Anion inhibition studies of two α-carbonic anhydrases from Lotus japonicus, LjCAA1 and LjCAA2

Journal of Inorganic Biochemistry 136 (2014) 67–72

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Anion inhibition studies of two α-carbonic anhydrases from Lotus japonicus, LjCAA1 and LjCAA2 Daniela Vullo a, Emmanouil Flemetakis b, Andrea Scozzafava a, Clemente Capasso c, Claudiu T. Supuran a,d,⁎ a

Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy Laboratory of Molecular Biology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece Istituto di Biochimica delle Proteine — CNR, Via P. Castellino 111, 80131 Napoli, Italy d Università degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy b c

a r t i c l e

i n f o

Article history: Received 10 January 2014 Received in revised form 27 March 2014 Accepted 27 March 2014 Available online 8 April 2014 Keywords: Carbonic anhydrase Anion α-Class enzyme Inhibitor Lotus japonicus

a b s t r a c t The model organism for the investigation of symbiotic nitrogen fixation in legumes Lotus japonicus encodes two carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-class, LjCAA1 and LjCAA2. Here we report the kinetic characterization and inhibition of these two CAs with inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, and phenylboronic/arsonic acids. LjCAA1 showed a high catalytic activity for the CO2 hydration reaction, with a kcat of 7.4 ∗ 105 s−1 and a kcat/Km of 9.6 ∗ 107 M−1 s−1 and was inhibited in the low micromolar range by N,N-diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic/arsonic acid (KIs of 4–62 μM). LjCAA2 showed a moderate catalytic activity for the physiologic reaction, with a kcat of 4.0 ∗ 105 s−1 and a kcat/Km of 4.9 ∗ 107 M−1 s−1. The same anions mentioned above for the inhibition of LjCAA1 showed the best activity against LjCAA2 (KIs of 7–29 μM). Nitrate and nitrite, anions involved in nitrogen fixation, showed lower affinity for the two enzymes, with inhibition constants in the range of 3.7– 7.0 mM. Halides and sulfate also behaved in a distinct manner towards the two enzymes investigated here. As LjCAA1/2 participate in the pH regulation processes and CO2 metabolism within the nitrogen-fixing nodules of the plant, our studies may shed some light regarding these complex biochemical processes. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Lotus japonicus is a wild legume originating from Central and Eastern Asia, belonging to the Fabaceae family [1,2]. It is used as a model organism for the investigation of symbiotic nitrogen fixation and other biochemical/physiologic processes of legumes, as it possesses a rather small genome of 470 Mb, which has recently been cloned, as well as a short life cycle of 2–3 months [1–3]. One of our groups [3] recently identified two members of the α-carbonic anhydrase (CA, EC 4.2.1.1) family [4–7] in this plant, LjCAA1 and LjCAA2, together with a β-CA [8], whereas the symbiont bacterium of this legume, Mesorhizobium loti, also encodes for an α-CA recently identified and investigated by the same group [9]. All these enzymes seem to be involved in nodule development and symbiotic nitrogen fixation by L. japonicus/M. loti, constituting thus a very interesting case for studying crucial physiologic processes of legumes and their microsymbionts [3,8,9]. CAs are in fact widespread enzymes in all life kingdoms with five distinct genetic families known to date, the α-, β-, γ-, δ- and ζ-CAs ⁎ Corresponding author at: Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. Tel.: +39 055 4573005; fax: +39 055 4573385. E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).

http://dx.doi.org/10.1016/j.jinorgbio.2014.03.014 0162-0134/© 2014 Elsevier Inc. All rights reserved.

