Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

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Bioorganic & Medicinal Chemistry

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

Anion inhibition study of the b-carbonic anhydrase (CahB1) from thecyanobacterium Coleofasciculus chthonoplastes (ex-Microcoleuschthonoplastes)

http://dx.doi.org/10.1016/j.bmc.2014.01.0260968-0896/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +39 055 4573005.E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).

Please cite this article in press as: Vullo, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.01.026

Daniela Vullo a, Elena V. Kupriyanova b, Andrea Scozzafava a, Clemente Capasso c, Claudiu T. Supuran a,d,⇑a Università degli Studi di Firenze, Dipartmento di Chimica Ugo Schiff, Via della Lastruccia 3, Rm. 188, 50019 Sesto Fiorentino (Florence), Italyb K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Str., Moscow 127276, Russiac Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italyd Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutriceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy

a r t i c l e i n f o

Article history:Received 9 December 2013Revised 15 January 2014Accepted 17 January 2014Available online xxxx

Keywords:Carbonic anhydraseb-ClassAnionEnzyme inhibitorCyanobacteriumCarbon concentrating mechanismCO2 capture

a b s t r a c t

We investigated the catalytic activity and inhibition of the b-class carbonic anhydrase (CA, EC 4.2.1.1)CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominatedMicrocoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO2 hydration, witha kcat of 2.4 � 105 s�1 and a kcat/Km of 6.3 � 107 M�1 s�1. A range of inorganic anions and small moleculeswere investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme(KIs >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with KIs of29.9–48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a rangeof heavy metal ions-containing anions were submillimolar–millimolar inhibitors (KIs in the range of0.15–0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide,phenylboronic acid and phenylarsonic acid, with KIs in the range of 8–75 lM, whereas acetazolamideinhibited the enzyme with a KI of 76 nM. This is the first kinetic and inhibition study of a cyanobacterialCA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentratingmechanism which assures substrate to RubisCO for the CO2 fixation by these organisms, a detailedkinetic/inhibition study may be essential for a better understanding of this superfamily of metalloen-zymes and for potential biotechnological applications in biomimetic CO2 capture processes.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction possesses activity for the CO2 hydration reaction (but no kinetic

Cyanobacteria, a phylum of bacteria which obtains energythrough photosynthesis, are among the most ancient organismson Earth, being documented already 3.5 billion years ago, beforethe expansion of eukaryotes.1–3 Through photosynthesis, they con-tributed to the formation of O2 in the terrestrial atmosphere, whichdramatically changed the forms of life on our planet, from thoseadapted to the primeval, reducing atmosphere to organisms ableto survive and thrive in oxidizing conditions.1–3 Chloroplasts,found in plants and algae (eukaryotes) probably also evolved fromcyanobacterial ancestors via endosymbiosis.4

Recently, one of our groups5 identified a b-class carbonic anhy-drase (CA, EC 4.2.1.1), CahB1, in the relict alkaliphilic cyanobacte-rium Coleofasciculus chthonoplastes (previous name Microcoleuschthonoplastes).6 This enzyme was cloned, expressed in Escherichiacoli and purified as recombinant protein, being shown that it

parameters were reported), that it is inhibited by the sulfonamideethoxzolamide, and its localization in the cell envelope of the cya-nobacterium has also been demonstrated.5

CahB1, as many other such CAs present in cyanobacteria/plantsare of great interest due to their involvement in the carbon concen-trating mechanisms (CCMs) used by many aquatic organisms (cya-nobacteria, diatoms, algae, etc.) to increase the CO2 concentrationat the site of fixation by D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), several-folds over its external concentra-tion.1,7,8 In fact RubisCO is a rather inefficient enzyme, as it has alow affinity for CO2, possesses a low catalytic rate, and O2 can actas an alternative substrate (to CO2).1 These inefficiencies of Rubi-sCO were ‘corrected’ during the evolution of life on earth by the ad-vent of mechanisms such as the CCM mentioned above, as well asby the encapsulation of RubisCO within carboxysomes togetherwith CAs which supply CO2 to the first enzyme and promote thecarboxylase reaction, suppressing at the same time the oxygenaseone.1,7,8 As around 50% of all CO2 on earth is converted into organiccompounds by cyanobacteria,1 the study of the enzymes involved

