Accepted Manuscript Cloning, characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii Sonia del Prete, Daniela Vullo, Andrea Scozzafava, Clemente Capasso, Claudiu T. Supuran PII: DOI: Reference:

S0968-0896(13)00919-X http://dx.doi.org/10.1016/j.bmc.2013.10.045 BMC 11196

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

17 September 2013 18 October 2013 29 October 2013

Please cite this article as: del Prete, S., Vullo, D., Scozzafava, A., Capasso, C., Supuran, C.T., Cloning, characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc. 2013.10.045

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Cloning, characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii Sonia del Prete,a Daniela Vullo,b Andrea Scozzafava,b Clemente Capasso*a and Claudiu T. Supuran*b,c a

Istituto di Biochimica delle Proteine – CNR, Via P. Castellino 111, 80131, Napoli, Italy.

b

Università degli Studi di Firenze, Dipartmento di Chimica Ugo Schiff, Via della Lastruccia 3, Rm.

188, 50019 - Sesto Fiorentino (Florence), Italy. c

Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutriceutical

Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy. Abstract. We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO2 hydration, its physiological reaction, with a kcat of 1.3 x 105 s-1 and a kcat/KM of 3.3 x 107 M-1 s-1. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (KIs > 200 mM) whereas other halides and pseudohalides were submillimolar – millimolar inhibitors (KIs in the range of 0.93 – 8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KIs in the range of 9 – 90 µM, whereas acetazolamide inhibited the enzyme with a KI of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO2 fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO2 capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms. _____ * Corresponding authors: Tel: +39-081-6132559, E-mail: [email protected] (CC): Tel +39-0554573005; Fax: +39-055-4573385; E-mail [email protected] (CTS).

1. Introduction Among the five genetically distinct classes of carbonic anhydrases (CAs, EC 4.2.1.1.) - the α-, β-, γ-, δ- and ζ-CAs1-6 - the δ-family is the least investigated one. Indeed, not only there is no Xray crystallographic structure for enzymes belonging to this family, but also the kinetic or inhibition data are virtually inexistent. Only very recently, a study7 reported the cloning and semi-quantitative kinetic data (i.e., specific activity of the enzyme for the physiologic, CO2 hydration reaction) from the marine diatom Thalassiosira weissflogii, comparing it to that of the bovine CA (bCA), an enzyme belonging to the α-CA class. This first study7 reported that the enzyme, denominated TWCA1, shows a rather low specific activity for the hydration of CO2 at pH 8.3, about 25 % that of bCA, but no kinetic parameters were measured, neither inhibition experiments have been reported as a control that the observed activity is indeed due to an enzymatic conversion of CO2 to bicarbonate. Indeed, on the SDS-PAGE of the reported protein two bands were observed, at 34 and 70 kDa, respectively, 7 whereas δ-CAs should be monomeric and not dimeric enzymes.8 Moreover, the diatom Thalassiosira weissflogii was very important for the research of novel CAs, as both the δ-8 and the ζ-class2 enzymes were discovered in this organism by Morel’s group in 1997 and 2008, respectively.2,8 Whereas the ζ-CA from T. weissflogii (also denominated CDCA1) is a well characterized enzyme (the X-ray crystal structure of the Zn(II) as well as Cd(II)-containing fragments - R1-R3 - of this pseudotrimeric enzyme were reported by Morel’s2 and our9 groups), the δ-CA was difficult to be cloned and investigated in detail up until now. However, Morel’s group showed by means of X-ray absorption spectroscopy at the Zn K-edge that the active site of TWCA1 is strikingly similar to that of mammalian enzymes belonging to the α-class. Indeed, the catalytically essential Zn(II) ion from the δ-CA seems to be coordinated by three His residues and a single water molecule, at a distance of 2 Å from the metal ion.10 However, more detailed information on this enzyme is not available, although the δ-CAs seem to be quite widespread in the marine phytoplancton, including haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes.11-13 CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate.2,8,9 Most of the phytoplankton operates a carbon concentrating mechanism (CCM) to increase the CO2 concentration at the site of fixation by D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), several folds over its external concentration, allowing the enzyme to function efficiently.2 Marine diatoms possess both external and internal CAs.14

It has been hypothesized in the model diatom T. weissflogii that the external CA catalyzes the dehydration of HCO3- to CO2 to increase the gradient of the CO2 diffusion from the external medium to the cytoplasm, and the internal CA in the cytoplasm catalyzes the rehydration of CO2 to HCO3- to prevent the leakage of CO2 to the external medium again.15 However one of the most remarkable findings regarding the ζ-CA from T. weissflogii was that this is a Cd(II)-containing enzyme, which can however also work with Zn(II) bound at the active site, and that there is a rather rapid metal exchange between zinc and cadmium, depending on the availability of metal ions in the marine environment.2 It is not yet known where this enzyme is localized in T. weissflogii or other diatoms,2 whereas for the δ-CA from this organism, Lee et al. hypothesized an extracellular localization of the enzyme.7 CCM is not limited only to marine phytoplankton performing photosynthesis, but is widespread to many other photosynthetic organisms. Its purpose is to support effective CO2 fixation in most plants and cyanobacteria, by enhancing favorable chemical conditions in the vicinity of the primary CO2-fixing enzyme RubisCO, but also promoting the carboxylase reaction and suppressing the oxygenase activity.16 In cyanobacteria and some proteobacteria, this is achieved by encapsulation of RubisCO within carboxysomes, which are examples of a group of proteinaceous bodies called bacterial microcompartments, in which various classes of CAs play a fundamental role.16 Considering our interest in the CAs and the fact that the literature on the δ-CAs is scarce and sometimes contradictory, we report here the cloning of the T. weissflogii enzyme as well as its complete biochemical characterization. The first kinetic data for a δ-class enzyme as well as its inhibition profile with anions are reported in this paper. As there are no more than one isoform of the δ-class enzyme in this organism, we denominated it TweCA, instead of TWCA1 as called earlier by other investigators.7,8

