii[END_REF] are conserved in mtCBS-PPase, even in the presence of the best metal cofactor, Co 2+ , mtCBS-PPase is less active than common family II PPases by three orders of magnitude. Therefore, the insert containing two CBS domains appears to severely disrupt the normal catalytic cycle.

Finally, the clustering

  of CBS-PPases near the center of the tree suggests that the insertion was acquired very early in the evolution of family II PPases. CBS-PPases are found in 16 of the 329 prokaryotic species with complete genomes sequences in the Kyoto Encyclopedia of Genes and Genomes (www.genome.jp/kegg/catalog/org_list.html). Thirteen of these 16 species also have a gene encoding a membrane-bound H + -translocating PPase (H + -PPase). Interestingly, none of the 83 prokaryotic species with a gene for H + -PPase has a gene for the common family II PPase. H + -PPase may be unnecessary when there is family II PPases because the latter are three orders of magnitude more active and would therefore deplete the cell of PP i , which H + -PPase uses to establish a transmembrane pH gradient. In contrast, the activities of CBS-PPase and membrane-bound-PPase in M. thermoacetica are similar (A. Malinen, unpublished work), permitting control of the PP i concentration by the levels of adenine nucleotides via mtCBS-PPase.

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20071017 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071017

Figure 1

 1 Figure 1 Effects of adenine nucleotides on Co 2+ /Mg 2+ -activated mtCBS-PPaseThe lines show the best fit to eqn 1.

Figure 2

 2 Figure 2 Lineweaver-Burk plots for mtCBS-PPase inhibition and activation by adenine nucleotides Top panel, Inhibition by AMP and activation by ATP. Numbers on the lines indicate AMP concentrations (in M). The line marked with ATP shows the effect of 10 M ATP. Bottom panel, Inhibition by ADP. Numbers on the lines indicate ADP concentrations (in M). The insets show the dependencies of K m,app (top panel) or 1/k cat,app and k cat,app /K m,app (bottom panel) on nucleotide concentration.

Figure 3

 3 Figure 3 Combined effects of ADP and ATP on the activity of mtCBS-PPase Top panel, Effect of various concentrations of ADP at several fixed concentrations of ATP. Bottom panel, Effect of various concentrations of ATP at several fixed concentrations of ADP. The numbers on the curves denote the fixed concentration of the second nucleotide in µM. The lines show the best fit to eqn 1. The insets show the dependences of K d,app value on the concentration of the corresponding fixed ligand. The lines in the insets show the best fit to eqn 2.

Figure 4

 4 Figure 4 SDS-PAGE analysis of mtCBS-PPaseLane a, molecular mass markers; lane b, intact enzyme; lane c, enzyme crosslinked with glutaraldehyde for 15 min; lane d, enzyme crosslinked with glutaraldehyde for 30 min.Protein load for lanes b-d was 1 µg per lane.

  To avoid inhibition of biosynthetic enzymes and to provide P i for ATP synthesis, this PP i must be efficiently degraded by PPase. In fact, during the mid-log phase, mtCBS-PPase is expected to be most active because of a high [ATP]/([ADP] + [AMP]) ratio. Interestingly, Co 2+ , the best cofactor for mtCBS-PPase, is required for the growth of M. thermoacetica[START_REF] Drake | Physiology of the thermophilic acetogen Moorella thermoacetica[END_REF][START_REF] Lundie | Development of a minimally defined medium for the acetogen Clostridium thermoaceticum[END_REF]. Metabolic stresses that decrease ATP production inhibit biosynthetic reactions and cell growth by decreasing the [ATP]/([ADP] + [AMP]) ratio. This, in turn, should inhibit mtCBS-PPase and allow accumulation of PP i , which would further inhibit biosynthetic reactions. Instead of or in conjunction with the action of H + -ATPase, the accumulated PP i is apparently used by H + -PPase to sustain the pH gradient across the cell membrane and therefore maintain cell vitality.

	This work was supported by Academy of Finland Grants 201611 and 114706, a grant for the
	National Graduate School in Informational and Structural Biology from the Ministry of
	Education and the Academy of Finland, and Russian Foundation for Basic Research Grant 06-
	04-48887. We thank P. V. Kalmykov for his assistance with analytical centrifugation.

This two-PPase system may allow the cell to combat stress more efficiently than cells with only soluble or membrane-bound PPases. Thus, CBS-PPase may act as a survival factor under conditions of low energy supply.

In summary, unlike other PPases, mtCBS-PPase is subject to strong inhibition by AMP and ADP as well as activation by ATP. Remarkably, this enzyme binds adenine nucleotides much tighter than other reported CBS proteins. Furthermore, an effect of ADP, which is the most efficient inhibitor of mtCBS-PPase, is uncommon in other CBS proteins. Further studies, including determination of the three-dimensional structure of mtCBS-PPase with a bound adenine nucleotide, are needed to elucidate the structural basis for nucleotide regulation of this interesting enzyme and other CBS proteins. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20071017 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071017 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1 Effects of nucleotides and nucleosides (100 µM) on Co 2+ /Mg 2+ -activated mtCBS- PPase The

 1 activity of mtCBS-PPase measured with no effector added (1.64 s -1 ) was taken as 100%.

	Effector

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20071017 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071017

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 Parameters describing the effects of nucleotides on Co 2+ -and Mn 2+ -bound mtCBS-PPase Co

 2 2+ /Mg 2+ -activated enzyme Mn 2+ /Mg 2+ -activated enzyme Enzyme NucleotideA +L /A -L (%) K d (µM) A +L /A -L (%) K d (µM)

	Source

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20071017 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071017 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society