Joonas Jämsen

Heidi Tuominen

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

Reijo Lahti
email: reijo.lahti@utu.fi

Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins

Keywords: inorganic pyrophosphatase, CBS domain, Moorella thermoacetica, allosteric regulation, mutagenesis, adenine nucleotides

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Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins

Joonas Jämsen, Heidi Tuominen, Alexander A Baykov, Reijo Lahti

INTRODUCTION

In Bacilli, Clostridia, and other bacterial lineages, including several human pathogens, the intracellular PP i level is controlled by family II pyrophosphatases (PPases) (EC 3.6.1.1) that convert PP i to P i [START_REF] Shintani | Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes[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]. Approximately one-quarter (67) of known family II PPase sequences, mostly from Clostridia and sulphate-reducing -proteobacteria, contain a large insert (~100-125 residues) composed of two CBS domains that form a Bateman fold [START_REF] Bateman | The structure of a domain common to archaebacteria and the homocystinuria disease protein[END_REF]. This structure, originally identified in cystathionine--synthase [START_REF] Bateman | The structure of a domain common to archaebacteria and the homocystinuria disease protein[END_REF], binds regulatory adenine nucleotides and S-adenosyl methionine and is present in a variety of proteins [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]. We recently expressed the CBS domain-containing PPase of Moorella thermoacetica (mtCBS-PPase) for the first time, and analysed enzyme regulation by kinetic and binding measurements [START_REF] Jämsen | A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides[END_REF][START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF]. The enzyme binds adenine nucleotides in the submicromolar range, is activated by ATP, and inhibited by AMP and ADP [START_REF] Jämsen | A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides[END_REF]. Stopped-flow kinetic measurements using a fluorescent AMP analogue revealed that nucleotide binding to mtCBS-PPase occurs in three steps with relaxation times from 0.01 to 100 s, implying conformational changes in the complex [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF]. Nucleotide binding was modulated by the substrate (PP i ), which additionally induced enzyme conversion to a more active form within a timescale of minutes [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF]. Recently, we expressed the CBS domain containing PPase of Clostridium perfringens (cpCBS-PPase) and determined the structure of the regulatory region comprising two CBS domains [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF].

According to the Pfam database [START_REF] Finn | The Pfam protein families database[END_REF], CBS domains have been identified in nearly 30,000 protein sequences from all kingdoms of life. In addition to the role played by CBS domains in cystathionine--synthase [START_REF] Bateman | The structure of a domain common to archaebacteria and the homocystinuria disease protein[END_REF], such domains are implicated in the control of intracellular trafficking by the chloride channel, CLC-5 [START_REF] Carr | A role for CBS domain 2 in trafficking of chloride channel CLC-5[END_REF]; gating of the osmoregulatory transporter, OpuA [START_REF] Bieman-Oldehinkel | A sensor for intracellular ionic strength[END_REF]12]; protein phosphorylation by AMP-dependent protein kinase [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF]; Mg 2+ transfer by the transporter, MgtE [START_REF] Ishitani | Mg 2+sensing mechanism of Mg 2+ transporter MgtE probed by molecular dynamics study[END_REF]; plant vacuolar nitrate transport by the transporter, AtCLCa [START_REF] De Angeli | ATP binding to the C terminus of the Arabidopsis thaliana nitrate/proton antiporter, AtCLCa, regulates nitrate transport into plant vacuoles[END_REF]; and adenylate nucleotide synthesis (IMP dehydrogenase) [START_REF] Pimkin | A regulatory role of the Bateman domain of IMP dehydrogenase in adenylate nucleotide biosynthesis[END_REF]. Single mutations in the A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

CBS domains of these proteins are linked to a variety of human hereditary disorders, including homocystinuria, Wolff-Parkinson-White syndrome, retinitis pigmentosa, and Bartter syndrome [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]. However, little is known about the mechanism underlying the transduction of regulatory inhibition or activation signals from CBS domains to the substratebinding regions of these proteins. Most crystal structures of CBS domains have been obtained for small proteins of unknown function containing only CBS domains, or separate regulatory regions of multi-domain proteins, which reveal no significant changes upon binding of nucleotides. Although the crystal structures of several complex proteins containing regulatory CBS domains are available [START_REF] Zhang | Characteristics and crystal structure of bacterial inosine-5'monophosphate dehydrogenase[END_REF][START_REF] Hattori | Mg 2+ -dependent gating of bacterial MgtE channel underlies Mg 2+ homeostasis[END_REF][18], pairs of structures for activated/inhibited or nucleotidebound/nucleotide-free forms have not been solved for any protein to date. Large structural differences in CBS domains have been observed in only two instances, dependent on the bound nucleotide: with the MJ0100 protein of unknown function from Methanocaldococcus jannaschii [START_REF] Lucas | Binding of Smethyl-5'-thioadenosine and S-adenosyl-L-methionine to protein MJ0100 triggers an open-toclosed conformational change in its CBS motif pair[END_REF] and the regulatory part of cpCBS-PPase [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF]. The cpCBS-PPase structures display marked conformational changes upon binding of activator (diadenosine tetraphosphate) compared to inhibitor (AMP) to the regulatory CBS domains, indicating that CBS-PPase is a promising model in which to explore the mechanism of regulation of CBS proteins.

Here, we report the effects of point mutations in the CBS domains of mtCBS-PPase on interactions with adenine nucleotides, with a view to identifying residues that determine the strength and selectivity of nucleotide binding as well as the direction of the effect (inhibition or activation). Additionally, we characterize the effects of substitutions on the substrate-and nucleotide-induced activity transitions observed in previous studies [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF]. Residues were selected for substitution based on approximate correspondence with physiologically disruptive mutations in other CBS domains and the X-ray structure of homologous cpCBS-PPase.

EXPERIMENTAL PROCEDURES

Reagents

Restriction enzymes and T4 DNA ligase were obtained from Fermentas (Lithuania) and Pfu Turbo DNA polymerase from Stratagene (USA). Oligonucleotides were purchased from MWG Biotech (Germany). AMP, ADP and ATP were from Fluka (Switzerland). [ 14 C]AMP was acquired from Moravek Biochemicals and Radiochemicals (USA). All other reagents and kits were obtained from Sigma (USA).

Site-directed mutagenesis, protein expression, and purification

Mutagenesis of the mtCBS-PPase gene was performed in pBluescript SK (Stratagene, USA) with a QuikChange kit (Stratagene). Primer sequences used are shown in Supplementary Table S1. Variant genes were cloned for expression into the pET36b+ plasmid (Novagen, USA) using NdeI and XhoI restriction sites. Gene integrity and presence of the desired point mutations were confirmed via DNA sequencing. Expression and purification of mutant proteins was performed as described for wild-type mtCBS-PPase [START_REF] Jämsen | A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides[END_REF]. Protein concentrations were determined from absorbance at 280 nm ( = 22,920 M -1 cm -1 , calculated using ProtParam [START_REF] Gasteiger | The Proteomics Protocols Handbook[END_REF]). Protein expression level and purity of the final preparation were analysed on 8-25% SDS-PAGE gels using the Phast system (GE Healthcare, USA).

Activity measurements

Unless otherwise stated, activity measurements were performed at 25°C in 25-40 ml of 100 mM Mops/KOH buffer (pH 7.2), including 0.1 mM CoCl 2 , 5 mM MgCl 2 and 0.16 mM PP i .

PP i hydrolysis was initiated by adding an aliquot (5-200 µl) of diluted enzyme solution to obtain 0.005-5 µg/ml final enzyme concentration. P i formation was monitored for 20-25 min A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. using a semi-automatic P i analyser [START_REF] Baykov | A simple and sensitive apparatus for continuous monitoring of orthophosphate in the presence of acid-labile compounds[END_REF]. Stock nucleotide solutions used in kinetic measurements were prepared immediately before use, and kept on ice.