2. Results and Discussion

2.1. Cloning, characterization and kinetic properties of TweCA We have cloned the full length TweCA from the synthetic gene encoding it (see Experimental for details). The enzyme contains 281 amino acid residues and has a molecular weight of 32.3 kDa (see Supplementary Fig. 1 for the SDS PAGE of the enzyme). The enzyme was purified to homogeneity by means of affinity chromatography and used thereafter to measure the kinetic properties at pH 7.5, by a stopped flow method monitoring the physiologic reaction, i.e., CO2 hydration to bicarbonate and protons.17

Table 1 here As shown from data of Table 1, in which other CAs belonging to all the genetic families were included, TweCA has efficient catalytic activity, with a kcat/Km of 3.2 × 107 M-1 s-1 and a kcat of 1.3 × 105 s-1. We stress here again that this is the first time that a δ-CA is characterized kinetically. Furthermore, this catalytic activity has been inhibited by acetazolamide (5-acetamido-1,3,4thiadiazole-2-sulfonamide) with a KI of 83 nM. This sulfonamide is an efficient inhibitor of all five classes of CAs known to date (see Table 1, where three α-CAs, two β-CAs, one γ- and two ζ-class enzymes ere included, for comparison reasons, together with the newly characterized δ-CA). Indeed, the human cytosolic isozymes hCA I and II and the bacterial SazCA (from Sulfurihydrogenibium azorense)5d,6 were the α-class CAs considered here, together with the β-class fungal enzyme Can2 from Cryptococcus neoformans

4b

and FbiCA1 from the plant Flaveria

bidentis.18 The γ-class enzyme was PgiCA from the anaerobic bacterium Porphyromonas gingivalis,19 whereas the ζ-class enzymes,

2,9

with either zinc or cadmium at the active site, were

from the same diatom (T. weissflogii) from which the δ-CA originates. It may be observed that acetazolamide is a weaker inhibitor of hCA I and PgiCA (KIs of 250-324 nM) but it strongly inhibits the remaining CAs with inhibition constants in the range of 0.9 – 83 nM (Table 1). Comparing the kcat/Km and kcat values of the enzymes from Table 1, it may be observed that TweCa has a medium catalytic efficiency, comparable to that of hCA I and Can2, enzymes known to play important physiological functions in humans and fungi, respectively.1-4 We should also stress that the measured catalytic activity in this work seems to be much higher compared to that reported in ref.7 for a TweCA preparation obtained by cloning the enzyme from genomic DNA isolated from the diatom itself. The differences may be accounted on the degree of purity of the two enzymes, as the one reported earlier seems to contain high amounts of a dimer.7 In addition, it seems to also contain an amino terminal fragment of 17 amino acid residues coming from the primer used for the cloning,7 which in fact are not present in the sequence of TweCA. Our enzyme has only the amino acid residues encoded by the Thalossosiria weissflogii δ-CA (GenBank accession number AAV39532.1).

2.2. Active site coordination of the metal ion, sequence homology and phylogenetic analysis of the δ-CAs All five genetic families encoding CAs are metalloenzymes and the apo-enzymes are totally devoid of any catalytic activity.1-6 Previous work from Morel’s group,10 showed by means of an elegant study that the δ-CA also has Zn(II) at its active site and that the metal ion coordination in the α- and

δ-CAs is similar, i.e., three His residues and a water molecule/hydroxide ion coordinate the catalytic metal ion. Later it became apparent that the same coordination pattern is present in the γ-class enzymes which also work with Co(II) or Fe(II) at the active site, instead of Zn(II) (Fig. 1A).3a-c This is quite different from the β- and ζ-CAs, in which coordination of the metal ion is achieved by two Cys and one His residues, whereas the fourth ligand is again a water molecule/hydroxide ion (Fig. 1B). In the ζ-CAs the Zn(II) is frequently replaced by Cd(II) and the enzymes possess high catalytic activity with both metal ions.1,2,9 Figs 1-4 here As there is no X-ray crystal structure of any δ-CAs at this moment, and in order to rationalize the kinetic data of Table 1, we performed a multi-alignment of the amino acid sequences from different plankton species reported to encode putative CAs (we stress again that no catalytic or other data are reported for these putative enzymes). Thus, the enzyme characterized here, TweCA_delta from T. weissflogii, (accession number: AAV39532.1); another one, TpsCA_delta from the related species Thalassiosira pseudonana (accession number: XP_002287620.1), together with LpoCA_delta, from Lingulodinium polyedrum (accession number: ABS87870.1) and Bpr_delta, from Bathycoccus prasinos (accession number: CCO20234.1) have been used in order to identify patterns of homology relevant for explaining the catalytic activity (Fig 2). These data showed that the putative Zn(II) ligands (assigned in the work of Cox et al.10 mentioned above) are conserved in all these sequences, being numbered as His114, His117 and His226. Furthermore, the quite efficient CO2 hydration activity of TweCA implies that it must possess one or more proton shuttling residues within its active site, which transfer the proton from the water coordinated to the metal ion to the buffer, assuring the formation of the nucleophilic, zinc hydroxide species of the enzyme.1-6 As seen from Fig. 2, at least four His residues at the amino terminal part, i.e., His 15, 17, 22 and 101 are conserved in all four putative δ-CAs considered here, and we propose that one or more of them may act as proton shuttling residues in the δ-CA family (Fig. 2). Considering that the α-, γ- and δ-CAs possess the same Zn(II) coordination pattern, we also performed a multi-alignment of the amino acid sequences of several such enzymes (Fig. 3). TweCA was

