Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

Abstract

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s−1 and kcat/Km of 3.5 × 107 M−1 ×s−1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

Activation Profile Analysis of CruCA4, an α-Carbonic Anhydrase Involved in Skeleton Formation of the Mediterranean Red Coral, Corallium rubrum

“…CruCA4 has a residue of Lys instead of His in position 64. This substitution, as demonstrated for other CAs, leads to a decrease in the k cat value [ 41 , 42 ]. Thus, the treatment of the enzyme with CAAs represents a very good strategy for stimulating the biomineralization process of the organism through the increase of the enzymatic activity of CruCA4.…”

Carbonic Anhydrase in Pacific Abalone Haliotis discus hannai: Characterization, Expression, and Role in Biomineralization

“…From protostomes to deuterostomes, CAs plays a critical role in various aspects of physiological processes ( Chegwidden and Carter, 2000 ). Several studies have been conducted on CA isozyme in bivalve and gastropod mollusk ( Le Roy et al, 2012 ; Ip et al, 2017 ; Perfetto et al, 2017 ). However, characterization and expression of CA in shell forming mantle tissues of Pacific abalone have not been reported yet.…”

Molecular Characterization of Carbonic Anhydrase II (CA II) and Its Potential Involvement in Regulating Shell Formation in the Pacific Abalone, Haliotis discus hannai

“…Carbonic anhydrases play an important role in many physiological processes by catalyzing the hydration reaction. In mollusks, CAs have been previously identified in Tridacna squamosa (Ip et al, 2017), Mytilus galloprovincialis (Perfetto et al, 2017), and H. tuberculata (Le Roy et al, 2012). To date, the identification and biomolecular characterization of CA II isoform from the Pacific abalone have not yet been reported.…”