Bioorganic & Medicinal Chemistry Letters 23 (2013) 1626–1630

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Kinetic and anion inhibition studies of a b-carbonic anhydrase(FbiCA 1) from the C4 plant Flaveria bidentis

0960-894X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.01.087

⇑ Corresponding authors. Tel.: +39 0812534583; fax: +39 0812534574 (S.M.M.);tel.: +39 0554573005; fax: +39 0554573385 (C.T.S.).

E-mail addresses: marmonti@unina.it (S.M. Monti), claudiu.supuran@unifi.it(C.T. Supuran).

Simona Maria Monti a,⇑, Giuseppina De Simone a, Nina A. Dathan a, Martha Ludwig b, Daniela Vullo c,Andrea Scozzafava c, Clemente Capasso d, Claudiu T. Supuran c,e,⇑a Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Napoli, Italyb School of Chemistry and Biochemistry [M310], The University of Western Australia, Crawley, Western Australia 6009, Australiac Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italyd Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131 Napoli, Italye Università degli Studi di Firenze, NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy

a r t i c l e i n f o

Article history:Received 22 December 2012Revised 17 January 2013Accepted 20 January 2013Available online 30 January 2013

Keywords:Carbonic anhydraseBeta-class enzymeAnionEnzyme inhibitorFlaveria bidentisPhotosynthesis

a b s t r a c t

Several b-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here wereport the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C4 plantFlaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has aninteresting CO2 hydrase catalytic activity (kcat of 1.2 � 105 and kcat/Km of 7.5 � 106 M�1 � s�1) and wasmoderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were alsodetected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acidand phenylarsonic acid (KIs in the range of 4–60 lM). Such inhibitors may be used as tools to betterunderstand the role of various b-CA isoforms in photosynthesis.

� 2013 Elsevier Ltd. All rights reserved.

The majority of land plants fix atmospheric CO2 into complexcarbohydrates using the C3 photosynthetic pathway. The primarycarboxylating enzyme of this pathway is ribulose-1,5-bisphos-phate carboxylase/oxygenase (RUBISCO), which uses atmosphericCO2 directly. However, O2 competes with CO2 for the active siteof RUBISCO, and the oxygenase activity of the enzyme catalyzesthe first step in the photorespiratory pathway, which can lead to25% of the fixed carbon being lost from C3 plants.1 A number ofplant lineages have evolved mechanisms that result in increasedconcentrations of CO2 around RUBISCO, decreasing its oxygenaseactivity and significantly reducing photorespiration. One of thesebiochemical CO2 concentrating mechanisms (CCM) is the C4 photo-synthetic pathway, which has evolved more than 60 independenttimes within the flowering plants from C3 ancestors.1,2 A combina-tion of changes in leaf anatomy and biochemistry are responsiblefor the increased efficiency of C4 plants, for example, the primarycarboxylating enzyme is phosphoenolpyruvate carboxylase (PEPC),

which requires inorganic carbon in the form of bicarbonate as asubstrate, and does not recognize O2.1,2

The genus Flaveria contains closely related species showing dif-ferent types of photosynthesis, including C3, C3–C4, C4-like and C4

photosynthesis.2 Flaveria spp. thus are an excellent model forexamining the molecular evolution of the C4 photosynthetic path-way.2 Work with the enzyme carbonic anhydrase (CA, EC 4.2.1.1),3–

8 which catalyzes the reversible hydration of CO2 to bicarbonate,the two forms of inorganic carbon used by C3 and C4 Flaveria cong-eners, respectively, has added significant insight into the under-standing of such processes.2 Three different b-CA isoforms havebeen identified in these plants: In Flaveria pringlei, a C3 species,CA 1 and CA 3 showed high expression in leaves (and their prod-ucts localized to the chloroplast).2 In contrast, while CA 1 was alsofound to be a chloroplastic enzyme in the C4 species, F. bidentis, CA3 transcripts were the most highly abundant CA mRNA in leavesand the enzyme localized to the cytosol of leaf mesophyll cells.CA 2 transcript levels were low in all organs examined in both F.pringlei and F. bidentis, and the gene was found to encode a cyto-solic enzyme.2