aligned

with

PgiCA_gamma

from

Porphyromonas

gingivalis

(accession

number

YP_004510261), an enzyme recently characterized by our groups,19 as well as with hCA I, Homo sapiens

(human

CA,

isoform

I,

accession

number:

NP_001729.1)

and

SspCA,

Sulfurohydrogenibium yellowstonense YO3AOP1 enzymes (accession number: ACD66216.1) belonging to the α-class.5d The numbering of all these enzymes is the original one reported for each class: for the δ-CAs it is the one used by Lee et al.,7 with the putative zinc ligands indicated in red. For the γ-CAs we used the CAM numbering system3 (the zinc ligands are shown again in red). For

α-CAs the human isoform CA I numbering system has been used (metal ion ligands were indicated in red; the gatekeeper residues were indicated in blue; and the proton shuttle residue His64 was indicated in pink). This multi-alignment allowed us to observe that in the α-class enzymes the zinccoordinating ligands are at positions x, x + 2 and x + 25, respectively (Fig. 3). For the γ-CAs these positions were x, x + 36 and x + 41, respectively, whereas for the δ-CAs, the putative zinc ligands are positioned as follows: x, x + 3 and x + 112, respectively. It is obvious that the α- and δ-CAs seem to be more similar to each other compared to the γ-class enzymes. First, the α- and δ-CAs are monomers whereas the γ-CAs are trimers.3,7,10 Then, in the first two types of enzymes the first two zinc ligands are closer to each other (at + 2 for the α-class, and +3, for the δ-CAs, with respect to the first residue coordinating the metal ion). The main difference between the α- and δ-CAs is the fact that the third zinc ligand is quite far away from the other two ones in the δ-CAs, at + 112 amino acid residues distance, whereas in the α-CAs it is much closer, at +25 amino acid residues distance from the first ligand. In the γ-CAs this pattern is more different with respect to the α- and δ-CAs, and it should be also noted that two residues come from one monomer whereas the third one from another monomer in the case of these trimeric enzymes.3,19 In fact, this conclusion was very much reinforced by looking at the phylogenetic tree of Fig. 4, in which enzymes belonging to the five different genetic families and from a rather high number of organisms all over the tree of life were considered (Fig. 4 and Table 2). The phylogenetic tree from Fig 4 clearly shows that the α-, γ- and δ-CAs cluster on one of the two main branches of the tree, whereas the β- and the ζ-CAs cluster on the second main branch (the lower one in Fig 4). Considering the first branch, it is also obvious that the α- and δ-CAs cluster again together, being thus evolutionarily more related, whereas the γ-CAs constitute their own different clade. As far as we know, this is the first time that a phylogenetic tree with all five classes of CAs is being built and discussed. These data clearly demonstrate in a very interesting manner the convergent evolution which led to the five genetic classes of CAs.

2.3. Inhibition studies of TweCA Anions and other small molecules, such as sulfamide, phenylboronic acid or phenylarsonic acid were highly investigated as CA inhibitors (CAIs) as they bind to the metal ion from the enzyme active site and impair the catalysis.1,20,21 Enzymes belonging to the α-, β-, γ- and ζ-CAs have been investigated up until now for their inhibition profiles with this class of small molecule,

anion inhibitor.1-3,9,18-21 Here we investigated for the first time the inhibition profile of a δ-CA with anions and similar small molecules (Table 3). Data of Table 3 show the inhibition of TweCA with a range of inorganic anions, some of which are abundant in sea water (chloride, sulfate, bromide, iodide, etc). Other investigated anions belong to the so-called metal poisons (cyanide, thiocyanate, azide, hydrogen sulfide) whereas others (sulfamide, sulfamic acid, phenyl boronic/arsonic acids) are known to inhibit CAs belonging to other classes, such as the α-, β-, γ- and ζ-CAs. The metal-binding function present in these compounds, the SO2NH- fragment, is in fact present in the main class of CAIs, the sulfonamides and their bioisosteres (sulfamides, sulfamates).19-21 The following should be noted regarding TweCA inhibition data of Table 3 (the inhibition data of the α-class human isoforms hCA I and II, as well as the Cd(II) and Zn(II) containing ζ-CAs – the R1 fragment – are also included in Table 3 for comparison reasons, as they were reported earlier by our groups,2,9,20 working in the same conditions as those used to screen the TweCA inhibitors): (i) A first group of anions, including chloride, sulfate, tetrafluoroborate and perchlorate, showed ineffective inhibition of TweCA, with no inhibitory activity observed up to concentrations as high as 200 mM (Table 3). This is not so much unexpected for tetrafluoroborate and perchlorate, as these anions show lower affinity for metal ions in solutions or metalloenzyme (see also the data for hCA I, II and the ζ-CAs from Table 3), but it is rather unexpected for chloride or sulfate. Indeed, for example the two ζ-CAs from the same organism showed submillimolar affinity for these anions, whereas the human isoforms are inhibited with KIs in the range of 6-200 mM by chloride, and much less by sulfate. These observations thus support the hypothesis