Membrane filtration

[ 14 C]AMP binding to mtCBS-PPase was determined with a filtration method [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF] using Multiscreen 96-well opaque filtration plates with an IP membrane (Millipore Corp., USA).

The membrane was washed with 20% ethanol using buffer supplemented with 1 mM unlabelled AMP, and twice with buffer without AMP. Next, 50 l of 10 µM mtCBS-PPase solution was added and incubated for 10 min. Finally, 14 C-labeled AMP (200-5,000 cpm, 100 l) was added, the complete mixture incubated for 10 min, and the wells emptied using a vacuum pump (time to empty about 1 s). Wells were washed twice with 0.1 mL of buffer, 0.2 mL of Ultima Gold XR scintillant added, and the radioactivity adsorbed on the membrane was determined with a Chameleon V multimode microplate reader (Hidex Ltd, Turku, Finland). Bovine serum albumin was added instead of enzyme to estimate non-specific [ 14 C]AMP binding.

Data analyses

All non-linear least-squares fittings were performed with the program Scientist (MicroMath, USA). Dependence of activity (A) on nucleotide concentration ([L]) at a fixed substrate concentration was fitted to eqn 1, where A +L and A L represent activities with and without bound nucleotide, respectively, and K i,app is the apparent nucleotide binding constant:

) / ] L [ 1 /( ) ( app i, L L L K A A A A (1)
Time-courses of PP i hydrolysis were fit to eqns 2 and 3 [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF], where A 0 , A and A lim

] E [ d / ] d[P A t (2) ])} P [ S] /([ 1 /{ ) S] /{ 1 )]( 1 )( ( [ 0 m 0 m 0 lim 0 tr K K e A A A A t k (3)

RESULTS

Selection and production of enzyme variants

Eight residues were selected for substitution in the two CBS domains of mtCBS-PPase, based on (a) the positions of disease-causing mutations in CBS proteins (Figure 1A and Supplementary Table S2), (b) the identity of the ligands binding to nucleotides in cpCBS-PPase (Figure 1B), and, (c) the residue conservation profiles of CBS-PPases and other CBS proteins (Figure 1A). Residues corresponding to Lys100, Tyr169, and Arg170 are nucleotide ligands in the cpCBS-PPase structure (Lys, Tyr, and Ser, respectively) (Figure 1) and are well conserved (61%, 69%, and 87%, respectively) in 67 putative CBS-PPase sequences.

Furthermore, residues at positions 168-170 form part of the RYRN sequence corresponding to the RYSN loop of cpCBS-PPase, which undergoes the most significant conformational change when the activator-and inhibitor-bound forms of cpCBS-PPase are compared [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF].

Residues 187-189 are part of a conserved CBS domain motif. Finally, Val99 is located at the Keeping in mind that replacement of disease-causing residues yields varying effects in different proteins (Figure 1A and Supplementary Table S2), Ala is commonly used as a substituting residue, to eliminate any effect of polar side-chain groups. In addition, Tyr169 was conservatively replaced with Phe. Two Arg residues were mutated to Gly, because such substitutions are known to affect the function of pig AMP-dependent protein kinase 3 linked to CBS domains [22]. For substitution of R168, the choice between Ala and Gly was made on the basis of protein solubility, as the R168G variant showed a tendency to aggregate during purification. Expression levels for all mtCBS-PPase variants (~ 10% of total cell protein) were similar to that for wild-type enzyme, and variants were easily separated from other proteins, including E. coli PPase. The final preparations were at least 90 % pure as estimated by SDS-PAGE analysis (Supplementary Figure S1).

Catalytic activity

Earlier studies on mtCBS-PPase revealed ~2-fold activation by substrate, with a half-time of approximately 40 s, during PP i hydrolysis [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF]. This effect was preserved in all variants studied, except for the Y169A and R170A proteins (Figure 2). Accordingly, steady-state rate was attained after several minutes of reaction. The corresponding activity (A lim ) was physiologically more relevant than was initial activity in estimation of the catalytic constants (k cat,lim values) and Michaelis constants (K m values) for variant enzymes. Based on the effect of mutations on k cat,lim , the variant enzymes were divided into three groups (Table 1): (1) those displaying k cat,lim values similar to that of the wild-type enzyme (K100G), (2) more active enzymes (V99A, R168A, Y169A, Y169F, Y188A, and H189A), and (3) less active enzymes (R170A and R187G). The effects of mutation on K m were smaller, and in most instances, did not exceed the error of determination.

Nucleotide inhibition

The effects of substitutions on activity regulation by AMP and ADP were significant (Figure 3). Substrate (PP i ) concentration was fixed at 160 µM for these measurements, ensuring at least 85% saturation of enzyme active sites, based on the K m values listed in Table 1. Thus the observed effects of nucleotides on activity mainly reflect an influence on k cat,lim .

Remarkably, AMP inhibited wild-type and the majority of variant enzymes, but activated the R168A and Y169A mutants (Figure 3, Table 1). Activation was particularly marked (2.8fold) for the R168A protein. In keeping with these observations, the maximal degree of inhibition, characterized by the residual activity of an enzyme-AMP complex, was increased in the Y188A and H189A variants compared with wild-type enzyme (Table 1). The AMPbinding affinity, estimated using K i,app , was not significantly affected in the Y169F and R170A mutants; somewhat decreased in the K100G and Y188A substituents; markedly less in R168A, Y169A, and, particularly, V99A; and significantly elevated in the R187G and H189A mutant proteins.

The effects of ADP were more uniform in that all variants were inhibited by more than 5fold (Table 1). However, the K i,app values showed more variation. Specifically, increases were seen for the V99A, Y169A, and R170A variants, and falls were noted for other variants, except for R168A and Y188A. In most instances, the effects of ADP on K i,app paralleled those of AMP.

Equilibrium AMP binding

Direct measurements of [ 14 C]AMP binding to mtCBS-PPase and variants thereof in the absence of substrate were performed using the membrane filtration technique (Figure 4). In an earlier study, we presented evidence that, in wild-type mtCBS-PPase, AMP binding to a dimeric enzyme is characterized by negative cooperativity [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF]. Unfortunately, the binding curves obtained from filtration assays showed some scatter, precluding accurate analysis of curve form, which was subsequently approximated as a simple, two-parameter hyperbola.

Furthermore, the membrane filtration assay may underestimate the binding affinity because of partial dissociation of the complex during the removal of unbound AMP, which occurs in about 1 s. This may explain why the K d values obtained were markedly larger than those of K i,app derived from kinetic assays. However, regardless of the actual significance of filtrationderived K d, values, a marked increase upon residue substitution, as observed with V99A, K100G, Y169A, Y188A, and H189A proteins (Table 1), is clearly indicative of altered (most likely, weakened) AMP binding. Another important finding is that the stoichiometry of AMP binding increased from 1 to 2 mol/mol monomer in the Y169F, R170A, R187G, and Y188A variants, implying occupancy of both tandem CBS domains.