In addition to catalyzing the first step in the C4 pathway, therole of CA in providing HCO�3 for photosynthesis has also been welldocumented in marine algae and cyanobacteria.9,10 However, an

Table 1Kinetic parameters for the CO2 hydration reaction catalysed by the human cytosolicisozymes hCA I and II (a-class CAs) at 20 �C and pH 7.5 in 10 mM HEPES buffer and20 mM Na2SO4, and the b-CAs Can2, CalCA (from C. neoformans and C. albicans,respectively), SceCA (from S. cerevisiae), and the Flaveria bidentis CA (FbiCA 1)measured at 20 �C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4

17

Isozyme Activity level kcat

(s�1)kcat/Km

(M�1 � s�1)KI (acetazolamide)(nM)

hCA I Moderate 2.0 � 105 5.0 � 107 250hCA II Very high 1.4 � 106 1.5 � 108 12Can2 Moderate 3.9 � 105 4.3 � 107 10.5CalCA High 8.0 � 105 9.7 � 107 132SceCA High 9.4 � 105 9.8 � 107 82FbiCA 1 Low 1.2 � 105 7.5 � 106 27

Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided.

Figure 1. Alignment of the predicted amino acid sequences of selected b-CAs from plantZinc ligands are indicated in red; amino acids involved in the enzyme catalytic cycle arebinding pocket are indicated in green. The asterisk (⁄) indicates identity at a position; thsubstitutions. Multiple alignment was performed with the program Clustal W, version 2.1VraCA_plant, Vigna radiata (Accession number: AAD27876); PsaCA_plant, Pisum sativumnumber: GI: 219109194); SceCA_yeast, Saccharomyces cerevisiae (Accession number:HpyCA_bacterium, Helicobacter pylori (Accession number: BAF34127.1).

S. M. Monti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1626–1630 1627

even more general CCM system seems to be present in most plants.This so-called basal CCM (bCCM) is proposed to be composed of amitochondrial CA belonging to the b-CA class, and a c-type CA do-main of the mitochondrial NADH dehydrogenase complex.5 To-gether, these two enzymes reduce leakage of CO2 from plant cellsand allow efficient recycling of mitochondrial CO2 for carbon fixa-tion in chloroplasts.11 Ultimately, CA functions in three primarymodes in photosynthetic systems: (a) to convert HCO�3 to CO2,for fixation by Rubisco; (b) to convert CO2 to HCO�3 for fixationby PEPC; and (c) to provide rapid equilibration between CO2 andHCO�3 so that facilitated diffusion of CO2 is enhanced.

CAs belonging to the a-,12 b-13 and c-CA14 families have been de-scribed from many photosynthetic organisms, including the cyano-bacteria Synechoccus spp.,12a and Microcoleus chthonoplastes,13c theunicellular algae Chlamydomonas reinhardtii11 and Coccomyxa,12b

, yeast, fungal and bacterial organisms. Pisum sativum numbering system was used.indicated in blue; amino acids forming the continuous hydrophobic surface in the

e symbol (:) designates conserved substitutions, while (.) indicates semi-conserved. Legend: FbiCA_plant, Flaveria bidentis, isoform I (Accession number: AAA86939.2);(Accession number: AAA33652); Can2_fungus; Cryptococcus neoformans (AccessionGAA26059); CalCA_fungus, Candida albicans (Accession number: XP_721672.1);