7,8

that TweCA is an extracellular

enzyme, which may be in contact with relatively high concentrations of these two anions (chloride and sulfate) from the sea water and in this way an evolutionary adaptation led to this insensitivity to inhibition by them. (ii) Other anions with low inhibitory activity towards TweCA were selenate, tellurate, tetraborate, perrhenate, peroxydisulfate, selenocyanide, iminodisulfonate and fluorosulfonate, which showed KIs in the range of 37.9 – 77.1 mM (Table 3). (iii) Anions showing millimolar affinity for TweCA were: the halides (except chloride discussed above), cyanide, azide, carbonate, nitrite, hydrogensulfite, and pyrophosphate, which showed KIs in the range of 1.9 – 8.8 mM. It may be observed that these are rather heterogeneous anions and no regularities for the inhibitory power were evidenced (as for example for the halide inhibition of hCA I and II, case in which the inhibitory power increased with the increase of the atomic weight of the halogen).

(iv) The following anions showed submillimolar affinity for TweCA (KIs in the range of 0.20 – 0.97 mM): cyanate, thiocyanate, bicarbonate, nitrate, stannate, divanadate, perruthenate, trithiocarbonate and diethyldithiocarbamate. Some of these anions are known for their high affinity for metal ions and potent complexing behaviour (e.g., cyanate, thiocyanate, trithiocarbonate and diethyldithiocarbamate) whereas other ones do not have at all this type of properties (e.g., stannate, divanadate, perruthenate). However, probably they share a common inhibition mechanism, i.e., by interacting with the metal core of the enzyme active site. (v) The most potent TweCa inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, which had KIs in the range of 9 – 90 µM. Together with acetazolamide (Table 1), which inhibited the enzyme with a KI of 83 nM, these were the most potent δ-CA inhibitors detected so far. HS- is known to have high affinity for metal ions and to act as potent CAI for enzymes belonging to all other CA classes, so this is now confirmed also for the δ-CAs (see also Table 3 for the affinity of this anion towards hCA I and II, or the Zn-R1 fragment of ζ-CA). Sulfamide and sulfamate incorporate the SO2NH- zinc binding group found in the main class of CAIs with clinical applications, i.e., the sulfonamides.22 This study thus confirms that the δ-CAs are also inhibited by this class of pharmacological agents (see the acetazolamide data of Table 1) and similar compounds. In addition, phenylboronic acid, with a KI of 9 µM, was the most potent nonsulfonamide CAI detected so far for the δ-CA TweCA (Table 3).

3. Conclusions In conclusion, we report here the biochemical characterization of the first δ-CA, TweCA, cloned from the genome of the diatom Thalassiosira weissflogii. The enzyme was an efficient catalysts for the CO2 hydration, physiological reaction, with a kcat of 1.3 x 105 s-1 and a kcat/KM of 3.3 x 107 M-1 s-1, similar to that of the α-class, physiologically relevant human isoform hCA I. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (KIs > 200 mM) whereas other halides and pseudohalides were submillimolar – millimolar inhibitors (KIs in the range of 0.93 – 8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KIs in the range of 9 – 90 µM, whereas acetazolamide inhibited the enzyme with a KI of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. These enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO2 fixation by sea organisms. A phylogenetic analysis in which all five genetic families of CAs also showed the α- and δ-CAs to be evolutionarily more related to

each other with respect to the γ-CAs, although these three families cluster all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO2 capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms.

4. Experimental

4.1. Construct preparation, protein expression and purification The synthetic gene encoding for the δ-CA from T. weissflogii and containing NdeI and XhoI restriction sites at the 5’ and the 3’ ends, has been ligated into the expression vector pET15-b (Novagen) to form the construct pET15-b/TweCA capable to produce the diatom δ-CA. Briefly, competent E. coli BL21 (DE3) cells were transformed with pET15-b/TweCA, grown at 37 °C and induced with 1 mM IPTG, as reported earlier for α-, β- and γ-CAs. 5d,6,18,19 After 30 min, ZnSO4 was added at the concentration of 0.5 mM. Cells were grown for an additional 4 hours, harvested and disrupted by sonication at 4 °C. The supernatant was loaded onto a HIS-Select HF Nickel Affinity Gel (Sigma–Aldrich, Milan, Italy) and the protein was eluted with 250 mM imidazole, as reported earlier for CAs belonging to the α-, β- and γ-CA class discovered by us.5d,6,18,19 At this stage of purification the enzyme was at least 95 % pure (Fig. S1).

4.2. CA activity and inhibition measurements An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 – 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 for maintaining constant the ionic strength, following the initial rates of the CAcatalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 µM were done thereafter with distilled-deionized water. Inhibitor and

enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver-Burk plots, as reported earlier,20,21 and represent the mean from at least three different determinations. All inhibitors (sodium salts of the anions from Table 3 and the several small molecules) were commercially available, highest purity compounds from Sigma-Aldrich (Milan, Italy) and were used as thus.

Acknowledgments. Research from our laboratories was financed by two FP7 EU projects (Metoxia and Dynano).