2 D. Vullo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

in these processes is crucial, also for the possible design of biomi-metic CO2 capture processes.9

CAs are in fact widepread in all life kingdoms, with five geneticfamilies known to date, the a-, b-, c-, d- and f-CAs.10–16 These en-zymes have as substrate a very simple molecule, CO2, which is effi-ciently converted into bicarbonate and protons. The reason whysuch enzymes are needed in all life kingdoms is that the spontane-ous CO2 hydration reaction is very slow at pH values at which mostorganisms live.16–18 Furthermore, some CAs possess also a certaincatalytic versatility, acting as esterases, phosphatases and even sul-fatases.19,20 Their inhibition was thoroughly investigated, at leastfor the a-CAs present in mammals,10 including humans, sincemany of the 15 isoforms known to date are drug targets forobtaining diuretics,10a,22b antiglaucoma,22c,d antiepileptic,14 anti-obesity15a,b or antitumor agents.15c

In this paper we report the kinetic characterization of CahB1,the interesting b-CA from the cyanobacterium C. chthonoplastes,5

as well as its inhibition profile with anions and other small mole-cules known to interact with zinc enzymes such as sulfamide,phenylboronic acid or phenylarsonic acid, investigated as CA inhib-itors (CAIs) against other such enzymes.21

2. Results and discussion

2.1. Kinetic properties of CahB

We have cloned and purified CahB1 by the method previouslydescribed by Kupriyanova et al.5a The enzyme contains 271 aminoacid residues and has a molecular weight of 30.4 kDa. The purifiedenzyme was used thereafter to measure the kinetic properties atpH 8.3, by a stopped flow assay method monitoring the physiologicreaction, that is, CO2 hydration to bicarbonate and protons.17

As shown from data of Table 1, in which other CAs belonging totwo genetic families (a- and b-) were included, CahB1 has efficientcatalytic activity, with a a kcat of 2.4 � 105 s�1 and kcat/Km of6.3 � 107 M�1 s�1. The human cytosolic isozymes hCA I and II, to-gether with the b-class fungal enzyme Can2 from Cryptococcus neo-formans,13b CalCA from Candida albicans,21d SceCA fromSaccharomyces cerevisiae,13c and FbiCA1 from the plant Flaveriabidentis,18 and CahB1 were included in the kinetic analysis for com-parison reasons (Table 1). Comparing the kcat/Km and kcat values ofthe enzymes from Table 1, it may be observed that CahB1 has amedium catalytic efficiency, comparable to that of hCA I andCan2, enzymes known to play important physiological functionsin humans and fungi, respectively.10,12e,13b,c The corresponding en-zymes from S. cerevisiae,13c and C. albicans,21d were slightly moreactive than CahB1, whereas the plant enzyme FbiCA 1 has a lowercatalytic activity compared to the cyanobacterial enzyme. hCA IIwas the most effective catalyst among the examined CAs, beingalso one of the most active enzymes known to date.10 Furthermore,CahB1 activity has been inhibited by acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) with a KI of 76 nM, confirming

Table 1Kinetic parameters for the CO2 hydration reaction catalysed by the human cytosolic isozymNa2SO4, and the b-CAs Can2, CalCA (from C. neoformans and C. albicans, respectively), SceCA8.3 in 20 mM TRIS buffer and 20 mM NaClO4

17

Isozyme Activity level kcat (s�1) kcat/K

hCA I Moderate 2.0 � 105 5.0 �hCA II Very high 1.4 � 106 1.5 �Can2 Moderate 3.9 � 105 4.3 �CalCA High 8.0 � 105 9.7 �SceCA High 9.4 � 105 9.8 �FbiCA 1 Low 1.2 � 105 7.5 �CahB1 Moderate 2.4 � 105 6.3 �

Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-t

Please cite this article in press as: Vullo, D.; et al. Bioorg. Med. Chem. (2

thus the earlier data regarding the sensitivity of this enzyme toinhibition by the sulfonamide CAI ethoxzolamide.5a These two sul-fonamides are in fact efficient inhibitors of all five classes of CAsknown to date, including the a- and b-CAs from Table 1.10–15

2.2. Sequence homology and phylogenetic analysis of CahB1

Kupriyanova et al.5a analyzed the sequence homology of CahB1with that of other b-CAs from cyanobacteria (e.g., Synechocystisspp., two such enzymes were considered, CcaA and IcfB), thebacterium E. coli (CynT enzyme) as well as the ArchaeonMethanobacterium thermoautotrophicum.12 Here we extend theprevious analysis, including yeast (C. albicans, S. cereviasiae)13 andplant (F. bidentis)18 such enzymes (Fig. 1).

As seen from data of Figure 1, CahB1 possesses, as all otherb-CAs described and characterized so far, all the structuralelements crucial for being able to perform efficiently CO2 hydrationto bicarbonate and protons: (i) the three zinc ligands, Cys39, His98and Cys101 (Fig. 1) which bind the metal ion in a tetrahedralgeometry, with the fourth ligand being a water molecule/hydroxide ion; (ii) the catalytic dyad, constituted by Asp41 andArg43, which is involved in the generation of the zinc hydroxide,nucleophilic species of the enzyme, and (iii) a not yet identifiedproton shuttling residue, which assures the high turnover of theseenzymes, avoiding accumulation of he reaction products withinthe enzyme active site.

It may be also observed that CahB1 has a length intermediatebetween the long, plant CA FbiCA 1 (330 amino acid residues)and the shorted fungal enzymes (Can2 for example has 243 resi-dues compared to the 271 of CahB1). It can be observed that FbiCA1 has a quite long amino terminal polypeptide which is absent inthe cyanobacterial or fungal enzymes, whereas the cyanobacterialprotein has a loop (towards the carboxy-terminal end) which is ab-sent both in the plant and the fungal b-CAs (Fig. 1).

In Figure 2 a phylogenetic tree of b-CAs of bacterial, cyanobac-terial, plant, algal and yeast/fungal origins is presented, in order totry to identify the evolutionary relationship of CahB1 with othersuch enzymes widespread in many types of organisms. It may beobserved that some bacterial b-CAs, such as those from Porphyro-monas gingivalis, Myroides injenensis, and Acinetobacter baumanniiare highly diverse (upper branch of the tree) from all other b-CAsof cyanobacterial, plant, algal and yeast origin (lower branches),although some bacterial/archaeal enzymes clustered together withthe eukaryotic b-CAs (e.g., Burkholderia thailandensis, Brucella suis,M. thermoautotrophicum). It should however be noted that thetwo cyanobacterial enzymes considered here, CahB1 and CcaA(from Synechocystis spp.) did not cluster with any of the plant,yeast or bacterial/archaeal CAs, but have their own distinct branch.Furthemore, they are not clusteriung together but they each pos-sess their own branch (Fig. 2) proving that the two cyanobacteriallineages (and their corresponding b-CAs) are not evolutionarilyhighly related to each other.

es hCA I and II (a-class CAs) at 20 �C and pH 7.5 in 10 mM HEPES buffer and 20 mM(from S. cerevisiae), the Flaveria bidentis CA (FbiCA 1) and CahB1 measured at 20 �C, pH

m (M�1 � s�1) KI (Acetazolamide) (nM) Ref.

107 250 10108 12 10107 10.5 13b107 132 21d107 82 13c106 27 18107 76 This work

hiadiazole-2-sulfonamide) are also provided.