[4,5,10–15]. They catalyze a simple but essential reaction, CO2 hydration to bicarbonate and protons [4–7]. This reaction as well as the three chemical entities involved in it, carbon dioxide, bicarbonate and protons, are important for the pH regulation and homeostasis of the organisms, CO2 and HCO− 3 transport coupled with several biosynthetic processes (involving either bicarbonate or CO2 as substrates), for the production of body fluids, bone resorption, tumorigenicity, and other such physiological processes in vertebrates, whereas in cyanobacteria, plants, diatoms and algae they are also involved in photosynthetic processes [3–7,10–15]. Plants encode CAs belonging to the α-, β-, and γ-classes and these enzymes started to be investigated in some detail recently [3,8,16–18]. In algae, diatoms and aquatic plants, CAs are involved in a carbon concentrating mechanism (CCM) which increases the CO2 concentration at the site of fixation by ribulose-1,5-bisphosphate carboxylase/ oxygenase (RUBISCO) several folds over its external concentration, allowing the enzyme to function efficiently [12]. Marine diatoms possess both external and internal CAs. It has been hypothesized in the model diatom Thalassiosira weissflogii that the external CA catalyzes the dehydration of HCO− 3 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 HCO− 3 to prevent the leakage of CO2 back to the external medium [12,15]. In land plants, Rubisco uses atmospheric CO2 for the fixation of inorganic carbon and

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D. Vullo et al. / Journal of Inorganic Biochemistry 136 (2014) 67–72

its conversion into complex carbohydrates [11,16]. However, O2 competes with CO2 for the active site of RUBISCO, and the oxygenase activity of the enzyme catalyzes the first step in the photo-respiratory pathway, which can lead to 25% of the fixed carbon being lost from C3 plants [19]. A number of plant lineages have evolved mechanisms that result in increased concentrations of CO2 around RUBISCO, decreasing its oxygenase activity and significantly reducing photorespiration. One of these biochemical CO2 CCMs, is the C4 photosynthetic pathway, which has evolved more than 60 independent times within the flowering plants from C3 ancestors [20]. In these rather intricate processes, CAs belonging to all classes mentioned above are involved in the various organisms performing photosynthesis, whereas an even more general CCM system seems to be present in most plants. This so-called basal CCM (bCCM) is proposed to be composed of a mitochondrial CA belonging to the β-CA class, and a γ-type CA domain of the mitochondrial NADH dehydrogenase complex [15,21]. Together, these two enzymes reduce leakage of CO2 from plant cells and allow efficient recycling of mitochondrial CO2 for carbon fixation in chloroplasts [15,21]. Ultimately, CA functions in three primary modes in photosynthetic systems: a) to convert HCO− 3 to CO2, for fixation by Rubisco; b) to convert CO2 to HCO− 3 for fixation by phosphoenolpyruvate carboxylase (PEPC); and c) to provide rapid equilibration between CO2 and HCO− 3 so that facilitated diffusion of CO2 is enhanced [15,21]. It is however unclear what is the role of αclass CAs in many plants in which such enzymes are present [5,11,20, 21]. An exception to this is constituted by the recent study from Flemetakis group [3] who showed that in L. japonicus, the two α-CAs identified so far, LjCAA1 and LjCAA2, are present in the nitrogen-fixing nodules, showing a particular developmental expression pattern, and being involved in biochemical processes both linked and not linked to the nitrogen fixation. In that study some kinetic data on these two new α-CAs were also obtained but no interaction of these enzymes with potential inhibitors have been presented. As α-CAs are sensitive to a rather large range of inhibitors, belonging to various chemical classes, from small inorganic anions [4–7], to sulfonamides and their isosteres [4–7], coumarins [22], polyamines [23], dithiocarbamates [24], xanthates [25], etc., it became of interest to investigate the inhibition profile of LjCAA1 and LjCAA2 with some of these inhibitors. Here we report the first inhibition study of the two enzymes from L. japonicus with a series of anions and other small molecules known to interfere with the activity of CAs. 2. Materials and methods 2.1. Chemistry All anions/small compounds used here were commercially available, highest purity reagents, from Sigma-Aldrich (Milan, Italy).

determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in distilleddeionized water and dilutions up to 0.01 nM 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 non-linear least-squares methods using PRISM 3, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver–Burk plots, as reported earlier [27–30], and represent the mean from at least three different determinations.