Activity transition

In the absence of substrate, wild-type mtCBS-PPase exists as a mixture of two conformations with different activities, and is converted to the more active conformation on the time-scale of the enzyme assay upon addition of substrate [START_REF] Jämsen | Nucleotide-and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica[END_REF]. Nucleotides bound to wild-type mtCBS-PPase slowed this transition. Similar analyses were performed for the variant enzymes. Fitting of eqns 2 and 3 to the time-courses of PP i hydrolysis (Figure 2) yielded both a ratio of initial to final activities (k cat,0 /k cat,lim ) (Table 2) and a rate constant for the substrate-induced transition (k tr ). For most variants, the k cat,0 /k cat,lim ratio was not significantly different from that of wild-type. The rate constant for transition in the absence of nucleotides was 1.5-3-fold less, compared with wild-type enzyme, and nucleotide addition further decelerated the transition (Figure 5), analogous to what was seen with wild-type PPase. Conversely, the Y169A and R170A variants exhibited no activity transition in the absence of nucleotides (k cat,0 /k cat,lim = 1). However, this transition was clearly accelerated in the presence of AMP or ADP. Another interesting finding was that ADP accelerated transition in the Y188A variant. The lack of effect of ADP on the k tr,lim value of the V99A variant appears to result from weak nucleotide binding (Table 1).

DISCUSSION

CBS Domain pair as an internal inhibitor in CBS-PPase

The specific activities of two CBS-PPases characterized to date, those of M. thermoacetica [START_REF] Jämsen | A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides[END_REF] and C. perfringens [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF], are 2-3 orders of magnitude lower than that reported for common family II PPases lacking CBS domains [START_REF] Shintani | Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes[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][START_REF] Parfenyev | Quaternary structure and metal ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii, and Streptococcus mutans[END_REF]. Previously, we hypothesized that CBS domains act as "intrinsic inhibitors" in CBS-PPases [START_REF] Jämsen | A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides[END_REF], supported by the observation that the activity of CBS-PPase and other CBS proteins may either decrease or rise, depending on the nature of the bound nucleotide. These allosteric effects are clearly mediated by conformational changes in CBS domains, and it is possible that other factors causing similar structural changes in CBS domains also affect PPase activity. The activation effect is particularly interesting, as it may provide relief against the "inhibition" induced by CBS domains.

The present results provide further support for the above theory. First, five of nine substitutions in CBS domains resulted in enhanced activity (as measured by k cat,lim values; Table 1). Although functional improvements in proteins after artificial mutation have been documented in the literature, function-disrupting mutations are far more common. This observation reflects the general dogma that functions tailored by nature are difficult to improve upon, but easy to damage. Our findings are in line with this belief if the apparent Second, the "internal inhibitor" mechanism is supported by reversal of the AMP effect from inhibition to activation in the R168A and Y169A substituents. To the best of our knowledge, no change in the direction of a nucleotide effect after amino acid substitution has ever been previously reported for any CBS protein. The possibility of such reversal implies high plasticity of the regulatory mechanism. Importantly, in the Y169A variant, the basal k cat,lim value in the absence of nucleotides (5.0 s -1 ) significantly exceeded that of wild-type mtCBS-PPase (2.2 s -1 ). The combined effect of mutation and AMP binding led to an increase in k cat,lim values by 2.8-3.5-fold in the R168A and Y169A variants, compared with the wildtype basal value. Most constructed mutations thus widen the range of activities attainable in the absence and presence of nucleotides, fully consistent with the "internal inhibitor" hypothesis.

Nucleotide-binding stoichiometry

Each CBS domain of the Bateman fold contains a potential binding site for nucleotides.

However, cpCBS-PPase structures [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF], most other reported structures [START_REF] Townley | Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase[END_REF][START_REF] Day | Structure of a CBS-domain pair from the regulatory gamma1[END_REF][26] and those found only in PDB (3LFR, 3FWR, 3FWS, 3FNA, 2YZQ, and 3DDJ) contain only one nucleotide molecule per two CBS domains. However, both sites are occupied by nucleotides in several reported protein structures [START_REF] Lucas | Binding of Smethyl-5'-thioadenosine and S-adenosyl-L-methionine to protein MJ0100 triggers an open-toclosed conformational change in its CBS motif pair[END_REF][27][28][29].

Wild-type mtCBS-PPase is similar to cpCBS-PPase in that the enzyme binds only one nucleotide per two CBS domains (Table 1). Remarkably, four substitutions in CBS2 elevated the AMP binding stoichiometry of mtCBS-PPase to two nucleotides per CBS domain pair (Table 1). Two of the replaced residues (Y169 and R170) correspond to cpCBS-PPase AMP ligands, and two (R187 and Y188) belong to a conservative CBS motif. One possible A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

explanation is that mutations in CBS2 induce conformational changes in this domain, inhibiting interactions with the CBS1-bound nucleotide and facilitating intrinsic nucleotide binding. Interestingly, the K i,app value was markedly decreased in the R187G variant, indicating tighter binding. This may indicate that, at least in this variant, the K i,app value is referable to the CBS2-bound nucleotide. Gómez-García et al. [29] recently reported unusual binding of AMP to the linker sequence connecting the two CBS domains in a CBS protein from Methanocaldococcus jannaschii. However, this type of binding is unlikely in mtCBS-PPase, because the linker sequence is relatively short.

Possible roles of mutated residues in regulation

The effects of substitutions on nucleotide binding affinity are easily explainable in terms of changes in nucleotide contact (group 1 residues), whereas effects on activity may be attributable to closure/opening of the CBS domain interface (group 2 residues), as seen in cpCBS-PPase structures [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF]. Group 1 residues include K100, Y169 and R170. The finding that AMP binding is weakened and ADP binding strengthened upon K100G substitution is consistent with the theory that K100 interacts with P1 of the nucleotides [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF]. Y169 stacks onto the adenine ring in cpCBS-PPase structures [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF], and alteration to Ala is thus expected to weaken binding, leading to malpositioning of the bound nucleotide, in turn facilitating opening of the CBS domain interface with concomitant enzyme activation. An opposite effect was predicted and indeed confirmed for the Y169F mutant. Based on the cpCBS-PPase structure [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF], R170 is expected to play a role in ADP P2 binding. Indeed, the K i,app values obtained for the R170A substitution indicate that this residue is clearly important for ADP, but not AMP, binding. Group 2 residues, which do not interact with bound nucleotide in the cpCBS-PPase structure [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF], include R168, R187, and Y188. R168 is located at the CBS domain interface within the RYRN sequence. The loop adopts markedly different conformations in activated and inhibited cpCBS-PPase, and may therefore play a central role in transmitting the regulatory signal to catalytic domains. This hypothesis is confirmed by the finding that mutation of both residues of this loop region reversed the effect of AMP from inhibition to activation. The importance of R187, a highly conserved (90%) residue in CBS-PPase, is explained by the interactions of this residue with the RYRN loop. Interestingly, the conformations adopted by R187 in inhibited and activated cpCBS-PPase structures [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF] are markedly different. Similar data were obtained for Y188, which is displaced from the domain interface to a hydrophobic cavity when a complex is formed with activating nucleotide. After Y188A substitution, the domain interface is expected to open, resulting in increased activity of the variant enzyme.

In summary, six CBS-domain residues, including four corresponding to disease-related residues of human proteins, are important in adenine nucleotide regulation of CBS-PPase.

Together with the previously determined structures of the inhibitor-and activator-bound forms of the regulatory domain of cpCBS-PPase, our results significantly clarify the mechanism underlying the regulatory effect of the CBS domain. Elucidation of the detailed mechanism of the cooperation between regulatory and catalytic domains is the next challenging step, and the current results will serve as a basis for this work.

ACKNOWLEDGEMENT

The authors wish to acknowledge Dr. Georgiy Belogurov for mutant design and the initial concept as well as Juho Vuononvirta and Minna Peippo for expression and purification of the mutant proteins. A c c e p t e d M a n u s c r i p t (B) Stereoview of the nucleotide-binding site of cpCBS-PPase with bound AMP [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF] showing the residues that were selected for mutation in the present study. Residue numbering is for cpCBS-PPase/mtCBS-PPase. The primed K100 belongs to the other subunit and forms an ion pair with P of AMP. S279/R170 is expected to interact with P of ADP, and Y278/Y169 forms a stacking interaction with the adenine moiety [START_REF] Tuominen | Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

selected variants

The assay medium contained 160 µM PP i . The reaction was initiated by adding an aliquot of enzyme solution pre-equilibrated at 25°C. Curve labels refer to variant identity and final enzyme concentration in nanograms per millilitre.