014), http://dx.doi.org/10.1016/j.bmc.2014.01.026

CahB_cyano ------------------------------------------------------------ Can2_yeast ------------------------------------------------------------ SceCA_yeast ------------------------------------------------------------ FbiCA_plan MSAASAFAMNAPSFVNASSLKKASTSARSGVLSARFTCNSSSSSSSSATPPSLIRNEPVF

CahB_cyano ------------------------------------------------------------ Can2_yeast -----------------------------PLGSMPFHAEPLKPSDEIDMDLGHSVAAQKF SceCA_yeast --------------------------------------MSATESSSI-FTLSHN------ FbiCA_plan AAPAPIITPNWTEDGNESYEEAIDALKKTLIEKGELEPVAATRIDQITAQAAAPDTKAPF

39 41 43CahB_cyano --MKKLLKGLRQFKSNYFSTHQELFEQL-SHGQKPRVLFITCSDSRIDPNLITNAEVGEL Can2_yeast KEIREVLEGNRYWARKVTSEEPEFMAEQ-VKGQAPNFLWIGCADSRVPEVTIMARKPGDV SceCA_yeast SNLQDILAANAKWASQMNNIQPTLFPDHNAKGQSPHTLFIGCSDSRYNEN-CLGVLPGEV FbiCA_plan DPVERIKSGFVKFKTEKFVTNPALYDEL-AKGQSPKFMVFACSDSRVCPSHVLDFQPGEA : : . : : : : :** *. : : *:*** *:

98 101 CahB_cyano FVIRNAGNIIPPFGAAN-GGEGAAVEYAVQALGVEHIIVCGHSHCGAMKGLL--KRSSLE Can2_yeast FVQRNVANQFKPEDDSS----QALLNYAIMNVGVTHVMVVGHTGCGG---CIAAFDQPLP SceCA_yeast FTWKNVANICHSEDLTL----KATLEFAIICLKVNKVIICGHTDCGGIKTCLTNQREALP FbiCA_plan FVVRNVANMVPPFDKTKYSGVGAAVEYAVLHLKVQEIFVIGHSRCGGIKGLM-----TFP *. .*..* . . : * :::*: : * ::: **: **. : .:

CahB_cyano DEMP----LVYEWLRHA--EATRQLLKENYSHLEGETLLEVAVAE-NVLTQIENLRTYPV Can2_yeast TEENPGGTPLVRYLEPI--IRLKHSLPE-GSDVND------LIKE-NVKMAVKNVVNSPT SceCA_yeast KVNC---SHLYKYLDDI--DTMYHEESQNLIHLKTQREKSHYLSHCNVKRQFNRIIENPT FbiCA_plan DEGPHSTDFIEDWVKVCLPAKSKVVAEHNGTHLDDQCVL--CEKE-AVNVSLGNLLTYPF : :: :. * . .: *

CahB_cyano IHSKLYR------NKLHLHAWIYHIETGEVLEYDSIRHDFVTPDENAMLEAYPVQCVLSL Can2_yeast IQGAWEQARKGEFREVFVHGWLYDLSTGNIVD---------------------------- SceCA_yeast VQTAVQN------GELQVYGLLYNVEDGLLQT---------------------------- FbiCA_plan VRDGLRN------KTLALKGGHYDFVNGTFEL---------------------------- :. . : : . * . * .

CahB_cyano PEDVEELVPGHRQSSAPAQTEVAAADRDAKMPWLSSEQVARIHGGSNGRR Can2_yeast --------------------------------LNVTQGPHPFVDDRVPRA SceCA_yeast --------------------------------VSTYTKVTPK-------- FbiCA_plan --------------------------------WALDFGLSSPTSV-----

Figure 1. Alignment of the amino acid sequences of selected b-CAs from plant, yeast, and cynobacterial organisms. Coleofasciculus chthonoplastes numbering system wasused. The zinc ligands are indicated in blue, whereas the catalytic dyad is shown in red. The asterisk (⁄) indicates identity at a position; the symbol (:) designates conservedsubstitutions, while (.) indicates semi-conserved substitutions. Multiple alignment was performed with the program Clustal W, version 2.1. Organisms and accessionnumbers used for the alignment are indicated in Figure 2.