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Enzyme Inhib. Med. Chem. 2012, 27, 759; c) Supuran, C.T. Bioorg. Med. Chem. 2013, 21, 1377; d) Bootorabi, F.; Jänis, J.; Hytönen, V.P.; Valjakka, J.; Kuuslahti, M.; Vullo, D.;Niemelä, O.; Supuran, C.T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2011, 26, 862; d) Vullo, D.; De Luca, V.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C.T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 6324; e) Vullo, D.; De Luca, V.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C.T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 7142. 6. a) Supuran, C.T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; b) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C.T.; De Simone, G. Chem. Rev. 2012, 112, 4421; c) Neri, D.; Supuran, C.T. Nat. Rev. Drug Discov. 2011, 10, 767; d) Del Prete, S.; Isik, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C.T.; Capasso, C. J. Med. Chem. 2012, 55, 10742; e) De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C.T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 5630; f) Wilkinson, B.L.; Bornaghi, L.F.; Houston, T.A.; Innocenti, A.; Supuran, C.T.; Poulsen, S.A. J. Med. Chem. 2006, 49, 6539.

7. Lee, R.B.Y.; Smith, J.A.C.; Rickaby, R.E.M. J. Phycol. 2013, 49, 170. 8. Roberts, S.B.; Lane, T.W.; Morel, F.M.M. J. Phycol. 1997, 33, 845. 9. a) Alterio, V.; Langella, E.; Viparelli, F.; Vullo, D.; Ascione, G.; Dathan, N.A.; Morel, F.M.; Supuran, C.T.; De Simone, G.; Monti, S.M. Biochimie 2012, 94, 1232; b) Viparelli, F.; Monti, S.M.; De Simone, G.; Innocenti, A.; Scozzafava, A.; Xu, Y.; Morel, F.M.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2010, 20, 4745. 10. Cox, E.H.; McLendon, G.L.; Morel, F.M.; Lane, T.W.; Prince, R.C.; Pickering, I.J.; George, G.N. Biochemistry 2000, 39, 12128. 11. Lapointe, M.; Mackenzie, T.D.; Morse, D. Plant Physiol. 2008, 147, 1427. 12. McGinn, P.J.; Morel, F.M. Physiol. Plant. 2008, 133, 78. 13. Soto, A.R.; Zheng, H.; Shoemaker, D.; Rodriguez, J.; Read, B.A.; Wahlund, T.M. Appl. Environm. Microbiol. 2006, 72, 5500. 14. Burkhardt, S.; Amoroso, G.; Riebesell, U.; Sultemeyer, D. Limnol. Oceanogr. 2001, 46, 1378. 15. Morel, F.M.M.; Cox, E.H.; Kraepiel, A.M.L.; Lane, T.W.; Milligan, A.J.; Schaperdoth, I.; Reinfelder, J. R.; Tortell, P.D. Funct. Plant Biol. 2002, 29, 301. 16. Rae, B.D.; Long, B.M.; Badger, M.R.; Price, G.D. Microbiol. Mol. Biol. Rev. 2013, 77, 357. 17. Khalifah, R.G. J. Biol. Chem. 1971, 246, 2561. 18. Monti, S.M.; De Simone, G.; Dathan, N.A.; Ludwig, M.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2013, 23, 1626. 19. Del Prete, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C.T.; Capasso, Bioorg. Med. Chem. Lett. 2013, 23, 4067. 20. Innocenti, A.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2009, 19, 1855 21. a) Innocenti, A.; Zimmerman, S.; Ferry, J.G.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2004, 14, 3327; b) Innocenti, A.; Lehtonen, J.M.; Parkkila, S.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2004, 14, 5435; c) Nishimori, I.; Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2007, 17, 1037; d) Innocenti, A.; Mühlschlegel, F.A.; Hall, R.A.; Steegborn, C.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett 2008, 18, 5066; e) Innocenti, A.; Hilvo, M.; Parkkila, S.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2009, 19, 1155. 22. a) Capasso C, Supuran C.T. Expert Opin. Ther. Pat. 2013, 23, 693; b) Carta, F.; Supuran, C.T. Expert Opin. Ther. Pat. 2013, 23, 681; c) Scozzafava, A.; Supuran, C.T.; Carta, F. Expert Opin. Ther. Pat. 2013, 23, 725; d) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229.

Table 1. Kinetic parameters for the CO2 hydration reaction catalysed by various CAs belonging to the various families.17 The α-class CAs were the human cytosolic isozymes hCA I and II and the bacterial one SazCA (from Sulfurihydrogenibium azorense).5d,6 The β-class includes the fungal enzyme Can2 from Cryptococcus neoformans

4b

and FbiCA1 the plant Flaveria bidentis.18 The γ-

class enzyme was PgiCA from the anaerobic bacterium Porphyromonas gingivalis,19 whereas the δ and ζ-class enzymes

2,9

(the last with zinc and cadmium at the active site) were from the diatom

Thalassiosira weissflogii. Inhibition data with the clinically used sulfonamide acetazolamide (5acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided. All data were obtained in the author’s laboratories.

