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Mar. Drugs 2017, 15(9), 270; doi:10.3390/md15090270

Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas)

1
Istituto di Bioscienze e Biorisorse, Consiglio Nazionale delle Ricerche (CNR), via Pietro Castellino 111, 80131 Napoli, Italy
2
Sezione di Scienze Farmaceutiche and Laboratorio di Chimica Bioinorganica, Polo Scientifico, Dipartimento Neurofarba, Università degli Studi di Firenze, Via U. Schiff 6, Sesto Fiorentino, 50019 Florence, Italy
3
Dipartimento di Biologia, Università degli Studi di Napoli, Federico II, via Cinthia 21, 80126 Napoli, Italy
4
Dipartimento di Agraria, Università degli Studi di Napoli, Federico II, via Università 100, 80055 Portici (Napoli), Italy
*
Authors to whom correspondence should be addressed.
Received: 28 July 2017 / Revised: 21 August 2017 / Accepted: 23 August 2017 / Published: 28 August 2017
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Abstract

The carbonic anhydrase (CA, EC 4.2.1.1) superfamily of metalloenzymes catalyzes the hydration of carbon dioxide to bicarbonate and protons. The catalytically active form of these enzymes incorporates a metal hydroxide derivative, the formation of which is the rate-determining step of catalytic reaction, being affected by the transfer of a proton from a metal-coordinated water molecule to the environment. Here, we report the cloning, expression, and purification of a particular CA, i.e., nacrein-like protein encoded in the genome of the Pacific oyster Magallana gigas (previously known as Crassostrea gigas). Furthermore, the amino acid sequence, kinetic constants, and anion inhibition profile of the recombinant enzyme were investigated for the first time. The new protein, CgiNAP2X1, is highly effective as catalyst for the CO2 hydration reaction, based on the measured kinetic parameters, i.e., kcat = 1.0 × 106 s−1 and kcat/KM = 1.2 × 108 M−1·s−1. CgiNAP2X1 has a putative signal peptide, which probably allows an extracellular localization of the protein. The inhibition data demonstrated that the best anion inhibitors of CgiNAP2X1 were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed a micromolar affinity for this enzyme, with KIs in the range of 76–87 μM. These studies may add new information on the physiological role of the molluskan CAs in the biocalcification processes. View Full-Text
Keywords: nacrein-like protein; carbonic anhydrase; molluskan; signal peptide; kinetic constants; phylogeny; anion inhibitors nacrein-like protein; carbonic anhydrase; molluskan; signal peptide; kinetic constants; phylogeny; anion inhibitors
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MDPI and ACS Style

Perfetto, R.; Del Prete, S.; Vullo, D.; Sansone, G.; Barone, C.M.A.; Rossi, M.; Supuran, C.T.; Capasso, C. Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas). Mar. Drugs 2017, 15, 270.

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