Figure 2. Phylogenetic tree of the b-CAs from selected prokaryotic and eukaryotic species. The tree was constructed using the program PhyML 3.0. Branch support values arereported at branch points. FbiCA_plant, VraCA_plant, PsaCA_plant, Can2_fungus, SceCA_yeast, CalCA_fungus and HpyCA_bacterium are indicated in the legend of Fig. 1;PgiCA_bacterium, Porphyromonas gingivalis ATCC 33277 (Accession number: YP_001929649.1); MinCA_bacterium, Myroides injenensis M09-0166 (Accession number:ZP_10784819); AbaCA_bacterium, Acinetobacter baumannii AB307-0294 (Accession number: YP_002326524); DmeCA_insect, Drosophila melanogaster (Accession number:NP_649849); Cab_bacterium, Methanobacterium thermoautotrophicum (Accession number: GI: 13786688); EcoCa_bacterium, Escherichia coli (Accession number: ACI70660);LpnCA_bacterium, Legionella pneumophila 2300/99 (Accession number: YP_003619232); DbrCA_yeast, Dekkera bruxellensis AWRI1499 (Accession number: EIF49256);OpaCA_fungus, Ogataea parapolymorpha DL-1 (Accession number: EFW97556); AfuCA_yeast, Aspergillus fumigatus Af293 (Accession number: XP_751704); SpoCA_yeast,Schizosaccharomyces pombe (Accession number: CAA21790); CspCA_alga, Coccomyxa sp. (Accession number: AAC33484.1); CreCA_alga, Chlamydomonas reinhardtii (Accessionnumber: XP_001699151.1); BsuCA_bacterium, Brucella suis 1330 (Accession number: NP_699962.1); BthCA_bacterium, Burkholderia thailandensis Bt4 (Accession number:ZP_02386321); TasCA_fungus, Trichosporon asahii CBS 8904 (Accession number: EKD04029); SmaCA_fungus, Sordaria macrospora (Accession number: CAT00781);AthCA_plant, Arabidopsis thaliana (Accession number: AAA50156); ZmaCA_plant, Zea mays (Accession number: NP_001147846.1); OsaCA_plant, Oryza sativa (Accessionnumber: AAA86943).

1628 S. M. Monti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1626–1630

and the land plants Arabidopsis thaliana,12b Lotus japonicus,12c Zeamays,13d and Pisum sativum.13e In most photosynthetic species, rep-resentatives belonging to several CA families have been found11–14

and typically multiple isoforms of the enzymes are present. Itshould be noted that few X-ray crystal structures are available forsuch enzymes. The a-CA from Chlamydomonas reinhardtii,12a as wellas the b-CAs from Coccomyxa,13a and Pisum sativum13e are amongthe few representatives for which detailed structural informationis available. Interestingly, for the Coccomyxa b-CA, adducts of theenzyme with several inhibitors, including iodide, thiocyanate andacetazolamide,13a showed an interesting inhibition pattern for thisb-class enzyme, thought earlier to be true only for a-CAs. For exam-ple, acetazolamide and thiocyanate coordinate the active site Zn(II)ion along with two Cys and one His residues from the protein, in atetrahedral geometry.13a This is reminiscent of the inhibition mech-anism of a-CAs with anion or sulfonamide inhibitors,3,4 althoughthiocyanate binds in a trigonal bipyramidal geometry with theZn(II) ion to human (h) hCA II. In this adduct, the Zn(II) is coordi-nated by the three His residues from the protein, one water mole-cule and the anion inhibitor. In the Coccomyxa b-CA, the mostinteresting inhibitor binding mode was observed for iodide, whichwas not coordinated with the Zn(II) ion, but anchored to the zinc-bound water/hydroxide ion, an inhibition mechanism observed

earlier (in the a-CAs) for phenols, polyamines and one ester.15,16

It is thus of great interest to investigate inhibition of such enzymeswith various classes of inhibitors, especially in that very few suchstudies have been done with plant enzymes. These studies may re-veal novel inhibition patterns and also help in understanding theinvolvement of these enzymes in photosynthesis and other biolog-ical mechanisms (e.g., calcification, in corals or other marineorganisms).1b

Although, as mentioned above, three b-CAs have been reportedin Flaveria spp., no detailed kinetic studies have been reported, andneither their catalytic activity nor their inhibition profile has beeninvestigated. Here we report the characterization of one isoform ofF. bidentis b-CA, FbiCA 1, with respect to its inhibition profile withanions and other small molecules known to interfere with theactivity of CAs. To our knowledge, this is the first detailed inhibi-tion study of a plant b-CA.17,18

Table 1 shows the kinetic parameters for the CO2 hydrationreaction catalyzed by FbiCA 1 as well as several well-investigatedCAs,17 including the two a-CAs of human (h) origin, hCA I andII,3,4 and b-CAs from the fungal pathogens Cryptococcus neoformans(Can2)7b and Candida albicans (CalCA),19 and the yeast from Saccha-romyces cerevisiae (SceCA).20 FbiCA 1 has significant catalytic activ-ity as a CO2 hydrase, with a kcat of 1.2 � 105 and a kcat/Km of

Table 2Inhibition constants of anionic inhibitors against a-CA isozymes derived from human(hCA II), and bacterial (SspCA) sources as well as the b-CA from a bacterium (H. pylori)HpyCA, one yeast Saccharomyces cerevisiae (SceCA) and the plant Flaveria bidentisisoform 1 (FbiCA 1), at 20 �C by a stopped flow CO2 hydrase assay17

Inhibitora KIb (mM)

hCA IIc SspCAd HpyCAe SceCAf FbiCA 1g

F� >300 41.7 0.67 2.85 0.71Cl� 200 8.3 0.56 0.85 0.74Br� 63 49 0.38 0.011 0.67I� 26 0.86 0.63 0.01 0.71CNO� 0.03 0.8 0.37 31.7 0.93SCN� 1.6 0.71 0.68 55.6 0.83CN� 0.02 0.79 0.54 16.8 0.62N�3 1.51 0.49 0.8 27.9 0.46HCO�3 85 33.2 0.5 0.78 0.66

CO2�3

73 39.3 0.42 0.76 0.84

NO�3 35 0.86 0.78 13.9 0.78NO�2 63 0.48 0.67 0.46 0.57HS� 0.04 0.58 0.58 0.33 0.86HSO�3 89 21.1 0.63 0.33 55.3

SnO2�3

0.83 0.52 0.48 nt 0.53

SeO2�4

112 0.57 0.65 nt 24.5

TeO2�4

0.92 0.53 0.45 nt 0.9

P2O4�7

48.5 0.69 0.75 nt 0.83

V2O4�7

0.57 0.66 0.18 nt 0.66

B4O2�7

0.95 0.67 0.68 nt 0.86

ReO�4 0.75 0.8 0.82 nt 0.52RuO�4 0.69 0.69 1.1 nt 26.1

S2O2�8

0.084 84.6 0.93 nt 0.87

SeCN� 0.086 0.07 0.97 nt 0.88

CS2�3

0.0088 0.06 0.21 nt 0.06

Et2NCS�2 3.1 0.004 0.0074 nt 0.008

SO2�4

>200 0.82 0.57 0.58 0.62

ClO�4 >200 >200 6.5 >200 >200BF�4 >200 >200 >200 >200 >200FSO�3 0.46 0.73 0.75 nt 0.69

NHðSO3Þ2�20.76 0.75 0.7 nt 50.9

H2NSO2NH2 1.13 0.009 0.072 0.0087 0.004H2NSO3H 0.39 0.042 0.094 0.84 0.005Ph-B(OH)2 23.1 0.041 0.073 38.2 0.008Ph-AsO3H2 49.2 0.005 0.092 0.4 0.006

a As sodium salt; nt = not tested.b Errors were in the range of 3–5% of the reported values, from three different

assays.c From Ref. 6a.d From Ref. 5c.e From Ref. 21.f From Ref. 20.g This work.

S. M. Monti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1626–1630 1629

7.5 � 106 M�1 � s�1. It is also highly sensitive to the clinically usedsulfonamide acetzolamide, with an inhibition constant of 27 nM.Among the investigated CAs shown in Table 1, FbiCA 1 is the mostinefficient catalyst, with other CA kcat/Km values in the range of107–108 M�1 � s�1. Nevertheless the value measured for FbiCA 1,even if outside this range, demonstrates that the enzyme is a goodcatalyst for the conversion of CO2 to bicarbonate and protons.