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204A c c e p t e d M a n u s c r i p t represent velocities at time zero, t, and infinity, respectively, [E] 0 is the total enzyme concentration, and K m is the Michaelis constant. Equation 3 assumes that activity increases with a first-order rate constant, k, to a constant level due to enzyme transition to a more active form during substrate conversion, and decreases upon substrate depletion. A 0 , A lim and k values were treated as adjustable parameters. The K m value, initially set at 10 µM at the initial round of calculation, was subsequently estimated from the calculated dependences of A lim on [S] 0 , and the calculation cycle repeated with the refined K m value until the results converged.

  0

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  same relative position in the second CBS domain of mtCBS-PPase compared to Y169 of the first domain (Figure1).

  Biochemical Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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"

  improvement" in function (activation) actually results in a disruption of the auto-inhibition mechanism underlying the regulatory activity of the CBS domains.

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  This work was supported by Academy of Finland Grant 114706, Russian Foundation for Biochemical Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Basic Research Grant 09-04-00869, and a grant from the Ministry of Education and the Academy of Finland (for the National Graduate School in Informational and Structural Biology). Biochemical Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204 A c c e p t e d M a n u s c r i p t subunit of human AMPK in complex with AMP and ZMP. Acta Crystallogr. D Biol. Crystallogr. 63, 587 596 26 Meyer, S., Savaresi, S., Forster, I. and Dutzler, R. (2007) Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5. Nat. Struct. Mol. Biol. 14, 60-67 27 King, N., Lee, T., Sawaya, M., Cascio, D. and Yeates, T. (2008) Structures and functional implications of an AMP-binding cystathionine beta-synthase domain protein from a hyperthermophilic archaeon. J. Mol. Biol. 380, 181 192 28 Xiao, B., Heath, R., Saiu, P., Leiper, F., Leone, P., Jing, C., Walker, P., Haire, L., Eccleston, J., Davis, C., Martin, S., Carling, D. and Gamblin, S. (2007) Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature, 449, 496 500 29 Gómez-García, I., Oyenarte, I. and Martínez-Cruz, L. A. (2010) The crystal structure of protein MJ1225 from Methanocaldococcus jannaschii shows strong conservation of key structural features seen in the eukaryal gamma-AMPK. J. Mol. Biol. 399, 53-70 Biochemical Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1

 1 Figure 1 Selection of residues for mutation

Figure 2

 2 Figure 2 Time-courses of PP i hydrolysis by wild-type mtCBS-PPase (WT) and

Figure 3

 3 Figure 3 Effects of AMP (A) and ADP (B) on the equilibrium activity of wild-type

Figure 4 [C 14 ]

 414 Figure 4 [C 14 ]AMP binding to wild-type mtCBS-PPase (WT) and selected

Figure 5

 5 Figure 5 Effects of AMP (A) and ADP (B) on the rate constant for substrate-

Table 1 Catalytic and nucleotide-binding parameters for wild-type and variant mtCBS-PPases Values

 1 showing large deviations from wild-type enzyme are in bold. Parameter values for catalytic activity and nucleotide inhibition refer to substrate-activated enzyme. Biochemical Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204	Catalytic activity AMP inhibition/activation a ADP inhibition a AMP binding b A c c e p t e d M a n u s c r i p t Binding stoichiometry (mol/mol) 0.94 ± 0.01 n.d. d 1.1 ± 0.2 1.06 ± 0.07 0.7 ± 0.3 1.8 ± 0.1 2.0 ± 0.1 1.6 ± 0.1 2.1 ± 0.2 Enzyme variant k cat,lim (s -1 ) K m (µM) K i,app (µM) Residual activity c (%) K i,app (µM) Residual activity c (%) K d (µM) WT 2.2 ± 0.2 8 ± 3 0.6 ± 0.1 6 ± 2 1.0 ± 0.4 14 ± 5 13 ± 1 V99A 7.3 ± 0.5 3.7 ± 1.6 >1000 n.d. d >1000 n.d. d >2000 K100G 1.9 ± 0.1 13 ± 4 1.7 ± 0.2 4 ± 2 0.13 ± 0.03 8 ± 3 340 ± 90 R168A 2.8 ± 0.3 11 ± 5 11 ± 5 280 ± 30 0.8 ± 0.2 12 ± 4 35 ± 8 Y169A 5.0 ± 0.7 9.4 ± 2.8 8 ± 3 122 ± 2 >1000 n.d. d 1000 ± 200 Y169F 7.1 ± 0.8 12 ± 6 0.39 ± 0.06 <2 0.017 ± 0.001 <2 50 ± 10 R170A 0.88 ± 0.06 7.9 ± 2.6 0.9 ± 0.1 2 ± 3 57 ± 36 <20 17 ± 1 Y188A R187G 0.98 ± 0.09 5.7 ± 2.4 0.051 ± 0.007 <4 0.009 ± 0.001 <2 27 ± 7 5.8 ± 0.9 15 ± 9 2.0 ± 0.7 37 ± 4 1.8 ± 0.8 5 ± 1 150 ± 50

c Activity at infinite nucleotide concentration versus activity at zero nucleotide concentration d n.d., not determined

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Table 2 Parameters describing activity transition in wild-type and variant mtCBS-PPases Values

 2 showing large deviations from wild-type enzyme are presented in bold.

	k tr,lim b

a k tr,lim values refer to >85% substrate saturation.

b Ratio of catalytic activities estimated with eqns 2 and 3 from the initial and final rates of substrate conversion in the absence of nucleotide. Biochemical Journal Immediate Publication. Published on 11 Nov 2010 as manuscript BJ20101204 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101204

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

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Christakis Panayiotou
email: christakis.panayiotou@ki.se

Nicola Solaroli

Yunjian Xu

Magnus Johansson

Anna Karlsson

The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes

Keywords: AK, adenylate kinase, TLC, thin layer chromatography, HPLC, highperformance liquid chromatography, GFP, green fluorescent protein adenylate kinase, nucleotide metabolism, nucleotide phosphorylation Biochemical

come

The characterization of human adenylate kinases 7 and 8

demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes

1

INTRODUCTION

Adenylate kinases (AK) [EC 2.7.4.3] are phosphotransferases that catalyze the interconversion of adenine nucleotides. The adenylate kinases are important in cellular energy homeostasis and the maintanance of high levels of ATP in a combined action of adenylate kinase activity and mitochondrial oxidative phosphorylation [START_REF] Dzeja | Adenylate kinase-catalyzed phosphotransfer in the myocardium: increased contribution in heart failure[END_REF]. The presence of several isoforms of adenylate kinases in mammalian tissues is already well established [START_REF] Dzeja | Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing[END_REF]. The presence of multiple isoforms is a specific feature of adenylate kinases that is only shared with the guanylate kinases among the nucleotide and nucleoside kinases. Seven different adenylate kinase isoenzymes have been identified in human tissues and six of them have been thoroughly characterized. The major cytosolic isoform AK1 is present at high levels in skeletal muscle, brain and erythrocytes, whereas the major mitochondrial isoform AK2 is expressed in the mitochondrial intermembrane space of tissues rich in mitochondria like liver, heart and skeletal muscle. AK3 and AK4 are located in the mitochondrial matrix, with AK3 having its highest expression levels in the liver, heart and skeletal muscle. AKs 1 -3 are expressed at higher levels compared to the other AK isoenzymes, AKs 4 -7, which seem to have more specialized functions in certain cell types. AK4 is expressed at low levels in brain, kidney, liver and heart tissues, and it is reported to contain an N-terminal mitochondrial import sequence that remains uncleaved after import into the mitochondria [START_REF] Panayiotou | Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4[END_REF]. AK5 is cytosolic, or both cytosolic and nuclear depending on the transcript variants, with two separate functional domains and, in contrast to the multi-tissue expression profiles of most other AKs, it is expressed almost exclusively in brain [START_REF] Solaroli | Identification of two active functional domains of human adenylate kinase 5[END_REF][START_REF] Van Rompay | Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein[END_REF]. As far as AK6 is concerned, fluorescence microscopy revealed a nuclear localization, but there is no confirmed expression profile available [START_REF] Ren | The crystal structure of human adenylate kinase 6: An adenylate kinase localized to the cell nucleus[END_REF]. AK7 seems to have a tissue-restricted expression and its activity has been associated with cilia function [START_REF] Fernandez-Gonzalez | Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia phenotype[END_REF].

