title: Cloning, characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii.
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 × 10(5)s(-1) and a k(cat)/K(M) of 3.3 × 10(7)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 (K(I)s in the range of 0.93-8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K(I)s in the range of 9-90 μM, whereas acetazolamide inhibited the enzyme with a K(I) 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.
authors.affiliation: Istituto di Biochimica delle Proteine and Institute of Bioscience and Bioresources (IBBR)-CNR, Via P. Castellino 111, 80131 Napoli, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Prete
authors.affiliation: Università degli Studi di Firenze, Dipartmento di Chimica Ugo Schiff, Via della Lastruccia 3, Rm. 188, 50019 Sesto Fiorentino (Florence), Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Vullo
authors.affiliation: Università degli Studi di Firenze, Dipartmento di Chimica Ugo Schiff, Via della Lastruccia 3, Rm. 188, 50019 Sesto Fiorentino (Florence), Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scozzafava
authors.affiliation: Istituto di Biochimica delle Proteine and Institute of Bioscience and Bioresources (IBBR)-CNR, Via P. Castellino 111, 80131 Napoli, Italy. Electronic address: c.capasso@ibp.cnr.it.
authors.forename: Clemente
authors.initials: C
authors.lastname: Capasso
authors.affiliation: Università degli Studi di Firenze, Dipartmento di Chimica Ugo Schiff, Via della Lastruccia 3, Rm. 188, 50019 Sesto Fiorentino (Florence), Italy; Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutriceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy. Electronic address: claudiu.supuran@unifi.it.
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
mesh_terms: D002257:Carbonic Anhydrase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000378:metabolism*; D017377:Diatoms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D013449:Sulfonamides; D002256:Carbonic Anhydrases
keywords: Anion; Carbon concentration mechanism; Carbonic anhydrase; Diatom; Sulfonamide; δ-Class enzyme