To rationalize the kinetic data in Table 1, an alignment of theamino acid sequence of FbiCA 1 with that of selected b-CAs fromother plants (VraCA, from Vigna radiata;14a PsaCA, from Pisum sat-ivum);13e fungi (Can2, from Cryptococcus neoformans;7b CalCA, fromCandida albicans);19 yeasts (SceCA, from Saccharomyces cerevisi-ae);20 as well as bacteria (HpyCA, from Helicobacter pylori)21 isshown in Figure 1. FbiCA 1 has all the amino acid residues involvedin catalysis, as is the case for the other members of the family (Fig1): (i) the three Zn(II) ligands, Cys160, His220 and Cys223 (Pisumsativum CA numbering system);13e (ii) the Asp162–Arg164 cata-lytic dyad, which forms a hydrogen bond network with the watercoordinated to the Zn(II) ion, enhancing its nucleophilicity;7b,13e

and (iii) a cluster of hydrophobic amino acid residues consideredto be involved in the binding of the substrate (and inhibitors),including those in position 151, 179 and 184.7b,13e Indeed, Q151,F179 and V184 are conserved in FbiCA 1 and in almost all theeukaryotic enzymes used in the alignment, irrespective of theirsource (plant, yeast, fungus).

Figure 2 presents a phylogenetic analysis of selected b-CAs forwhich predicted amino acid sequence is available in data bases.We included sequences from a number of plants, algae, yeasts,fungi, and bacteria, even if the corresponding enzymes werenot yet characterized completely, to get an overview on the phy-logenetic relationship of this family of CAs. FbiCA 1 clusters withcorresponding enzymes from the other land plants, and mosttightly with the CAs from other dicotyledonous species (Pisumsativum, Vigna radiata, Arabidopsis thaliana). The enzymes fromthe fungi Trichosporon asahii (TasCA) and Sordaria macrospora(SmaCA) are sister to the plant CAs. Some algal, bacterial andfungal enzymes cluster in a sister clade while the yeast CAsand most of the bacterial enzymes are more distantly relatedto FbiCA1. It is interesting to note that the only arthropod b-CA characterized so far, the enzyme from Drosophila melanogas-ter DmeCA,21 clustered with the bacterial and archaeal enzymes.This may not be so much surprising considering the fact thatDmeCA is a mitochondrial enzyme,22 and mitochondria origi-nated from bacteria which have been ‘captured’ by other cellsduring evolution of the Eukaryotes.

Table 2 shows the inhibition data for FbiCA 1 with respect to arather wide range of simple and complex inorganic anions as wellas several small molecules observed in previous studies to act asCA inhibitors (CAIs).5,6,20,21 The analogous data for the inhibitionof hCA II and the bacterial enzyme SspCA (from the extremophileSulfurihydrogenibium yellostonense) 5c—belonging to the a-CAclass—as well as HpyCA and SceCA (b-CAs) are provided for com-parison. The inhibition profile of FbiCA 1 is distinct from that ofa- or other b-CAs as follows:

(i) The anions with the lowest propensity to coordinate metalions, perchlorate and tetrafluroborate, were not inhibitorsof FbiCA 1 up to concentrations of 200 mM. The same is truefor the human isoform hCA II, as well as the bacterial/yeastenzymes tested (Table 2).

(ii) Several other anions, such as hydrogensulfite, selenate, perr-hutenate and iminidisulfonate were also weak inhibitors ofthe plant enzyme FbiCA 1, with inhibition constants in therange of 24.5–55.3 mM. It should be noted that all of thesecompounds are much better inhibitors of the a- or otherb-CAs investigated earlier.6a