The existence of several AKs with different subcellular localizations, expression profiles, substrate specificities and kinetic properties provides evidence of specialized functions in specific cellular processes. Our studies aim at getting a complete picture of all AKs expressed in mammalian cells and their role in the homeostasis and synthesis of adenine nucleotides that are required for a variety of cellular metabolic processes. The AKs are also important for DNA and RNA synthesis and may contribute to the activity of pharmacologically active nucleoside and nucleotide analogs.

In this study we have expressed and characterized AK7 and determined its substrate specificity, kinetic properties and subcellular localization. In addition, a novel member of the AK family with two functional domains has been identified and characterized and it was assigned the name AK8. By the identification and characterization of AK7 and AK8 we believe that the identification of members of the AK family of isoenzymes is close to being completed. Our results demonstrate that in addition to differences in tissue expression, subcellular localization and structural organization, the AKs show differences in kinetic parameters with both high and low substrate affinity enzymes.

EXPERIMENTAL

Cloning and sequencing

The IMAGE clone 4828427 (GenBank ID: BC035256) for AK7 was purchased from RZPD and the IMAGE clone 5744517 (GenBank ID: BC050576) for AK8 was purchased from the American Type Culture Collection.

AK7 cDNA was cloned into Nde1/BamH1 sites of the pET16b plasmid vector (Novagen) using the primers: AK7 forward, 5´- AK8p1 forward and AK8p2 reverse primers were used for the cloning of the full length AK8. For cloning of full length AK8 into Xho1/EcoR1 sites of pEGFP-N1 the oligonucleotides AK8GFP forward, 5´-AGATCTCGAGCTGGAAGTTCTGTTCCAG-3´with Xho1 site and AK8GFP reverse, 5´-GTGAGAATTCGGGGGATTTTCTTGG-3´ with EcoR1 site were used. The plasmids were sequenced by MWG-Biotech (Ebersberg Germany) to verify the DNA sequences.

Expression and purification

The plasmids were transformed into the Escherichia coli strain BL21 (DE3) pLysS (Stratagene) and single colonies were inoculated into LB-medium supplemented with 100 µg/ml ampicillin and 34 µg/ml chloramphenicol. Bacteria were grown at 37 °C and protein expression was induced at OD 600 0.9 with 1 mM isopropyl-1-thio-β-D-galactopyranoside for 12 h at 27 °C. The expressed proteins were purified using TALON Metal Affinity Resin (Clontech) as described by the manufacturer. The size and purity of the recombinant proteins were verified by SDS-PAGE and the protein concentration was determined with Bradford Protein Assay (BIO-RAD) using BSA as standard. The proteins were aliquoted and stored at -80 °C.

In vitro translation

AK8, AK8p1 and AK8p2 were synthesized with PURExpress ® In Vitro Protein Synthesis Kit (NEB) in the presence of [ 35 S] methionine (Perkin Elmer), following the manufacturer's instructions. 2 µl of each reaction and 5 µl of Rainbow [ 14 C] methylated protein molecular weight marker (Amersham) were loaded on a NuPAGE 4-12% Bis-Tris gel (Invitrogen). The gel was washed with Gel-Dry drying solution, dried with Dry Ease Mini Cellophane (Invitrogen) and used for autoradiographic detection.

Enzyme assays

The substrate specificity of the recombinant AK7, the full length AK8 and its two domains were assayed by thin layer chromatography (TLC) as described before [START_REF] Lust | Contributions of anion-exchange thin-layer chromatography to the study of free nucleotides in tissues[END_REF]. The nucleoside monophosphates and triphosphates were purchased from Sigma. [γ-32 P] ATP/GTP and [α-32 P] UTP/CTP/TTP were purchased from Perkin Elmer. All assays were performed in 10 µl-volume reactions containing 50 mM Tris-HCl pH 7.6, 5 mM MgCl 2, 1 mM unlabeled NTPs, 0.1 µCi/µl radiolabeled NTPs (3000 Ci/mmol), 1 mM NMPs or dNMPs and 0.5 µg of recombinant enzyme. After incubation for 1 h at 37 °C, 2 µl of the reaction products were To determine the kinetic properties of AK1, AK7 and AK8, the non-radiolabeled products were separated and quantified by reversed-phase high-performance liquid chromatography (HPLC) using a Chromolith TM column (RP-18e, 100-4.6 mm) (Merck) as described before [START_REF] Curbo | The Drosophila melanogaster UMP-CMP kinase cDNA encodes an N-terminal mitochondrial import signal[END_REF]. All assays were performed in 25 µl-volume reactions containing 5 mM of phosphate donor, 50 mM Tris-HCl pH 7.6, 10 mM MgCl 2, 5 mM DTT and 0.5 mg/ml BSA. 25 ng of AK7, 20 ng of AK8 and 10 ng of AK1 and different concentrations of substrate (from 1 µM to 10 mM) were used to determine the K m and V max values and thereafter the V max / K m ratios were calculated. Human AK1 was purchased from Abcam.

Cell culture and transfection

HeLa cells were seeded in µ-Slides (Ibidi) and cultured in Dulbecco's Modified Eagle's Medium with 10% (v/v) fetal calf serum (Gibco BRL, Carlsbad, CA, USA), 100 U/ml penicillin and 0.1 mg / ml streptomycin. Cells were grown at 37 °C in a humidified incubator with 5 % CO 2 . Plasmids were transfected into cells using FuGENE HD transfection reagent (Roche) as described in the manufacturer's protocol. The cells were stained 30 min with 100 nM MitoTracker Red (Molecular Probes) 48 h after transfection. Cell fluorescence was imaged using a Leica DMI6000B with Leica Application Suite AF software v1.8.

RESULTS

Expression of AK7 and the different forms of AK8

The cDNA predicted to encode AK7 is longer than previously identified sequences of single-domain AKs (figure 1A). The AK7 encoding cDNA contained an N-terminal 368 amino acid sequence with no clear homology to any known sequence. The full length AK7 cDNA was expressed and shown to produce a 656 amino acid protein with a molecular weight of ∼ 74 kDa. According to the databases, the gene product was expected to be 723 amino acid residues but the presence of a stop codon at the end part of the gene product cloned in this study suggests that there may exist more than one transcript variants of AK7. The gene is localized on chromosome 14 and consists of 18 exons with a total length of 96.67 kb. The AK domain of AK7 showed high conservation with the other single domain AKs, AK1-4 and AK6 (figure 1B).