Figure 2. Phylogenetic tree of the b-CAs from bacteria, cyanobacteria, fungi, algae and plants. The tree was constructed using the program PhyML 3.0. Organisms andaccession numbers in the table attached on the right hand of the figure follow the same order of the cryptonyms used in the phylogenetic tree.

D. Vullo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx 3

2.3. Inhibition studies of CahB

Anions and other small molecules, such as sulfamide, phenylbo-ronic acid or phenylarsonic acid were much investigated as CAIs asthey bind to the metal ion from the enzyme active site and impaircatalysis.10,12–16,20,21 Enzymes belonging to the a-, b-, c-, d andf-CA families, from a variety of organisms, from diatoms, corals,sponges, bacteria, plants and mammals have been investigatedup until now for their inhibition profiles with this class of smallmolecule, anion inhibitor.10,12–16,20–23 Here we investigated forthe first time the inhibition profile of a b-CA from a cyanobacteriawith anions and other small molecules (Table 2).

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Data of Table 2 show the inhibition of CahB1 with a range ofinorganic anions, some of which are abundant in sea water orthe soda lakes in which C. chthonoplastes lives (e.g., chloride, sul-fate, bromide, iodide, etc.). Other investigated anions belong tothe so-called metal poisons (cyanide, thiocyanate, azide, hydrogensulfide) whereas others (sulfamide, sulfamic acid, phenyl boronic/arsonic acids) are known to inhibit CAs belonging to other classes.The metal-binding function present in some of these compounds,the SO2NH� fragment, is in fact present in the main class of CAIs,the sulfonamides and their bioisosteres (sulfamides, sulfamates).10

The following should be noted regarding CahB1 inhibition data ofTable 2 (the inhibition data of the a-class human isoform hCA II,

014), http://dx.doi.org/10.1016/j.bmc.2014.01.026

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, the plant Flaveria bidentis isoform 1 (FbiCA 1), and the CahB1 enzyme at 20 �C,pH 8.3, by a stopped flow CO2 hydrase assay17

Inhibitor§ KI# (mM)

hCA IIa SspCAb HpyCAc FbiCA 1d CahB1e

F� >300 41.7 0.67 0.71 0.53Cl� 200 8.30 0.56 0.74 0.74Br� 63 49.0 0.38 0.67 0.63I� 26 0.86 0.63 0.71 0.66CNO� 0.03 0.80 0.37 0.93 0.35SCN� 1.60 0.71 0.68 0.83 0.42CN� 0.02 0.79 0.54 0.62 0.44N�3 1.51 0.49 0.80 0.46 0.47HCO�3 85 33.2 0.50 0.66 0.61

CO2�3

73 39.3 0.42 0.84 0.68

NO�3 35 0.86 0.78 0.78 1.86NO�2 63 0.48 0.67 0.57 1.38HS� 0.04 0.58 0.58 0.86 0.26HSO�3 89 21.1 0.63 55.3 0.51

SnO2�3

0.83 0.52 0.48 0.53 0.59

SeO2�4

112 0.57 0.65 24.5 29.9

TeO2�4

0.92 0.53 0.45 0.90 0.41

P2O4�7

48.50 0.69 0.75 0.83 0.90

V2O4�7

0.57 0.66 0.18 0.66 0.40

B4O2�7

0.95 0.67 0.68 0.86 5.4

ReO�4 0.75 0.80 0.82 0.52 4.3RuO�4 0.69 0.69 1.10 26.1 7.6

S2O2�8

0.084 84.6 0.93 0.87 0.79

SeCN� 0.086 0.07 0.97 0.88 48.6

CS2�3

0.0088 0.06 0.21 0.06 0.15

Et2NCS�2 3.1 0.004 0.0074 0.008 0.008

SO2�4

>200 0.82 0.57 0.62 7.9

ClO�4 >200 >200 6.50 >200 >200BF�4 >200 >200 > 200 >200 >200FSO�3 0.46 0.73 0.75 0.69 6.8

NHðSO3Þ2�20.76 0.75 0.70 50.9 0.73

H2NSO2NH2 1.13 0.009 0.072 0.004 0.009H2NSO3H 0.39 0.042 0.094 0.005 0.010Ph-B(OH)2 23.1 0.041 0.073 0.008 0.075Ph-AsO3H2 49.2 0.005 0.092 0.006 0.008

§ As sodium salt.# Errors were in the range of 3–5% of the reported values, from three different

assays.a From Ref. 6a.b From Ref. 5c.c From Ref. 20c.d From Ref. 19a.e This work.