(iii) Most of the investigated anions, including the halides,pseudohalides (cyanide, cyanate, thiocyanate, azide), bicar-bonate, carbonate, nitrate, nitrite, hydrogensulfide, stannate,tellurate, diphosphate, divanadate, tetraborate, perrhenate,peroxydisulfate, selenocyanide, sulfate, and fluorosulfate,inhibited FbiCA 1 in the sub-millimolar range, with KIs of0.46–0.93 mM. This is a rather unexpected, flat inhibitionprofile, considering the data for the a- and b-CAs shown inTable 2. For example, hydrogensulfide was 21.5 times a bet-ter hCA II than FbiCA 1 inhibitor. The small variation in inhi-bition of FbiCA 1 seen between the halides andpseudohalides is interesting and contrasts with thatobserved for other enzymes, especially those belonging tothe a-class, for example, between fluoride and iodide, orbetween the light halogenides (F�, Cl�) and pseudohaloge-nides, such as cyanide, cyanate or azide (Table 2). Thus,anions known to easily complex metal ions in solution (orwithin the active sites of metalloenzymes), such as cyanide,azide, thiocyanate, or anions with less strong such properties

1630 S. M. Monti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1626–1630

(e.g., sulfate, fluorosulfate) showed similar inhibitory effectson FbiCA 1. The significance of this activity is not clear at thistime, largely because of the lack of an X-ray crystal structureof the plant enzyme.

(iv) A group of anions/molecules, such as trithiocarbonate, dieth-yldithiocarbamate, sulfamide, sulfamate, phenylboronic acidand phenylarsonic acid, showed low micromolar affinity forFbiCA 1, with KIs in the range of 4.0–60 lM. The best inhib-itors of FbiCA 1 were sulfamide, sulfamate and phenylarson-ic acid, with KIs of 4–6 lM (Table 2). Some of thesecompounds, for example, phenylboronic acid, are muchweaker inhibitors of the yeast and human enzymes SceCA/hCA II, but they effectively inhibited other such enzymes(SspCA, HpyCA).

In conclusion, we report the first detailed biochemical charac-terization of a b-CA isoform, FbiCA 1, from the plant F. bidentis,which has been cloned, purified and characterized as recombinantprotein. FbiCA 1 has an interesting CO2 hydrase catalytic activity(kcat of 1.2 � 105 and kcat/Km of 7.5 � 106 M�1 � s�1) and was mod-erately inhibited by most simple/complex inorganic anions. Morepotent FbiCA 1 inhibitors were also detected, such as trithiocar-bonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenyl-boronic acid and phenylarsonic acid (KIs in the range of 4–60 lM). Such inhibitors may be used as tools to better understandthe role of various b-CA isoforms in photosynthesis.

Acknowledgments

This work was financed in part by two FP7 EU project (Metoxiaand Dynano), by a Grant from the ‘Accordo di Programma, CNR-MSE, PAR 2009-2010’.

References and notes

1. (a) Cousins, A. B.; Badger, M. R.; von Cammerer, S. Plant Physiol. 2006, 141, 232;(b) Bertucci, A.; Moya, A.; Tambutté, S.; Allemand, D.; Supuran, C. T.; Zoccola, D.Bioorg. Med. Chem. 2012. http://dx.doi.org/10.1016/j.bmc.2012.10.024; (c)Sinetova, M. A.; Kupriyanova, E. V.; Markelova, A. G.; Allakhverdiev, S. I.;Pronina, N. A. Biochim. Biophys. Acta 2012, 1817, 1248.

2. (a) Ludwig, M. Plant Cell Environ. 2012, 35, 22; (b) Ludwig, M. J. Exp. Bot. 2011,62, 3071; (c) Tanz, S. K.; Tetu, S. G.; Vella, N. G.; Ludwig, M. Plant Physiol. 2009,150, 1515; (d) Tetu, S. G.; Tanz, S. K.; Vella, N.; Burnell, J. N.; Ludwig, M. PlantPhysiol. 2007, 144, 1316.

3. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem.Rev. 2012, 112, 4421; (b) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (c)Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3233; (d) Neri, D.; Supuran, C. T. Nat.Rev. Drug Disc. 2011, 10, 767.

4. (a) Eriksson, A. E.; Jones, T. A.; Liljas, A. Proteins 1988, 4, 274; (b) Pastorekova,S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19,199; (c) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146.