3. Results and discussions The inhibition of CAs is a well understood process, with most classes of inhibitors (anions, sulfonamides, dithiocarbamates and xanthates) binding to the metal center [4–7,10,24,25], either by substituting the fourth, non-protein zinc ligand, leading thus to tetrahedral Zn(II) geometries, or by addition to the coordination sphere and leading to trigonalbipyramidal Zn(II) ions [4,5,10]. However the polyamines, similar to the phenols, anchor to the zinc-coordinated water molecule/hydroxide ion [23] whereas the coumarins (and their derivatives) act as prodrug-type inhibitors being hydrolyzed by the esterase CA activity to 2-hydroxycinnamic acid derivatives which bind at the entrance of the active site cavity, occluding it [22], and inhibit these enzymes by an alternative, completely novel mechanism of action. Anions are of interest to be investigated as CA inhibitors (CAIs) due to the fact that they can offer important details regarding the catalytic/ inhibition mechanism but also for the drug design of organic, more potent inhibitors with biomedical or environmental applications [4,5,10]. With very few exceptions, most of the zinc-binding inhibitors investigated so far act as monodentate ligands when coordinating to the metal ion within the CA active site [4,10,31]. Adducts of sulfonamide (as anions) or inorganic anions such as hydrogen sulfide, halides, bisulfite, azide, and thiocyanate have been characterized in great detail by means of spectroscopic and X-ray crystallographic techniques (reviewed in Refs. [4,10]). We have measured the kinetic parameters for the CO2 hydration reaction of LjCAA1 and LjCAA2 and compared them with those of other α- and β-CAs, such as the human (h) hCA I and II, and the enzyme from the extremophilic bacterium Sulfurihydrogeninbium yellowstonensis, SspCA [10] which is one of the most stable and catalytically effective CA known to date, at 20 °C and pH 7.5 in 10 mM HEPES buffer (and in the presence of 20 mM NaClO4). The kinetic data for the plant β-CAs FbiCA 1 form Flaveria bidentis, FbiCA1 [15,16] are also shown in Table 1 for comparison reasons, being measured in slightly different conditions (i.e., at 20 °C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4) due to the fact that most β-CAs show low or no activity at pH values lower that 8.3 [34].

2.2. Enzymology LjCAA1 and LjCAA2 were recombinant enzymes obtained in-house as described earlier [3]. 2.3. CA catalytic activity and inhibition assay An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity [26]. 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, for α-CAs) or TRIS (pH 8.3 for β-CAs) as buffers, and 20 mM NaClO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed 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

Table 1 Kinetic parameters for the CO2 hydration reaction [26] catalyzed by the human cytosolic isozymes hCA I and II, the bacterial one SspCA (from S. yellowstonensis) and L. japonicus α-CA (LjCAA1 and LjCAA2), at 20 °C and pH 7.5 in 10 mM HEPES buffer, the presence of 20 mM NaClO4. The kinetic data for the plant β-CAs FbiCA 1 form F. bidentis (measured at 20 °C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4) are also included for comparison. Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido1,3,4-thiadiazole-2-sulfonamide) are also provided.

X

Isozyme

Class

Activity level

kcat (s−1)

kcat/Km (M−1 × s−1)

hCA I hCA II SspCA FbiCA 1 LjCAA1 LjCAA2

α α α β α α

Moderate Very high Very high Low High Moderate

2.0 × 105 1.4 × 106 9.35 × 105 1.2 × 105 7.4 × 105 4.0 × 105

5.0 1.5 1.1 7.5 9.6 4.9

This study.

× × × × × ×

107 108 108 106 107 107

KI (acetazolamide) (nM)

Ref

250 12 4.5 27 51 24

[4,5] [4,5] [32] [15] x x

D. Vullo et al. / Journal of Inorganic Biochemistry 136 (2014) 67–72

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Fig. 1. Alignment of the amino acid sequences of α-CAs from different sources (primates, eudicots, aquifecales and sponges). The proton shuttle residue (His64 in red), the zinc ligands (His94, 96 and 119, in black bold) and the gatekeeper residues (Glu106 and Thr199, in blue) are conserved in all these enzymes. The hCA I numbering system was used. The asterisk (*) indicates identity at all aligned positions; the symbol (:) relates to conserved substitutions, while (.) means that semi-conserved substitutions are observed. Multialignment was performed with the program Muscle, version 3.7. Legend: LjCAA1, Lotus japonicus, isoform 1 (Accession number: CAM59682.1); LjCAA2, Lotus japonicus, isoform 2 (Accession number: CAM59683.1); TccA, Theobroma cacao (Accession number: EOY34415.1); AwiCA, Astrosclera willeyana (Accession number: ABR53887.1); SspCA, Sulfurihydrogenibium yellowstonense YO3AOP1 (Accession number: ACD66216.1); SazCA, Sulfurihydrogenibium azorense (Accession no.: ACN99362.1); hCA I, Homo sapiens, isoform I (Accession number: NP_001158302.1); and hCA II, H. sapiens, isoform II (Accession number: AAH11949.1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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D. Vullo et al. / Journal of Inorganic Biochemistry 136 (2014) 67–72