Isozyme

Class Organism

kcat

kcat/Km

-1

-1 -1

KI (acetazolamide)

(s )

(M .s )

(nM)

hCA Ia

α

human

2.0x105

5.0x10 7

250

hCA IIa

α

human

1.4x106

1.5x10 8

12

SazCAb

α

bacterium

4.4x106

3.5x10 8

0.9

Can2 c

β

fungus

3.9x105

4.3x10 7

10.5

1.2x10

5

7.5x10

6

27

4.1x10

5

5.4x10

7

324

6

FbiCA1 PgiCA

d

β

e

CdCA1-R1

γ f

plant bacterium

82

ζ

diatom

1.5x10

ZnCA1-R1 f ζ

diatom

1.4x106

1.6 x10 8

58

TweCA g

diatom

1.3x105

3.3 x10 7

83

δ

a

Data from ref.1a

b

Data from ref.1

c

Data from ref.5d

d

Data from ref.18

e

Data from ref.19

f

Data from ref.9

g

This work.

1.4 x10

8

Table 2: CA class, organism (belonging to the Bacteria, Archaea and Eukarya kingdoms), accession numbers and cryptonyms of the sequences used in the phylogenetic analysis of Fig. 4. CA class

Organism

Accession number

Cryptonym

alpha

Helicobacter pylori J99 Homo sapiens, isoform II Homo sapiens, isoform I Sulfurihydrogenibium yellowstonense YO3AOP1 Streptococcus salivarius PS4 Neisseria gonorrhoeae

NP_223829.1 AAH11949.1 NP_001158302.1 ACD66216.1 EIC81445.1 CAA72038.1

HpylCA_alpha HumCAII_alpha humCAI_alpha SspCA_alpha SsalCA_alpha NgonCA_alpha

beta

Schizosaccharomyces pombe Brucella suis 1330 Burkholderia thailandensis Bt4 Coccomyxa sp. Chlamydomonas reinhardtii Acinetobacter baumannii Porphyromonas gingivalis Myroides injenensis Zea mays Vigna radiata Flaveria bidentis, isoform I

CAA21790 NP_699962.1 ZP_02386321 AAC33484.1 XP_001699151.1 YP_002326524 YP_001929649.1 ZP_10784819 NP_001147846.1 AAD27876 AAA86939.2 AAA50156 BAF34127.1 YP_003619232 ACI70660 GI:13786688 GAA26059 EIF49256

SpoCA_beta BsuCA_beta BthCA_beta CspCA_beta CreCA_beta

ZP_10427314.1

Arabidopsis thaliana Porphyromonas gingivalis

ACQ57353.1 XP_001703237.1 NP_564091.1 YP_001929649.1

PseCA_gamma BglCA_gamma CAM_gamma CreCA_gamma AthCA_gamma PgiCA_gamma

delta

Thalassiosira weissflogii Thalassiosira pseudonana Emiliania huxleyi Bathycoccus prasinos Lingulodinium polyedrum

AAV39532.1 XP_002287620.1 ABG37687.1 CCO20234.1 ABS87870.1

TweCA_delta TpsCA_delta EhuCA_delta Bpr_delta LpoCA_delta

zeta

Thalassiosira weissflogii Micromonas pusilla CCMP1545

AAX08632.1 XP_003063214.1

TweCA_zeta, CDCA1 MpuCA_zeta

Legionella pneumophila 2300/99 Escherichia coli Methanobacterium thermoautotrophicum Saccharomyces cerevisiae Dekkera bruxellensis AWRI1499 gamma

Pseudomonas sp. PAMC 25886 Burkholderia gladioli BSR3

PgiCA_beta MinCA_beta ZmaCA_beta VraCA_beta FbiCA_beta AthCA_beta HpyCA_beta LpnCA_beta EcoCa_beta Cab_beta SceCA_beta DbrCA_beta

Table 3: Inhibition constants of anionic inhibitors against human α-class isozymes hCA I, II, the ζclass ones with Cd(II) or Zn(II) at their active site, as well as the δ-CA TweCA, for the CO2 hydration reaction, at 20 °C and pH 7.5. The last three enzymes are from the diatom T. weissflogii.

KI #[mM]

Inhibitor hCA Ia

hCA IIa

CdCA-R1b ZnCA-R1 b TweCAc

> 300

>300

0.53

0.36

5.8

Cl-

6

200

0.76

0.41

>200

-

4

63

0.85

0.53

8.3

0.3

26

1.12

0.61

1.9

0.0007

0.03

0.10

0.11

0.93

SCN-

0.2

1.6

0.089

0.10

0.93

CN-

0.0005

0.02

0.11

0.10

4.9

N3 -

0.0012

1.5

0.84

0.11

7.5

FBr -

I

CNO

-

HCO3-

12

85

0.12

0.10

0.89

CO3

2-

15

73

0.13

0.11

2.5

NO3

-

7

35

0.82

0.21

0.97

NO2

-

8.4

63

0.88

0.58

3.1

HS-

0.0006

0.04

0.70

0.15

0.090

HSO3-

18

89

0.63

0.34

8.2

SnO32-

0.57

0.83

nt

nt

0.65

SeO42-

118

112

nt

nt

77.1

TeO42-

0.66

0.92

nt

nt

68.7

4-

25.8

48.5

nt

nt

8.8

4-

0.54

0.57

nt

nt

0.78

2-

0.64

0.95

nt

nt

43.9

ReO4-

0.11

0.75

nt

nt

69.0

RuO4-

0.10

0.69

nt

nt

0.86

S2O82-

0.11

0.084

nt

nt

37.9

0.085

0.086

nt

nt

31.4

0.0087

0.0088

nt

nt

0.58

0.00079

3.1

nt

nt

0.20

P2O7

V2O7 B4O7

SeCN CS3

-

2-

Et2NCS2

-

SO42-

63

>200

0.48

0.24

>200

>200

>200

>200

>200

>200

>200

>200

>200

>200

>200

NH(SO3)22-

nt

0.76

nt

nt

75.2

FSO3-

nt

nt

nt

43.0

H2NSO3 H*

0.021

0.39

0.010

0.072

0.079

H2NSO2NH2

0.31

1.13

0.065

0.060

0.048

PhB(OH)2

58.6

23.1

0.69

0.61

0.009

PhAsO3 H2 *

31.7

49.2

0.60

0.52

0.076

BF4

-

ClO4

#

-

Errors were in the range of 3-5 % of the reported values, from three different assays.