5. (a) Smith, K. S.; Ferry, J. G. FEMS Microbiol. Rev. 2000, 24, 335; (b) Cronk, J. D.;Rowlett, R. S.; Zhang, K. Y.; Tu, C.; Endrizzi, J. A.; Lee, J.; Gareiss, P. C.; Preiss, J. R.Biochemistry 2006, 45, 4351; (c) De Luca, V.; Vullo, D.; Scozzafava, A.; Rossi, M.;Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 5630.

6. (a) De Simone, G.; Supuran, C. T. J. Inorg. Biochem. 2012, 111, 117; (b) Supuran,C. T. Front. Pharmacol. 2011, 2, 34; (c) Supuran, C. T. Future Med. Chem. 2011, 3,1165; (d) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467.

7. (a) Domsic, J. F.; Avvaru, B. S.; Kim, C. U.; Gruner, S. M.; Agbandje-McKenna, M.;Silverman, D. N.; McKenna, R. J. Biol. Chem. 2008, 283, 30766; (b) Schlicker, C.;Hall, R. A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C. T.; Mühlschlegel, F.A.; Steegborn, C. J. Mol. Biol. 2009, 385, 1207; (c) Zimmerman, S. A.; Ferry, J. G.;Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 901.

8. (a) Xu, Y.; Feng, L.; Jeffrey, P. D.; Shi, Y.; Morel, F. M. Nature 2008, 452, 56; (b)Viparelli, F.; Monti, S. M.; De Simone, G.; Innocenti, A.; Scozzafava, A.; Xu, Y.;

Morel, F. M. M.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 4745; (c)Alterio, V.; Langella, E.; Viparelli, F.; Vullo, D.; Ascione, G.; Dathan, N. A.; Morel,F. M. M.; Supuran, C. T.; De Simone, G.; Monti, S. M. Biochimie 2012, 94, 1232.

9. Aizawa, K.; Miyachi, S. FEMS Microbiol. Rev. 1986, 39, 215.10. (a) Ferrier, J. M. Plant Physiol. 1980, 66, 1198; (b) Badger, M. R.; Price, G. D.

Annu. Rev. Plant Physiol. Plant Mol. Biol. 1994, 45, 369.11. Zabaleta, E.; Martin, M. V.; Braun, H. P. Plant Sci. 2012, 187, 97.12. (a) Suzuki, K.; Yang, S. Y.; Shimizu, S.; Morishita, E. C.; Jiang, J.; Zhang, F.;

Hoque, M. M.; Sato, Y.; Tsunoda, M.; Sekiguchi, T.; Takénaka, A. ActaCrystallogr., Sect. D Biol. Crystallogr. 2011, 67, 894; (b) Burén, S.; Ortega-Villasante, C.; Blanco-Rivero, A.; Martínez-Bernardini, A.; Shutova, T.; Shevela,D.; Messinger, J.; Bako, L.; Villarejo, A.; Samuelsson, G. PLoS ONE 2011, 6,e21021; (c) Tsikou, D.; Stedel, C.; Kouri, E. D.; Udvardi, M. K.; Wang, T. L.;Katinakis, P.; Labrou, N. E.; Flemetakis, E. Biochim. Biophys. Acta 2011, 1814,496.

13. (a) Huang, S.; Hainzl, T.; Grundström, C.; Forsman, C.; Samuelsson, G.; Sauer-Eriksson, A. E. PLoS ONE 2011, 6, e28458; (b) Long, B. M.; Rae, B. D.; Badger, M.R.; Price, G. D. Photosynth. Res. 2011, 109, 33; (c) Kupriyanova, E. V.; Sinetova,M. A.; Markelova, A. G.; Allakhverdiev, S. I.; Los, D. A.; Pronina, N. A. J.Photochem. Photobiol., B. 2011, 103, 78; (d) Tems, U.; Burnell, J. N. Plant Physiol.Biochem. 2010, 48, 945; (e) Kimber, M. S.; Pai, E. F. EMBO J. 2000, 19, 1407.