The cDNA of AK8 was identified in the same genomic library in the open reading frame 98 of chromosome 9 and its expression produced a 479 amino acid protein. Since the AK8 polypeptide contains two p-loops, we expressed its possible two domains by inserting: i) a stop codon before the second p-loop to translate only the first domain (268 amino acids) and ii) a start codon before the second p-loop to translate only the second domain (216 amino acids) (figure 2A). Our previous studies of other AKs suggest that a short N-terminal amino acid fragment before the p-loop structure is crucial for the correct folding and activity of the enzyme. A start codon was therefore introduced in a way that an N-terminal fragment, consisting of amino acid residues MAPFTP, is present before the p-loop of AK8p2. The twodomain AK protein sequence of AK8 is similar to the previously characterized two-domain sequence of AK5 (figure 2B). The full length AK8 and its two domains were synthesized with a cell-free in vitro translation system in order to verify their expression and their sizes that were ∼53, 33 and 27 kDa for full length AK8, AK8p1 and AK8p2 respectively (figure 3). A phylogenetic tree analysis suggests that AK7 and AK8 are more distantly related to AK1 than the previously characterized AKs (figure 4).

Substrate specificity of AK7 and the different forms of AK8

An initial screening of phosphotransferase activity was performed using TLC. AMP, CMP, GMP, UMP, dAMP, dCMP, dGMP, dTMP and dUMP were tested as phosphate acceptors at 1 mM concentrations with 1 mM of the phosphate donors ATP, CTP, GTP, TTP or UTP. Similar substrate specificity was observed for all of the investigated enzymes. With ATP as phosphate donor AK7 (data not shown), AK8, AK8p1 and AK8p2 phosphorylated AMP, CMP, dAMP and dCMP and in addition to that they phosphorylated AMP, CMP and dCMP when GTP was used as phosphate donor (figure 5). There was no phosphorylation of substrates using CTP, TTP or UTP as phosphate donors. The phosphorylation reactions were confirmed by using the in vitro translated enzymes to exclude the possibility of bacterial contamination of the recombinant enzyme preparations (data not shown).

Kinetic properties of AK7 and AK8

The substrates and phosphate donors identified to be most efficiently utilized by AK7 and AK8 in the initial TLC screening were used to determine the kinetic properties of the enzymes. AK7 showed the highest affinity for AMP with ATP as phosphate donor with a K m of 1 µM and a V max of 1130 pmol/µg/min (table 1). The affinity for dAMP and CMP was also high with a K m of 28 µM and a V max of 860 pmol/µg/min for dAMP and a K m of 1.2 µM and a V max of 150 pmol/µg/min for CMP. The affinity of dCMP for AK7 with ATP as phosphate donor was too low for kinetic determinations. The best substrate for AK8 was AMP when ATP was used as phosphate donor, followed by CMP (table 2). AK8 was less efficient in dAMP phosphorylation, as compared to AK7, with a K m of 630 µM and a V max of 1360 pmol/µg/min. As judged by the TLC assays AMP was the preferred substrate for both AK7 and AK8 also when using GTP as phosphate donor but the affinity of the substrates were too low to obtain accurate values for kinetic determinations.

A comparison of the kinetic parameters of AMP as substrate for AK7 and AK8 with the more abundant AK1 showed that the K m of AK1 was in the millimolar range whereas the K m of AK7 and AK8 were in the micromolar range (table 3). Also the V max of AK1 was in a different range as compared to the V max of AK7 and AK8. The ratio V max / K m , that is used to define the overall efficiency of an enzyme, was higher for AK7 and AK8 as compared to AK1.

Subcellular localization of AK7 and AK8

The subcellular prediction softwares used did not indicate any targeting signal within the amino acid sequences of the investigated enzymes. Nevertheless, the highest probability, according to the softwares, was a cytosolic localization for both enzymes. This was confirmed by in vivo expression of AK7 and full length AK8 as fusion proteins with the Green Fluorescent Protein (GFP) demonstrating that both proteins had a cytosolic localization (figure 6).

DISCUSSION

With the data of the present study the human AK family of enzymes now has 8 members. These enzymes have high sequence similarity with distinguishable conserved AK domains and catalyze similar reversible phosphoryl transfer reactions between adenine nucleotides. In this study AK7 and AK8 were expressed and characterized. Both AK7 and AK8 were shown to have the highest affinity for AMP as substrate but both these enzymes also recognized dAMP, CMP and dCMP as substrates. The preferred substrate of all AKs is A c c e p t e d M a n u s c r i p t 5 AMP and their main phosphate donor is ATP, although some can phosphorylate several other substrates and use other NTPs as phosphate donors. AK1 and AK2 can utilize all NTPs as phosphate donors but they specifically recognize AMP as substrate and, as we show in the present study, AK1 also phosphorylates dAMP [START_REF] Wilson | Adenylate kinases in man: evidence for a third locus[END_REF]. AMP is also the main phosphate acceptor of AK3, but the phosphate donor specificity is restricted to GTP and ITP [START_REF] Noma | Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix[END_REF]. AK4 phosphorylates AMP, dAMP, CMP and dCMP with ATP and GTP as phosphate donors, a pattern of substrate and phosphate donor specificity that AK4 shares with AK5, AK7 and AK8 with some differences in kinetics [START_REF] Panayiotou | Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4[END_REF][START_REF] Van Rompay | Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein[END_REF]. AK6 can use all phosphate donors to phosphorylate AMP, dAMP, CMP and dCMP [START_REF] Ren | The crystal structure of human adenylate kinase 6: An adenylate kinase localized to the cell nucleus[END_REF]. Taken together, the common feature of the different AKs is to participate in AMP conversion and ATP homeostasis. Whether the additional substrate affinities have any physiological importance has not yet been shown.

There are significant differences in the phosphorylation efficiency among the AKs demonstrated both in the present and earlier studies. When the kinetic properties of AK7 and AK8 were determined, AK1 was included as a reference enzyme since AK1 is the major AK in human cells. Both AK7 and AK8 showed a higher affinity for AMP as substrate, as compared to AK1, but a much lower maximal catalytic rate. When both substrate affinity and maximum velocity of AMP phosphorylation were combined and expressed as V max / K m , both AK7 and AK8 showed higher efficiency in AMP phosphorylation as compared to AK1. This feature could compensate the low expression levels of these enzymes compared to the expression profile of AK1. The high AMP affinity of AK7 and AK8 may relate to specific functions in certain cell types, such as kinocilia-bearing cells where AK7 was initially identified [START_REF] Fernandez-Gonzalez | Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia phenotype[END_REF]. There may be a special demand on high local ATP production in kinocilia expressing cells as well as in other cells with similar high energy demanding functions. Of the previously characterized human AK4, AK5 and AK6, all of these enzymes have different kinetic parameters that may indicate that they contribute to adenosine nucleotide homeostasis in different microenvironments and have specific cellular functions [START_REF] Panayiotou | Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4[END_REF][START_REF] Van Rompay | Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein[END_REF][START_REF] Ren | The crystal structure of human adenylate kinase 6: An adenylate kinase localized to the cell nucleus[END_REF].

All previously reported AKs with a single AK domain (AKs 1-4 and AK6) have a size of around 200 amino acids whereas the presently studied AK7 is substantially larger. However, the AK domain of AK7 has approximately the size of the single-domain AKs. The first N-terminal 368 amino acid domain of AK7 does not contain any of the conserved AK sequences and other indications of its potential function were not suggested by blast searches of this N-terminal sequence. Whether this domain encodes a functional polypeptide or not, there is no evidence for any contribution to the AK activity of AK7. In a previous study human AK5 was identified to have two enzymatically active AK domains and we now demonstrate the existence of a second human AK with two complete AK domains. This enzyme was named AK8 because it was the eighth human AK isoenzyme identified. Although AK8 has a similar two-domain structure as found in AK5, AK8 is different from AK5 in that it apparently has only one transcript whereas the AK5 open reading frame has two transcripts, which differ in a 27 amino acid Tag sequence [START_REF] Solaroli | Identification of two active functional domains of human adenylate kinase 5[END_REF]. However, when the two AK domains of AK8 were expressed separately they both showed similar AK activity. The AK8 protein thus may harbor two functionally active domains in the same polypeptide chain, something that is supported by the activity data presented in our study. Whether different AK isoenzymes can enhance their combined activity by formation of functional complexes with multiple sequential catalytic domains is an important issue for future investigations. As of today only monomer forms of AKs are known, but future studies may reveal if oligomerisation is of physiological significance for regulating the activity of the AK isoenzymes. According to databases and the literature there are at least two AKs in lower species with more than one possible catalytic domain; a Drosophila melanogaster predicted AK that consists of 562 amino acids with two domains and a sea urchin sperm flagellar AK (920 amino acids) with triplicated domains [12]. With the picture getting completed regarding A c c e p t e d M a n u s c r i p t 6 the members of the human AK family, the question addressed was how many AKs exist in other organisms. Escherichia coli is known to encode for one single AK and interestingly when searching the databases the number of AK isoenzymes appeared to increase with more complex organisms. According to the databases Saccharomyces cerevisiae encodes 3 AKs, Caenorhabditis elegans encodes 4 AKs and Drosophila melanogaster encodes 6 different AK isoenzymes.

There are correlations found between AK expression and certain human disorders although conclusive confirmation of genetic AK alterations as the cause of the disease may still be lacking. In humans, AK1 deficiency caused by a nonsense homozygous mutation, has been associated with mild chronic haemolytic anaemia and psychomotor impairment [START_REF] Bianchi | A case of complete adenylate kinase deficiency due to a nonsense mutation in AK-1 gene (Arg 107 --> Stop, CGA --> TGA) associated with chronic haemolytic anaemia[END_REF]. Reticular dysgenesis, the most severe form of human inborn immunodeficiencies, is characterized by complete absence of granulocytes and lymphocytes and has been linked to mutations of the AK2 gene [14]. The mitochondrial localization of AK2 implies a central role in providing the energy required for the proliferation of hematopoietic precursors and in controlling cell apoptosis [15,[START_REF] Lee | AK2 activates a novel apoptotic pathway through formation of a complex with FADD and caspase-10[END_REF]. Increased AK4 protein levels have been detected in cultured cells exposed to hypoxia and in an animal model of amyotrophic lateral sclerosis, a neurodegenerative disease in which oxidative stress is implicated [START_REF] Liu | Enzymatically inactive adenylate kinase 4 interacts with mitochondrial ADP/ATP translocase[END_REF]. A recent study showed that pancreatic β-cells express AK1 and AK5 that play an important role in regulating K-ATP channel activity and thereby control insulin secretion [START_REF] Stanojevic | Cytosolic adenylate kinases regulate K-ATP channel activity in human beta-cells[END_REF]. Autoantibodies targeted against AK5 have been traced in two cases of non-viral immune-mediated limbic encephalitis [19]. The identification of the nuclear-localized AK6 gave evidence on nucleotide metabolism in the nucleus and the energy supply routes between the mitochondria and the nucleus [START_REF] Dzeja | Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer[END_REF]. The involvement of AK7 in human disease has been reported in recent studies where it was related to the expression of AK7 in tissues rich in epithelium with cilia [START_REF] Fernandez-Gonzalez | Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia phenotype[END_REF]. The AK8 gene (C9ORF98) was identified to be one of the genes that have a negative regulatory role in epithelial cell migration [START_REF] Simpson | Identification of genes that regulate epithelial cell migration using an siRNA screening approach[END_REF]. Both AK1 and AK7 have been deleted in mice models and found to have specific phenotypic alterations. In a study of AK1-deficient mice they were shown to have low AMP-activated protein kinase (AMPK) phosphorylation in skeletal muscle, consistent with limited AMP production [START_REF] Hancock | Contraction-mediated phosphorylation of AMPK is lower in skeletal muscle of adenylate kinase-deficient mice[END_REF]. In addition, deletion of the AK1 gene accelerated the loss of cardiac contraction on ischemic challenge [START_REF] Pucar | Adenylate kinase AK1 knockout heart: energetics and functional performance under ischemia-reperfusion[END_REF]. An AK7-deficient mouse model presented pathological signs of microtubular defects, decreased ciliary beat frequency, hydrocephalus, abnormal spermatogenesis, mucus accumulation and acute respiratory responses upon allergen challenge [START_REF] Fernandez-Gonzalez | Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia phenotype[END_REF]. In summary, the vast literature on AK associated alterations and disorders supports the importance of AK activity for many vital processes in humans and in other organisms.

The number of AK isoenzymes present in different subcellular localizations and with different tissue and cell expression illustrates the importance of maintaining a sufficient AK activity for proper cell and organism functions. All of the 8 human AKs that now have been characterized catalyze the reversible reaction ATP + AMP ↔ 2ADP and their contribution to energy transfer and metabolic signaling has been demonstrated to be central for cell viability and functions. The present study contributes to complete the picture of the family of human AK isoenzymes and should be an important contribution to clarify their role in human health and disease. Figure 4 The human adenylate kinase isoenzymes A phylogenetic tree of the human adenylate kinases shows that AK7 and AK8 are more distantly related to AK1 as compared to AK2-AK6.

Biochemical

  Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101443 A c c e p t e d M a n u s c r i p t 3 separated on poly (ethyleneimine)-cellulose F chromatography sheets (Merck); soaked in methanol prior to use. The nucleotides were separated in 0.5 M ammonium formate pH 3.5 for 2 h. TLC sheets were then autoradiographed by phosphorimaging plates (BAS 1000, Fuji Photo Film).

  Biochemical Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101443 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101443

Biochemical

  Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101443

Figure 1

 1 Figure 1 Human adenylate kinase 7 (A) Map of human AK7. (B) Alignment of human AK7 with the other one-domain human AKs (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 2 Human adenylate kinase 8 (A) Map of the two-domain human adenylate kinases, AK8 and AK5. (B) Alignment of AK8 and AK5 (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 3 SDS-PAGE of the in vitro synthesized proteinsLane 1; AK8p1, lane 2; AK8p2 and lane 3; full length AK8. Figure4The human adenylate kinase isoenzymes A phylogenetic tree of the human adenylate kinases shows that AK7 and AK8 are more distantly related to AK1 as compared to AK2-AK6. Figure 5 Substrate specificity of human adenylate kinase 8 and its two domains Screening of ribo-and deoxyribonucleoside monophosphate specificity of full length AK8, AK8p1 and AK8p2. 1mM of NMPs and dNMPs were used as substrates with 0.5 µg of enzyme. (A) Unlabeled ATP and [γ-32 P] ATP was used as phosphate donor. (B) Unlabeled GTP and [γ-32 P] GTP was used as phosphate donor. (C 1 : control without enzyme or substrate; C 2 : control without substrate). Figures shown here are representative of at least three repetitions of each experiment.Figure 6 Cytosolic localization of human adenylate kinases 7 and 8 Fluorescent microscopy of HeLa cells expressing GFP (control), fusion protein AK7 with GFP and fusion protein full length AK8 with GFP. The mitochondria were contra-stained with Mitotracker.