nt = not tested. a

Data from ref.20

b

Data from ref.9

c

This work.

* As sodium salt.

-

-

2+ Zn N N

N H

N H A

O

OH

OH

2+ Zn

NH

N

S

S N H

NH N

O N H

B

Fig. 1: Metal ion coordination in the CAs: A. α-, γ- and δ-CAs have three His residues and a water molecule/hydroxide ion coordinating the metal ion (for the γ-class the metal ion can also be Co(II) or Fe(II), and as the enzyme is a trimer, two His residues come from one monomer and the third from another one); B. β- and ζ-CA coordination of the metal ion, with two Cys and one His residues, whereas the fourth ligand is again a water molecule/hydroxide ion. In the ζ-CAs the Zn(II) is frequently replaced by Cd(II) and the enzymes possess high catalytic activity with both metal ions.1,2,9

TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta

----------------------------------MEVDVVPNTKN------------------------------------------------MEVDVVPNTKN------------------------MVARLMLAASVLLVRAWGTGCPDDPEVDLCSETTTDESGTGTGTEEVNVN MSGVQYSNVEKSSSSGMMSKVLVALGVVTFCGVFANVGLVHQMQKDVDHIVSGMEPEHAP :*.: : .

TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta

----------------------------------------------------------------------------------------------------------------------GAMRTRTSLMPMLXLAGVFRSKNALFALPLLGXPLAAEAAAAAGTSGPSTCGAVKDMYKE MSTKSPMS----------------------------------------------------

TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta

----------------------------------------------------------------------------------------------------------------------QGCCGRPDKELDVVIVPKPTKRLFGANICEGKQPVHATPGDNYFKNVDCLNGTTLQVLEQ ------------------------GTNTCEGQKKTIETYFNGALDNGSCADKLVAGV-EQ

TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta

15 17 22 ------------------------YWQSSMCPVNVHWHLGTEHYSVGEYDENGSGPN--------------------------YWQSSMCPVNVHWHLGTEHYSVGEYDENGSGPN--AGANVTLGYRGRLDASSRTPILTPYWQNGLCPVNVHWHLGTEHYSKGQFDEHGTGPDIAA AGGNVTVGYEGGLDVGTDEPIKEPYYKQKLCPVNVHWHLGAEHLSVGQYDETGSGPG--*::. :**********:** * *::** *:**. 101 114 117 --GNVGVPYRRTLAEGEVQDGFRCHHYDPDDEAYTRPYEWKHCIGMEVGETYEVHWPHSG --GNVGVPYRRTLAEGEVQDGFRCHHYDPDDEAYTRPYEWKHCIGMEVGETYEVHWPHSG EEDAEGEADSRRLAVA--RRGYRCSKYDAKDAKFTTEYNWQHCEGMHVGETYEVHWPHSA -----SASHRKLLAEG-ARLGGRCHKYDSSVEMYTKEYDWQHCVGMKVGETYEVHWPHSS . . . ** . . * ** :**.. :* *:*:** ** ************.

TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta

AGACGTTYQYQTPFYDGVFCNLDMETLQT-LAPQDIANAVGVQGQIFTIVNDDTYYYPDL AGACGTTYQYQTPFYDGVFCNLDMETLQT-LAPQDIANAVGVQGQIFTIVNDDTYYYPDL AGACGTPYQYQTPFYDGVFC-VDG--IVS-LSPLNTYMKIGVQSQVYTIVNDETYYYPEM VGACGTPNQYQTPFYDGVLCDIDGPTLATVISGNQLHLNVGVQAQIFTIVNDENYYYPDL .*****. **********:* :* : : :: : :***.*::*****:.****::

TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta

IRGWIVDEEMGMGQDIAMYTGSTTGESRSNEICSSYSPITWQVDRKCHKISASSFDKLCY IRGWIVDEEMGMGQDIAMYTGSTTGESRSNEICSSYSPITWQVDRKCHKISASSFDKLCY IKGMIVDGH--YGQDIAKYTGSTTGTSRDNEVCSRYTPITWQVDRKCHLISASSFDKMCA MRGMIVDGD--KGSEITYYTGSTTGTSRDNSVCSAWSPITWQVDRTCHLISASTFDKMCA :.* *** *.:*: ******* **.*.:** ::********.** ****:***:*

TweCA_delta TpsCA_delta LpoCA_delta Bpr_delta

DMKMQRDDMSDDLYAHGSRELVTPEYVANNQQTRRLTEKHEHNHSHGHSHVRGHQHHQWF DMKMQRDDMSDDLYAHGSRELVTPEYVANNQQTRRLTEKHEHNHSHGHSHVRGHQHHQWF DMKNQHDDMSSDLHAHGSRVLVDRNFTGNNFHRRM------------------------DMKSQRDDMSDDLYAHGSREVTTDTITANNQDFNGLAP---------------------*** *.****.**:***** :. ..** .