14. (a) Klodmann, J.; Braun, H. P. Phytochemistry 2011, 72, 1071; (b) Gawryluk, R.M.; Gray, M. W. BMC Evol. Biol. 2010, 10, 176.

15. (a) Nair, S. K.; Ludwig, P. A.; Christianson, D. W. J. Am. Chem. Soc. 1994, 116,3659; (b) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.;Supuran, C. T. J. Med. Chem. 2010, 53, 5511.

16. (a) Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.; Mühlschlegel, F. A.; Vullo,D.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1682; (b)Martin, D. P.; Cohen, S. M. Chem. Commun. 2012, 48, 5259.

17. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An applied photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydrationactivity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5,for a-CAs) or 20 mM TRIS (pH 8.3 for the b-CAs) as buffers, and 20 mM NaClO4

(for maintaining constant the ionic strength), following the initial rates of theCA-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 thekinetic parameters and inhibition constants. For each inhibitor at least sixtraces of the initial 5–10% of the reaction have been used for determining theinitial velocity. The uncatalyzed rates were determined in the same mannerand subtracted from the total observed rates. Stock solutions of inhibitor(10 mM) were prepared in distilled–deionized water and dilutions up to0.01 lM were done thereafter with the assay buffer. Inhibitor and enzymesolutions were preincubated together for 15 min at room temperature prior toassay, in order to allow for the formation of the E–I complex or for the eventualactive site mediated hydrolysis of the inhibitor. The inhibition constants wereobtained by non-linear least-squares methods using PRISM 3, as reportedearlier,15b,16a and represent the mean from at least three differentdeterminations. All CA isoforms were recombinant ones obtained in-house asreported earlier.15b,16a.

18. A truncated version of FbCAI, without the chloroplast transit peptide, to renderit more stable, was cloned in pETM11 (EMBL), with an N-terminal 6�His tag.Recombinant protein was expressed in BL21(DE3) 16 h at 20 �C, 225 rpm.Purification was performed on 1 ml HisTrapFF (GE Healthcare) in 20 mM Tris,8.0, 500 mM NaCl, 1 mM DTT, TEV-digested 20 h in dialysis at 4 �C and theresulting cleaved protein passed over MonoQ in a linear salt gradient, at pH 8.0.Purity and homogeneity of FbCA 1 was assessed by SDS/PAGE and LC-ESI-MS.

19. (a) Mogensen, E. G.; Janbon, G.; Chaloupka, J.; Steegborn, C.; Fu, M. S.; Moyrand,F.; Klengel, T.; Pearson, D. S.; Geeves, M. A.; Buck, J.; Levin, L. R.; Mühlschlegel,F. A. Eukaryot. Cell 2006, 5, 103; (b) Bahn, Y. S.; Mühlschlegel, F. A. Curr. Opin.Microbiol. 2006, 9, 572; (c) Innocenti, A.; Mühlschlegel, F. A.; Hall, R. A.;Steegborn, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18,5066.

20. (a) Isik, S.; Kockar, F.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A.; Supuran, C. T.Bioorg. Med. Chem. Lett. 2008, 18, 6327; (b) Isik, S.; Kockar, F.; Aydin, M.; Arslan,O.; Ozensoy Guler, O.; Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17,1158.

21. (a) Nishimori, I.; Minakuchi, T.; Kohsaki, T.; Onishi, S.; Takeuchi, H.; Vullo, D.;Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 3585; (b)Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C. T. Curr. Pharm. Des. 2008, 14,622; (c) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C.T. J. Enzyme Inhib.Med. Chem. 28, in press.

22. Syrjänen, L.; Tolvanen, M.; Hilvo, M.; Olatubosun, A.; Innocenti, A.; Scozzafava,A.; Leppiniemi, J.; Niederhauser, B.; Hytönen, V. P.; Gorr, T. A.; Parkkila, S.;Supuran, C. T. BMC Biochem. 2010, 11, 28.