It may be observed that both Lotus enzymes possess significant catalytic activity for the CO2 hydration reaction, with a kcat of 7.4 × 105 s−1 and a kcat/Km of 9.6 × 107 M−1 s−1 in the case of LjCAA1, and a kcat of 4.0 × 105 s−1 and a kcat/Km of 4.9 × 107 M−1 s−1, for LjCAA2. The first isoform thus shows a high catalytic activity, intermediate between that of the widespread human isoform hCA I and the bacterial, highly effective SspCA. Only hCA II showed a higher catalytic activity then LjCAA1 among the enzymes investigated here. LjCAA2 showed a lower catalytic activity compared to LjCAA1, which is in fact quite similar (considering the kcat/Km values) to that of hCA I. Furthermore, the two Lotus enzymes are around one order of magnitude better catalysts compared to the plant β-CAs FbiCA 1, which possesses important physiological functions in the CCM of the C3 plant F. bidentis [16,17,20]. LjCAA1 and LjCAA2 were also inhibited by the sulfonamide, clinically used compound acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) [4,5], with inhibition constants of 24–51 nM (Table 1). It should be noted that the enzyme with the lower catalytic activity, LjCAA2, showed higher affinity for the sulfonamide. Of note is also the fact that other α- and β-CAs investigated here were inhibited by this compound better (hCA II, SspCA or FbiCA 1) or less (hCA I) compared to the two L. japonicus enzymes (Table 1). In order to explain these data we have aligned the amino acid sequences of LjCAA1 and LjCAA2 with those of other α-CAs, from plants (Theobroma cacao, TccA), sponges (Astrosclera willeyana CA, AwiCA), bacteria (Sulfurihydrogenibium yellowstonense SspCA and, Sulfurihydrogenibium azorense SazCA) as well as the highly investigated human isoforms hCA I, hCA II, (Fig. 1) [32–36]. It may be observed that the zinc-binding ligands (His94, 96 and 119, hCA I numbering system), as well as the catalytic dyad involved in the orientation of the substrate/ zinc-coordinated water molecule in the proper way for the nucleophilic attack (Glu106–Thr199) [4,5,10] are all conserved among these mammalian, bacterial and plant α-CAs (Fig. 1). However a striking difference between the two L. japonicus enzymes exists, which has already been noticed in the first paper in which the two enzymes were reported [3]. LjCAA1 possesses the proton shuttle residue His64, similar to hCA I and II, or SspCA. This residue facilitates the rate-determining step of the catalytic cycle, by transferring protons from the zinc-coordinated water molecule to the external buffer and allows the formation of the zinc hydroxide, the catalytically active species of the enzyme [4,5,35]. However, LjCAA2 does not possess His in position 64 but a Phe residue. Thus, the proton transfer processes (and the possible residue(s) involved in it) are presently not understood for this isoform. The absence of His64 in LjCAA2 may explain the lower catalytic activity of this isoform compared to LjCAA1, and was only rarely observed in other α-CAs (e.g., in astrosclerin, an α-CA from the living fossil sponge Astrosclera willeyana [36] or in a similar enzyme from the pathogenic protozoan Trypanosoma cruzi, TcCA [37]) characterized earlier by one of these groups. However, similar to LjCAA2, these enzymes (astrosclerin and TcCA) possess a significant catalytic activity, leaving open the question of their catalytic mechanism compared to that of the canonical α-CAs which incorporate a His64 as the proton shuttle residue. Fig. 2 shows a phylogenetic tree of these α-CAs in which enzymes of mammalian, plant, bacterial and protozoan origin were included. It may be observed that the three plan enzymes cluster together on two different but nearby branches of the tree, which are distinct from the bacterial, sponge or mammalian enzymes. However, LjCAA1 and LjCAA2 are not on the same branch of the tree, showing that evolutionarily they are not closely related. In fact LjCAA2 is more related to the cocoa tree enzyme TccA than to the other isoform from L. japonicus, LjCAA1. This interesting evolutionary relationship between the two Lotus isoforms is not completely understood at this moment. Inhibition data of the two plant CAs investigated here, LjCAA1 and LjCAA2 with a wide range of inorganic anions and small molecule compounds known to interact with the CA family of proteins [10] are shown in Table 2. Inhibition of the two other α-CAs, hCA II (highly abundant

