Sulfonamide inhibition studies of the c-carbonic anhydrase from the Antarctic cyanobacterium Nostoc commune Daniela Vullo a , Viviana De Luca b , Sonia Del Prete a,b , Vincenzo Carginale b , Andrea Scozzafava a , Clemente Capasso b,⇑ , Claudiu T. Supuran a,c,⇑ a Università degliStudi di Firenze, Dipartimento Di Chimica, Laboratorio di ChimicaBioinorganica, Polo Scientifico, Via della Lastruccia3, 50019 Sesto Fiorentino, Florence, Italy b Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 81, Napoli, Italy c Università degliStudi di Firenze, DipartimentoNeurofarba, Sezione di ScienzeFarmaceutiche, Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy article info Article history: Received 10 January 2015 Revised 11 February 2015 Accepted 24 February 2015 Available online 4 March 2015 Keywords: Carbonic anhydrase Metalloenzymes Hydratase activity Antarctic carbonic anhydrase Cyanobacteria abstract A carbonic anhydrase (CA, EC 4.2.1.1) belonging to the c-class has been cloned, purified and characterized from the Antarctic cyanobacterium Nostoc commune. The enzyme showed a good catalytic activity for the physiologic reaction (hydration of carbon dioxide to bicarbonate and a proton) with the following kinetic parameters, k cat of 9.5 Â 10 5 s À1 and k cat /K M of 8.3 Â 10 7 M À1 s À1 , being the c-CA with the highest cat- alytic activity described so far. A range of aromatic/heterocyclic sulfonamides and one sulfamate were investigated as inhibitors of the new enzyme, denominated here NcoCA. The best NcoCA inhibitors were some sulfonylated sulfanilamide derivatives possessing elongated molecules, aminobenzolamide, aceta- zolamide, benzolamide, dorzolamide, brinzolamide and topiramate, which showed inhibition constants in the range of 40.3–92.3 nM. As 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and c-CAs are clo- sely associated in carboxysomes of cyanobacteria for enhancing the affinity of RubisCO for CO 2 and the efficiency of photosynthesis, investigation of this new enzyme and its affinity for modulators of its activ- ity may bring new insights in these crucial processes. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Carbonic anhydrases (CAs, EC 4.2.1.1) are biological catalysts for the interconversion of CO 2 and water to bicarbonate (HCO 3 À ) and protons (H + ). 1–18 CAs are multifunctional enzymes which play a central role in different physiological and biochemical processes, such as respiratory gas exchange; acid–base homeostasis; elec- trolytes secretion; biosynthetic reactions (e.g., ureagenesis, gluconeogenesis, synthesis of fatty acids); ionic transport; muscu- lar contraction (in vertebrates) and photosynthesis (in cyanobac- teria, plants and algae). 19–21 Recently many studies revealed that CAs are also widely distributed in prokaryotes where their role may be much more important than previously thought. Six different, genetically distinct CA families are known to date, the a-, b-, c-, d-, f- and g-CAs. 22–39 Except for d- and g-CAs, the three-dimensional structure of all the other CA classes has been resolved by X-ray diffraction techniques. 26,28,40–51 a-CAs are normally monomers and rarely dimers; b-CAs are dimers, tetra- mers or octamers; c-CAs are trimers, whereas the d- and f-CAs are less well understood at this moment. For example, the most investigated f-CA (from the marine diatom Thalassiosira weissflogii) has three slightly different active sites on the same polypeptide chain. 52 Bacteria encode for enzymes belonging to the a-, b-, and c-CA classes. 53,54 All these enzymes contain a zinc ion (Zn 2+ ) in their active site, coordinated by three histidine residues and a water molecule/hydroxide ion (in the a- and c-CAs) or by two Cys and one His residues (in the b class), with the fourth ligand being a water molecule/hydroxide ion acting as nucleophile in the cat- alyzed reactions. 48,53,55–58 Few data are available in the literature on CAs from Antarctic organisms 59–61 and most such data deal with CAs isolated from mammals, prokaryotes or other mesophilic sources, these organ- isms living at physiological temperatures of around 37 °C. 15,22,24,25,27,29–31,33–39,62–67 Recently, our group studied CAs from extremophiles, microorganisms living at temperatures rang- ing from 70 °C to 110 °C, demonstrating that these enzymes are thermostable, thermoactive and stable to the common enzyme denaturants. 41,68–73 http://dx.doi.org/10.1016/j.bmc.2015.02.045 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Tel./fax: +39 0816132559 (C.C.); tel./fax: +39 055 4573729 (C.T.S.). E-mail addresses: clemente.capasso@ibbr.cnr.it (C. Capasso), claudiu.supuran@ unifi.it (C.T. Supuran). Bioorganic & Medicinal Chemistry 23 (2015) 1728–1734 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc