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

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 react...

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Bibliographic Details
Published in:Marine Drugs
Main Authors: Perfetto, Rosa, SANSONE, GIOVANNI, BARONE, CARMELA MARIA ASSUNTA, ROSSI, MOSE', CAPASSO, CLEMENTE, Del Prete, Sonia, Vullo, Daniela, Supuran, Claudiu T
Other Authors: Sansone, Giovanni, Barone, CARMELA MARIA ASSUNTA, Rossi, Mose', Capasso, Clemente
Format: Article in Journal/Newspaper
Language:English
Published: 2017
Subjects:
anion inhibitor
carbonic anhydrase
kinetic constant
molluskan
nacrein-like protein
phylogeny
signal peptide
Pacific
Crassostrea gigas
Pacific oyster
Online Access:http://hdl.handle.net/11588/683350
https://doi.org/10.3390/md15090270
Description
Summary: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.

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