Fig. 2. Phylogenetic trees of the sequences of the α-CAs shown in Fig. 1. The radial tree was constructed using the program PhyML 3.0.

protein with important physiological functions [4,5]), and the bacterial enzyme SspCA (from the extremophile S. yellowstonensis) [32,33] as well as the plant, β-CA FbiCA1 from F. bidentis [16,17], is also provided in Table 2, for comparison reasons. The following should be noted regarding the inhibition data of Table 2: (i) Against LjCAA1, several anions such as perchlorate and tetrafluoroborate showed no inhibitory activity (KIs N 200 mM), a situation frequently observed [10] with these anions less prone to coordinate metal ions from the enzyme active sites when assayed against other α- or β-CAs [30–34]. Other anions with weak affinity for LjCAA1 were bromide, iodide, and peroxydisulfate, which had KIs in the range of 37.5–76.9 mM (Table 2). (ii) A large number of anions, among which bicarbonate, nitrate, nitrite, bisulfite, stannate, selenite, tellurate, diphosphate, divanadate, perrhenate, perrhutenate, and sulfate were more effective, millimolar LjCAA1 inhibitors, with KIs in the range of 3.1–8.4 mM (Table 1). Among them which are of great interest are bicarbonate, nitrate, and nitrite, as the first one is also a substrate for the enzyme, whereas nitrate and nitrite are involved in the nitrogen fixation processes in the nodules of the plant where these enzymes are abundant. Probably the relatively low affinity of the enzyme for these anions is an evolutionary adaptation to work in the presence of relatively high amounts of these anions. (iii) Submillimolar inhibition of LjCAA1 was observed for the following anions: fluoride, chloride, (thio)cyanate, cyanide, azide, carbonate, hydrogensulfide, tetraborate, selenocyanate, trithiocarbonate, fluorosulfonate and iminodisulfonate, which showed KIs in the range of 0.36–0.89 mM. One can observe an interesting trend of the weakening of the LjCAA1 inhibition with the halogenide, with an increase in the halogen atomic weight. This is quite unusual, for example considering the hCA II data. For this isoform, the light halogenides are either not inhibitory (fluoride) or very weak CAIs (chloride) and the inhibitory power is greatly increasing with the increase of the atomic weight of the halogen. For SspCA the same is true except the bromide data which is totally different compared to the other halides [32,33]. The pseudohalides with strong affinity for metal ions in solution ((thio)cyanate, cyanide, azide, selenocyanide) also show a rather compact behavior of medium-potency ljCAA1 inhibitors. Similar potency was observed for carbonate and trithiocarbonate. Thus the difference of inhibitory power between carbonate and bicarbonate is rather important, with carbonate being 12 times a better LjCAA1 inhibitor compared to bicarbonate (Table 2). (iv) The most effective LjCAA1 inhibitors were N,N-diethyldithiocarbamate, sulfamide, sulfamic acid, and phenylboronic/arsonic

D. Vullo et al. / Journal of Inorganic Biochemistry 136 (2014) 67–72 Table 2 Inhibition constants of anionic inhibitors against α-CA isozymes derived from human (hCA II), and bacterial (SspCA) sources as well as the plant L. japonicus α-CAs (LjCAA1 and LjCAA2) at 20 °C and pH 7.5 in 10 mM HEPES buffer by a stopped flow CO2 hydrase assay [26]. Inhibition data of the β-CA from the plant F. bidentis isoform 1 (FbiCA 1) are also provided for comparison reasons. Inhibitora