4 D. Vullo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

as well as the bacterial one from Sulfurihydrogenibium yellowston-ensis SspCA,14e and the b-CAs from Helicobacter pylori13a and theplant F. bidentis18 are also included in Table 2 for comparison rea-sons, as they were reported earlier by our group,10,13,14,18 workingin the same conditions as those used to screen the CahB1inhibitors):

(i) A first group of anions, including tetrafluoroborate and per-chlorate, showed ineffective inhibition of CahB1, with noinhibitory activity observed up to concentrations as high as200 mM (Table 2). This is not so much unexpected, as theseanions show lower affinity for metal ions in solution ormetalloenzyme (see also the data for all other a- andb-CAs from Table 2).

(ii) Other anions with low inhibitory activity towards CahB1were selenate and selenocyanide, which showed KIs in therange of 29.9–48.61 mM (Table 2). Selenate is also a weakhCA II and FbiCA 1 ihibitor, whereas other such enzymes,among which SspCA and HpyCA are inhibited in the submil-limolar range by this anion. Selenocyanide on the other hand

Please cite this article in press as: Vullo, D.; et al. Bioorg. Med. Chem. (2

is an effective CAI for all the enzymes from Table 2 exceptCahB1.

(iii) The following anions showed millimolar affinity for CahB1:nitrate, nitrite, tetraborate, perrhenate, perruthenate, sulfateand fluorosulfonate, with KIs in the range of 1.38–7.9 mM(Table 2). The most interesting case is constituted by sulfate,which has no affinity for hCA II but is a submillimolar CAI forother bacterial (SspCA and HpyCA) or plant (FbiCA 1)enzymes (Table 2).

(iv) Anions showing submillimolar affinity for CahB1 were: thehalides and pseudohalides such as cyanate, thiocyanate, cya-nide, azide, but also bicarbonate, hydrogen sulfide, hydrogensulfite, with KIs in the range of 0.26–0.74 mM (Table 2). Themost surprinsing fact is that all these quite diverse anionsshow a very similar behavior of medium potency CahB1inhibitors, even the halidfes and bicarbonate/carbonate,which presumably are abundant in the environmentalniches where C. chthonoplastes lives. Furthermore, a largenumber of heavy metal ion-containing anions, such as stan-nate, tellurate, and pyrovanadate also belong to this cate-gory of medium potency CahB1 inhibitors, with KIs in therange of 0.40–0.59 mM. Pyrophosphate, peroxydisulfate, tri-thiocarbonate, and iminodisulfonate are also mediumpotency CahB1 inhibitors, with KIs ranging from 0.15 to0.90 mM (Table 2). It may be observed that these are ratherheterogeneous anions and no regularities for the inhibitorypower were evidenced (as for example for the halide inhibi-tion of hCA I and II, a case in which the inhibitory powerincreased with the increase of the atomic weight of the hal-ogen). For CahB1 not clear trend of this type was observed,and even the anions with potent complexing properties suchas cyanide and azide showed comparable inhibition with thehalides (Table 2).

(v) The most potent CahB1 inhibitors were N,N-diethyldithio-carbamate, sulfamate, sulfamide, phenylboronic acid andphenylarsonic acid, which had KIs in the range of 8–75 lM.Together with acetazolamide (Table 1), which inhibited theenzyme with a KI of 76 nM, these were the most potentinhibitors detected so far for the cyanobacterial enzymestudied here. N,N-diethyldithiocarbamate is known to havehigh affinity for metal ions and to act as potent CAI forenzymes belonging to all other CA classes, so this is nowconfirmed also for the cyanobacterial enzyme. Sulfamideand sulfamate (KIs of 9 and and 10 lM, respectively) incor-porate the SO2NH� zinc binding group found in the mainclass of CAIs with clinical applications, that is, the sulfona-mides.10 This study thus confirms that CahB1 is also inhib-ited by this class of pharmacological agents and similarcompounds, such as phenylboronic acid, with a KI of75 lM,or phenylarsonic acid, with a KI of 8 lM (equipotentto N,N-diethyldithiocarbamate).

3. Conclusions

In conclusion, we report here the kinetic characterization ofCahB1, a cyanobacterial b-CA from C. chthonoplastes. The enzymewas an efficient catalysts for the CO2 hydration, physiological reac-tion, with a kcat of 2.4 � 105 s�1 and a kcat/Km of 6.3 � 107 M�1 s�1.A range of inorganic anions and small molecules were investigatedas inhibitors of CahB1. Perchlorate and tetrafluoroborate did notinhibit the enzyme (KIs >200 mM), with selenate and selenocya-nide being ineffective inhibitors too, with KIs of 29.9–48.61 mM.The halides, pseudohalides, carbonate, bicarbonate, trithiocarbon-ate and a range of heavy metal ions containing anions were subm-illimolar–millimolar inhibitors (KIs in the range of 0.15–0.90 mM).The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfa-

014), http://dx.doi.org/10.1016/j.bmc.2014.01.026

D. Vullo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx 5

mate, sulfamide, phenylboronic acid and phenylarsonic acid, withKIs in the range of 8–75 lM, whereas acetazolamide inhibited theenzyme with a KI of 76 nM. This is the first kinetic and inhibitionstudy of a cyanobacterial CA. As these enzymes are widespreadin many cyanobacteria, being crucial for the carbon concentratingmechanism which assures substrate to RubisCO for the CO2 fixa-tion by these organisms, a detailed kinetic/inhibition study maybe essential for a better understanding of this superfamily ofmetalloenzymes and for potential biotechnological applicationsin biomimetic CO2 capture processes.

4. Experimental

4.1. CahB1 cloning and purification

The enzyme has been cloned and purified as described earlierby Kupriyanova et al.5a

4.2. CA activity and inhibition measurements

An Applied Photophysics stopped-flow instrument has beenused for assaying the CA catalysed CO2 hydration activity. Phenolred (at a concentration of 0.2 mM) has been used as indicator,working at the absorbance maximum of 557 nm, with 10–20 mMHepes (pH 7.5) as buffer, and 20 mM Na2SO4 for maintaining con-stant the ionic strength, following the initial rates of the CA-cata-lyzed CO2 hydration reaction for a period of 10–100 s.17 The CO2

concentrations ranged from 1.7 to 17 mM for the determinationof the kinetic parameters and inhibition constants. For each inhib-itor, at least six traces of the initial 5–10% of the reaction have beenused for determining the initial velocity. The uncatalyzed rateswere determined in the same manner and subtracted from the to-tal observed rates. Stock solutions of inhibitors (10 mM) were pre-pared in distilled–deionized water and dilutions up to 0.01 lMwere done thereafter with distilled-deionized water. Inhibitorand enzyme solutions were preincubated together for 15 min atroom temperature prior to assay, in order to allow for the forma-tion of the E–I complex. The inhibition constants were obtainedby non-linear least-squares methods using PRISM 3 and theCheng–Prusoff equation. Kinetic parameters for the uninhibitedenzymes were from Lineweaver–Burk plots, as reported ear-lier,20,21 and they represent the mean from at least three differentdeterminations. All inhibitors (sodium salts of the anions fromTable 2 and several small molecules) were commercially available;highest purity compounds from Sigma–Aldrich (Milan, Italy) andwere used by such.

Acknowledgments

Research from our laboratories was financed by two FP7 EUprojects (Metoxia and Dynano). E.V.K. was supported by the Grantfrom Russian Foundation for Basic Research No. 13-04-00193a.

References and notes

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