Protonography and anion inhibition profile of the α-carbonic anhydrase (CruCA4) identified in the Mediterranean red coral Corallium rubrum
Graphical abstract
Introduction
The precious red coral, Corallium rubrum is an octocoral famous since the ancient time. This feature is due to its beauty and the occult powers supposedly played by its intense colored red axial skeleton [1]. Nowadays, it is still highly valued and used to produce jewelry and ornaments. C. rubrum is mostly found in the Mediterranean Sea, but it also exists in the neighboring eastern Atlantic coasts [2], [3]. Some populations of C. rubrum are threatened by local over-harvesting [4], [5] and global change including ocean acidification, which has been shown to affect axial skeleton formation [6], [7]. The calcification process, which results in the formation of the high-magnesium calcium carbonate (calcite) axial skeleton was first studied by Lacaze-Duthiers in 1864 [8]. This work was completed more than one century later by data obtained with various approaches including microscopy [9], physiology [10], crystallography [11], [12], biochemistry [13], [14] and chemistry [15]. Interestingly a superfamily of metalloenzymes known as carbonic anhydrases (CA, EC 4.2.1.1) has been molecularly characterized for their pivotal role in the calcification process of the hard corals. The CA superfamily includes seven known families: α-, β-, γ-, δ-, ζ-, η- and θ-CAs [16], [17], [18], which efficiently catalyze the hydration of carbon dioxide to bicarbonate and protons [19], [20], [21], [22], [23]. The catalytically active form of CAs is the metal hydroxide derivative [16], [17], [18], the velocity of the catalytic reaction is determined by the transfer of a proton from the metal-coordinated water molecule from the enzyme active site, to the surrounding solvent [17], [18], [19], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. The metal ion in the active site and CAs distribution in living organisms is very diverse. The metal ion is coordinated by three His residues in the α-, γ-, δ- and, probably, θ-classes; by one His, and two Cys residues in β- and ζ-CAs or by two His and one Gln residues in the η-class, with the fourth ligand being a water molecule/hydroxide ion acting as the nucleophile in the catalytic cycle of the enzyme [16], [20], [21], [22], [33], [34]. The α-, β-, δ-, η- and, perhaps θ-CAs are characterized by a Zn(II) ion in the active site. γ-CAs are probably Fe(II) enzymes, but they are active also with bound Zn(II) or Co(II) ions [35], [36], [37], [38], [39], [40], [41], [42]. ζ-CAs are active both with Cd(II) or Zn(II) [43], [44], [45]. α-CAs are normally active as monomers or dimers; β-CAs are active only as dimers, tetramers or octamers, whereas γ-CAs must be trimers for accomplishing their physiological function [36], [37], [40], [46]. Intriguing, α- and η-CAs also catalyze the hydrolysis of esters/thioesters, while no esterase activity was detected for the other five CA families [16], [47]. CAs distribution in living organisms is very variegated. Animals contain α-class [48], [49], plants and algae have α-, β-, γ-, δ- and θ-classes; fungi encode for α- and β-CAs; protozoa for α-, β- and/or η-CAs; bacteria for α-, β- and γ-CA classes [19], [20], [21], [22], [27], [50], [51]; and in metazoans, the α-CAs are largely represented [52], [53]. Recently, a series of studies coupled with molecular biology and in vivo cell imaging data have allowed the deciphering of the calcification mechanisms in C. rubrum through the identification and characterization of different isoforms of CAs [54], [55]. Interesting, one of them named CruCA4, has been identified as being involved in coral calcification [53], [54]. CruCA4 belongs to the α-CA family. Both by qPCR and Western-blotting, it has been shown that this isozyme is specifically localized and expressed in the calcifying tissues [54]. As for other calcifying corals this enzyme, which is responsible for the reversible hydration/dehydration of carbon dioxide into bicarbonate could help catalyzing the supply of inorganic carbon at the site of calcification [56]. Indeed, this enzyme could help transforming the metabolic carbon dioxide into bicarbonate, which is then transformed into carbonate that reacts with calcium to precipitate under calcium carbonate [54], [55]. We have recently determined the kinetic properties of the recombinant CruCA4 and shown that this enzyme has a “moderate activity” level with a kcat of 4.1 × 105 s−1 and a kcat/Km of 5.2 × 107 M−1s−1 [53]. The present work aimed at using the recently developed technique of protonography to detect in a simple way the activity of CruCA4. We have also investigated the inhibition profile of CruCA4 with one major class of CA inhibitors, the inorganic anions and compared it to data obtained for other corals.
Section snippets
CruCA4 features
Based on our previous biochemical studies, multiple sequence alignment and phylogenetic analysis, in Fig. 1 we have been graphically represented the characteristics of the metalloenzyme indicated with the acronym CruCA4 and identified in the genome of the precious red coral, Corallium rubrum. It has been observed that: (i) the CruCA4 nucleotide sequence consists of an open reading frame encoding for 284 amino acid polypeptide chain, and 5′-and 3′-untranslated regions; (ii) CruCA4 is a hydratase
Conclusion
CAs are involved in biomineralization both in invertebrates as well as in vertebrates, even if the mechanisms of this biomineralization are still poorly understood. Corals, which precipitate calcium carbonate (CaCO3) skeletons use CAs for the generation of bicarbonate from carbon dioxide [56]. Six α-CAs were isolated from the red coral Corallium rubrum [54]. The isoenzymes were indicated with the acronyms CruCA1 to 6. Among them, the isoform CruCA3 is a cytosolic isoenzyme; CruCA2 is considered
Gene synthesis and cloning
The identification of the gene encoding for the Corallium rubrum protein (CruCA4) was performed running the ‘‘Protein BLAST’’ program and using as query sequence the amino acid sequence of the CruCA4 with the accession number KU557746.1. The GeneArt Company (Thermo Fisher Scientific), specialized in gene synthesis, designed the synthetic CruCA4 gene without the peptide signal and having at the 5′ end four base-pair sequences (CACC) necessary for the directional cloning in the pMK-T vector
Acknowledgements
Part of this work was funded by the Government of the Principality of Monaco and the Foundation Paul Hamel.
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