−

F Cl− Br− I− CNO− SCN− CN− N− 3 HCO3− CO2− 3 NO− 3 NO− 2 − HS HSO− 3 SnO2− 3 SeO42− TeO2− 4 P2O4− 7 V2O4− 7 B4O2− 7 ReO− 4 RuO− 4 2− S2O8 SeCN− CS2− 3 Et2NCS− 2 SO2− 4 − ClO4 BF− 4 FSO− 3 NH(SO3)2− 2 H2NSO2NH2 H2NSO3H Ph-B(OH)2 Ph-AsO3H2 a b c d e f

KI [mM]b hCA IIc

SspCAd

FbiCA 1e

LjCAA1f

LjCAA2f

N300 200 63 26 0.03 1.60 0.02 1.51 85 73 35 63 0.04 89 0.83 112 0.92 48.50 0.57 0.95 0.75 0.69 0.084 0.086 0.0088 3.1 N200 N200 N200 0.46 0.76 1.13 0.39 23.1 49.2

41.7 8.30 49.0 0.86 0.80 0.71 0.79 0.49 33.2 39.3 0.86 0.48 0.58 21.1 0.52 0.57 0.53 0.69 0.66 0.67 0.80 0.69 84.6 0.07 0.06 0.004 0.82 N200 N200 0.73 0.75 0.009 0.042 0.041 0.005

0.71 0.74 0.67 0.71 0.93 0.83 0.62 0.46 0.66 0.84 0.78 0.57 0.86 55.3 0.53 24.5 0.90 0.83 0.66 0.86 0.52 26.1 0.87 0.88 0.06 0.008 0.62 N200 N200 0.69 50.9 0.004 0.005 0.008 0.006

0.73 0.87 41.1 76.9 0.55 0.36 0.68 0.77 5.4 0.45 5.2 4.1 0.40 5.3 5.1 4.8 5.6 3.1 3.5 0.50 8.4 6.5 37.5 0.89 0.49 0.004 7.1 N200 N200 0.66 0.46 0.062 0.048 0.006 0.005

7.3 8.7 6.4 7.6 0.45 0.73 0.42 0.54 3.9 6.1 7.0 3.7 0.36 7.4 5.0 0.62 0.51 3.9 7.6 6.2 8.3 7.9 62.3 0.76 0.20 0.007 51.9 N200 N200 0.69 55.9 0.009 0.027 0.009 0.008

As sodium salt. Errors were in the range of 3–5% of the reported values, from three different assays. From Ref. [10]. From Ref. [32]. From Ref. [15]. This work.

71

divanadate, tetraborate, perrhenate, and perrhutenate, which have inhibition constants ranging between 3.7 and 8.7 mM (Table 2). It should be noted that all halides had very similar KIs, a net difference between this isoform and LjCAA1 discussed above. Another difference between the two Lotus isoforms regards the bicarbonate/carbonate inhibition data. It may be observed that unlike the situation of LjCAA1 discussed above, LjCAA2 carbonate was a weaker inhibitor than bicarbonate. (vii) Submillimolar LjCAA2 inhibitors were the following anions: the pseudohalides, hydrogensulfide, selenite, tellurate, selenocyanate, trithiocarbonate and fluorosulfonate, which possessed KIs in the range of 0.20–0.76 mM (Table 1). Again the pseudohalides showed a very similar inhibitory pattern against LjCAA2 (as for LjCAA1), whereas trithiocarbonate was more than two times a better LjCAA2 than LjCAA1 inhibitor. (viii) As for LjCAA1, the best LjCAA2 inhibitors were N,N-diethyldithiocarbamate, sulfamide, sulfamic acid, and phenylboronic/arsonic acid, which showed KIs in the range of 7–27 μM (Table 2). (ix) The inhibition profile of the two Lotus isoforms is rather different from each other (except the most ineffective and the most effective inhibitors), and also different from those of other α- or β-CAs of different origins. As for most CAs investigated so far, we propose that the anion inhibition mechanism of the two Lotus isoforms involves either the binding of the anion to the zinc ion (in tetrahedral or trigonal-bipyramidal geometry of the metal ion) or its anchoring to the zinc-coordinated water molecule (as discovered recently for the inhibition of the Coccomyxa β-CA with iodide, which is not coordinated to the metal ion but anchored to its non-protein ligand) [38]. Although the physiological roles of LjCAA1 and LjCAA2 are not completely understood yet, a previous study showed that their developmental expression is distinct and correlates with biochemical functions both connected and not connected to the nitrogen fixation in the nodules of the legume [3]. Considering the fact that there are two α- and one β-CAs in L. japonicus, together with another α-CA in the bacterial symbiont of the plant, M. loti, finding specific and selective inhibitors for all these enzymes (as we tried to do in this first study) may help a better understanding of their role in the biochemical and physiologic processes connected with nitrogen fixation and CO2 metabolism in plants. 4. Conclusions

