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

Rosa Perfetto,Giovanni Sansone,Claudiu Supuran,Mosè Rossi,Daniela Vullo,Carmela Barone,Sonia Del Prete,Clemente Capasso

doi:10.3390/md15090270

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.

Highlights

The genome of Eubacteria, Archaea, and Eukarya encodes for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1)
By translated inspection of the Crassostrea gigas genome, we identified a gene encoding for a nacrein-like protein, which is indicated with the acronym CgiNAP2X1 in this article
It is possible to inspecting other molluskan nacrein or nacrein-like proteins deposited in the protein data bank and observe, that theused clade of nacrein has arisen from an ancestral sequence, which led before to the bacterial for this phylogenetic analysis with SignalP, we found that nacreins, generally, are characterized α-CAs characterized by a signal and molluskan clade

Summary

Introduction

The genome of Eubacteria, Archaea, and Eukarya encodes for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). The CA superfamily includes seven known families: α-, β-, γ-, δ-, ζ-, η- and θ-CAs [1,2,3], which represent a fashionable example of convergent/divergent protein evolution. CA families aforementioned efficiently catalyze the hydration of carbon dioxide to bicarbonate and protons [4,5,6,7,8]; the catalytically active form of the enzyme is the metal hydroxide derivative [1,2,3]; the velocity of the catalytic reaction depends on the formation of a metal hydroxide species, which is formed by the transfer of a proton from the metal-coordinated water molecule from the enzyme active site to the surrounding solvent [2,3,4,6,7,8,9,10,11,12,13,14,15,16,17]. Taking into account the three-dimensional structure of the CA families, the seven genetic families are characterized by a different arrangement of the active site, with the metal ion playing an important part, as the apoenzymes are devoid of any catalytic activity

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Conclusion