226

Figure 2. Multialignment of the amino acid sequences δ-CAs from different species was performed with the program CLUSTAL W, version 2.1. Legend: The asterisk (*) indicates identity at all aligned positions. The symbol (:) relates to conserved substitutions, while (.) means that semiconserved substitutions are observed. TweCA_delta, Thalossosiria weissflogii, (delta CA, accession number AAV39532.1); TpsCA_delta, Thalassiosira pseudonana (accession number XP_002287620.1); LpoCA_delta, Lingulodinium polyedrum (accession number: ABS87870.1); Bpr_delta, Bathycoccus prasinos (accession number: CCO20234.1). Numbering system for delta CAs has bee used. The putative zinc ligands are indicated in red and the putative proton shuttling residues in orange.

TweCA_delta PgiCA_gamma SspCA_alpha HumCAI_alpa

-------------------------MEVDVVPNTKNYWQSSMC-----------PVNVHW -------------------------MAQR---ENSDYLTTKMA--LIQSVRGFTPIIGED MRKILISAVLVLSSISISFAEHEWSYEGE---KGPEHWAQLKPEFFWCKLKNQSPINIDK ------------------MASPDWGYDDK---NGPEQWSKLYP---IANGNNQSPVDIKT : : *:

HLGTEHYSV-----------GEYDENGSGPNGNVGVPYRRTLAEGEVQDGFRCHHYDPDDEAYTRPYEW-------81

TFLAENATIVGDVVMGKGCSVWFNAVLRGDVNSIRIGDNVNIQDGSILHTL---------------YQ--------64

94 96

KYKVKANLP--------KLNLYYKTAKESEVVNNGHTIQINIKEDNTLNYL-----GEKYQLKQFHFHTPS-----SETKHDTSL-------KPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGS :. . . .: :.. : : 114 117

KHCIGMEVGETYEVHWPH-----------SGAGACGTTYQYQTPFYDGVFCNLDMETLQTLAPQDIANAVGVQGQIFT 117

122

KSTIEIGDNVSVGHNVVI-----------HGAKICDYALI-------------GMGAVVLDHVVVGEGAIVAAGS--106

119

EHTIE-KKSYPLEIHFVH-------------KTEDGKILV------VGVMAKLGKTNKELDKILNVAPA--EEGE--EHTVD-GVKYSAELHVAHWNSAKYSSLAEAASKADGLAVI-------GVLMKVGEANPKLQKVLDALQAIKTKGK--: : . : . . * *. 226

IVNDDTYYYPDLIRGWIVDEEMGMGQDIAMYTGSTTGESRSNEICSSYSPITWQVDRKCHKISASSFDKLCYDMKMQR VVLTGTQIEPNSI-----------------YAGA---------------PARFI-----KKVDPEQSREMNFRIA--199

-KILDKNLNLNNL--------IPKDKRYMTYSGSLTTP-------PCTEGVRWIVLKKPISISKQQLEKLKSVMVN--RAPFTNFDPSTL--------LPSSLDFWTYPGSLTHP-------PLYESVTWIICKESISVSSEQLAQFRSLLSNVE . . : *.*: : .:. .. :: : DDMSDDLYAHGSRELVTPEYVANNQQTRRLTEKHEHNHSHGHSHVRGHQHHQWF ---------------------HNYRM-------------YASWFKDESSEIDNP ---------------------PNNRPVQEI---------NSRWIIEGF-----GDNAVPMQ-------------HNNRPTQPL---------KGRTVRASF-----* . .

Figure 3. Multialignment of the amino acid sequences of α, γ and δ- CAs was performed with the program CLUSTAL W, version 2.1. Legend: The asterisk (*) indicates identity at all aligned positions. The symbol (:) relates to conserved substitutions, while (.) means that semiconserved substitutions are observed. TweCA_delta, Thalassiosira weissflogii, (delta CA, accession number AAV39532.1); PgiCA_gamma, Porphyromonas gingivalis (gamma CA, accession number YP_004510261); HumCAI, Homo sapiens (human CA, isoform I, accession number: NP_001729.1); SspCA, Sulfurohydrogenibium yellowstonense YO3AOP1 (alpha CA, accession number: ACD66216.1). Numbering arrangement for delta CA is refereed to the δ system used by Lee et al. (the putative zinc ligands are indicated in red). For γ-CAs we used the CAM numbering system (the zinc ligands in red); For α-CAs the human CA I numbering system has been used (metal ion ligands are indicated in red; the gatekeeper residues are indicated in blue; the proton shuttle residue is indicated in pink.

Figure 4. Phylogenetic tree of the α- , β- , γ- , δ- and ζ-CAs from selected prokaryotic and eukaryotic species. The tree was constructed using the program PhyML 3.0. Branch support values have been reported at branch points. Organisms, accession numbers and cryptonyms of the sequences used in the alignment have been indicated in Table 2.

Cloning, characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii Sonia del Prete,a Daniela Vullo,b Andrea Scozzafava,b Clemente Capasso*a and Claudiu T. Supuran*b,c -

-

2+ Zn N N

N H

N H A

O

OH

OH

2+ Zn

NH

N

S

S N H

NH N

O N H

B