acid, which showed KIs in the range of 4–62 μM (Table 1). It may be observed that phenylboronic/arsonic acids are quite effective CAIs of the plant CAs both belonging to the α-class (LjCAA1) or β-class (FbiCA1), being much less effective against the human enzyme hCA II. N,N-Diethyldithiocarbamate on the other hand is a very potent CAI for bacterial and plant, α- and β-CAs (Table 2). (v) The second isoform, LjCAA2 showed a rather different inhibition pattern compared to LjCAA1. The exception regards the two anions with low affinity for metal ions in metalloenzymes, perchlorate and tetrafluoroborate, which showed no inhibitory activity (KIs N 200 mM) against this isoform too. The other anions with weak LjCAA2 inhibitory power were peroxydisulfate, sulfate and iminodisulfonate, with KIs in the range of 51.9–62.3 mM (Table 1). It is very interesting to note the huge difference in the inhibition pattern of iminodisulfonate against the two Lotus enzymes. This anion is in fact 120.9 times a weaker LjCAA2 than LjCAA1 inhibitor. For sulfate this difference is by a factor of 7.3. Interestingly, iminodisulfonate is also a weak FbiCA1 inhibitor but it acts as a submillimolar CAI against hCA II and SspCA (Table 2). (vi) A range of different anions showed an inhibitory power against LjCAA2 in the millimolar range. They include the halides, bicarbonate, carbonate, nitrate, nitrite, bisulfite, stannate, diphosphate,

We evaluated a series of inorganic simple/complex anions and other small molecules known to bind to metalloenzymes (sulfamide, sulfamic acid, phenylboronic/arsonic acids), for the inhibition of two α-CAs from the model legume L. japonicus, LjCAA1 and LjCAA2. LjCAA1 showed a high catalytic activity for the CO2 hydration reaction, with a kcat of 7.4 × 105 s−1 and a kcat/Km of 9.6 × 107 M− 1 s−1 and was inhibited in the low micromolar range by N,N-diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic/arsonic acid (KIs of 4–62 μM) whereas bromide, iodide and sulfate were weak inhibitors (KIs of 7.1–76.9 mM). LjCAA2 showed a moderate catalytic activity for the physiologic reaction, with a kcat of 4.0 × 105 s− 1 and a kcat/Km of 4.9 × 107 M−1 s−1. The same anions mentioned above for the inhibition of LjCAA1 showed the best activity against LjCAA2 (KIs of 7–29 μM), whereas sulfate, iminodisulfonate and peroxydisulfate were weak inhibitors (KIs of 51.9–62.3 mM). Nitrate and nitrite, anions involved in nitrogen fixation, showed lower affinity for the two enzymes, with inhibition constants in the range of 3.7–7.0 mM. Halides and sulfate also behaved in a distinct manner towards the two enzymes investigated here. As both enzymes LjCAA1/2 participate in the pH regulation processes and CO2 metabolism within the nitrogen-fixing nodules of the plant, our studies may shed some light regarding these complex biochemical processes.

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Abbreviations CA carbonic anhydrase CAI carbonic anhydrase inhibitor CCM carbon concentrating mechanism FbiCA 1 Flaveria bidentis carbonic anhydrase, isoform 1 hCA I, hCA II human carbonic anhydrase, isoform I, II HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid LjCAA 1/2 Lotus japonicus α-carbonic anhydrase, isoform ½ PEPC phosphoenolpyruvate carboxylase RUBISCO ribulose-1,5-bisphosphate carboxylase/oxygenase SspCA Sulfurihydrogenibium yellowstonensis CA

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