Figure 2

 2 Figure 1 Human adenylate kinase 7 (A) Map of human AK7. (B) Alignment of human AK7 with the other one-domain human AKs (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 2 Human adenylate kinase 8 (A) Map of the two-domain human adenylate kinases, AK8 and AK5. (B) Alignment of AK8 and AK5 (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 3 SDS-PAGE of the in vitro synthesized proteinsLane 1; AK8p1, lane 2; AK8p2 and lane 3; full length AK8. Figure4The human adenylate kinase isoenzymes A phylogenetic tree of the human adenylate kinases shows that AK7 and AK8 are more distantly related to AK1 as compared to AK2-AK6. Figure 5 Substrate specificity of human adenylate kinase 8 and its two domains Screening of ribo-and deoxyribonucleoside monophosphate specificity of full length AK8, AK8p1 and AK8p2. 1mM of NMPs and dNMPs were used as substrates with 0.5 µg of enzyme. (A) Unlabeled ATP and [γ-32 P] ATP was used as phosphate donor. (B) Unlabeled GTP and [γ-32 P] GTP was used as phosphate donor. (C 1 : control without enzyme or substrate; C 2 : control without substrate). Figures shown here are representative of at least three repetitions of each experiment.Figure 6 Cytosolic localization of human adenylate kinases 7 and 8 Fluorescent microscopy of HeLa cells expressing GFP (control), fusion protein AK7 with GFP and fusion protein full length AK8 with GFP. The mitochondria were contra-stained with Mitotracker.

Figure 3

 3 Figure 1 Human adenylate kinase 7 (A) Map of human AK7. (B) Alignment of human AK7 with the other one-domain human AKs (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 2 Human adenylate kinase 8 (A) Map of the two-domain human adenylate kinases, AK8 and AK5. (B) Alignment of AK8 and AK5 (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 3 SDS-PAGE of the in vitro synthesized proteinsLane 1; AK8p1, lane 2; AK8p2 and lane 3; full length AK8. Figure4The human adenylate kinase isoenzymes A phylogenetic tree of the human adenylate kinases shows that AK7 and AK8 are more distantly related to AK1 as compared to AK2-AK6. Figure 5 Substrate specificity of human adenylate kinase 8 and its two domains Screening of ribo-and deoxyribonucleoside monophosphate specificity of full length AK8, AK8p1 and AK8p2. 1mM of NMPs and dNMPs were used as substrates with 0.5 µg of enzyme. (A) Unlabeled ATP and [γ-32 P] ATP was used as phosphate donor. (B) Unlabeled GTP and [γ-32 P] GTP was used as phosphate donor. (C 1 : control without enzyme or substrate; C 2 : control without substrate). Figures shown here are representative of at least three repetitions of each experiment.Figure 6 Cytosolic localization of human adenylate kinases 7 and 8 Fluorescent microscopy of HeLa cells expressing GFP (control), fusion protein AK7 with GFP and fusion protein full length AK8 with GFP. The mitochondria were contra-stained with Mitotracker.

Figure 5

 5 Figure 1 Human adenylate kinase 7 (A) Map of human AK7. (B) Alignment of human AK7 with the other one-domain human AKs (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 2 Human adenylate kinase 8 (A) Map of the two-domain human adenylate kinases, AK8 and AK5. (B) Alignment of AK8 and AK5 (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 3 SDS-PAGE of the in vitro synthesized proteinsLane 1; AK8p1, lane 2; AK8p2 and lane 3; full length AK8. Figure4The human adenylate kinase isoenzymes A phylogenetic tree of the human adenylate kinases shows that AK7 and AK8 are more distantly related to AK1 as compared to AK2-AK6. Figure 5 Substrate specificity of human adenylate kinase 8 and its two domains Screening of ribo-and deoxyribonucleoside monophosphate specificity of full length AK8, AK8p1 and AK8p2. 1mM of NMPs and dNMPs were used as substrates with 0.5 µg of enzyme. (A) Unlabeled ATP and [γ-32 P] ATP was used as phosphate donor. (B) Unlabeled GTP and [γ-32 P] GTP was used as phosphate donor. (C 1 : control without enzyme or substrate; C 2 : control without substrate). Figures shown here are representative of at least three repetitions of each experiment.Figure 6 Cytosolic localization of human adenylate kinases 7 and 8 Fluorescent microscopy of HeLa cells expressing GFP (control), fusion protein AK7 with GFP and fusion protein full length AK8 with GFP. The mitochondria were contra-stained with Mitotracker.

Figure 6 Cytosolic localization of human adenylate kinases 7 and 8

 68 Figure 1 Human adenylate kinase 7 (A) Map of human AK7. (B) Alignment of human AK7 with the other one-domain human AKs (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 2 Human adenylate kinase 8 (A) Map of the two-domain human adenylate kinases, AK8 and AK5. (B) Alignment of AK8 and AK5 (black boxes indicate completely conserved amino acid residues and different shades of grey indicate different levels of conserved residues).Figure 3 SDS-PAGE of the in vitro synthesized proteinsLane 1; AK8p1, lane 2; AK8p2 and lane 3; full length AK8. Figure4The human adenylate kinase isoenzymes A phylogenetic tree of the human adenylate kinases shows that AK7 and AK8 are more distantly related to AK1 as compared to AK2-AK6. Figure 5 Substrate specificity of human adenylate kinase 8 and its two domains Screening of ribo-and deoxyribonucleoside monophosphate specificity of full length AK8, AK8p1 and AK8p2. 1mM of NMPs and dNMPs were used as substrates with 0.5 µg of enzyme. (A) Unlabeled ATP and [γ-32 P] ATP was used as phosphate donor. (B) Unlabeled GTP and [γ-32 P] GTP was used as phosphate donor. (C 1 : control without enzyme or substrate; C 2 : control without substrate). Figures shown here are representative of at least three repetitions of each experiment.Figure 6 Cytosolic localization of human adenylate kinases 7 and 8 Fluorescent microscopy of HeLa cells expressing GFP (control), fusion protein AK7 with GFP and fusion protein full length AK8 with GFP. The mitochondria were contra-stained with Mitotracker.

Figure 1 Biochemical

 1 Figure 1

Figure 2 Biochemical

 2 Figure 2

Figure 3 Biochemical

 3 Figure 3

Figure 4 Biochemical

 4 Figure 4

Figure 5 Biochemical

 5 Figure 5

Figure 6 Biochemical

 6 Figure 6

Table 1 .

 1 HPLC-based kinetic properties of the recombinant human adenylate kinase 7 with ATP as phosphate donor. Values are presented as mean ± standard deviation from three independent experiments. NM, not measurable.

	Biochemical Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443
		K m (µM)	V max (pmol/µg/min)	V max / K m
	Substrate
	AMP	1 ± 0.2	1130 ± 17	1130
	dAMP	28 ± 12	860 ± 19	31
	CMP	1.2 ± 0.8	150 ± 59	125
	dCMP	NM	NM	-

Table 2 .

 2 HPLC-based kinetic properties of the recombinant human adenylate kinase 8 with ATP as phosphate donor. Values are presented as mean ± standard deviation from three independent experiments. NM, not measurable.

	Biochemical Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443
		K m (µM)	V max (pmol/µg/min)	V max / K m
	Substrate
	AMP	4.1 ± 0.6	2400 ± 460	585
	dAMP	630 ± 36	1360 ± 71	2.2
	CMP	1.4 ± 0.9	190 ± 79	136
	dCMP	NM	NM	-

Table 3 .

 3 HPLC-based kinetic properties of the recombinant human adenylate kinase 1 with ATP as phosphate donor. Values are presented as mean ± standard deviation from three independent experiments. NM, not measurable.

	Biochemical Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443
		K m (µM)	V max (pmol/µg/min)	V max / K m
	Substrate
	AMP	2400 ± 840	156000 ± 12000	65
	dAMP	18100 ± 1500	265000 ± 11000	14.6

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

FUNDING

This work was supported by grants from the Swedish Cancer Society, the Swedish Research Council and the Karolinska Institute. Biochemical Journal Immediate Publication. Published on 16 Nov 2010 as manuscript BJ20101443 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101443


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