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.
Highlights
Carbonic anhydrases superfamily (CAs, EC 4.2.1.1) are metalloenzymes, which have been found in all the three domains of life (Eubacteria, Archaea, and Eukarya) and represent a very interesting example of convergent/divergent evolution phenomenon with seven known families: a, b, c, d, f, g, and h-CAs1–3
The result of Basic Local Alignment Search Tool (BLAST) analysis showed that the first amino acid sequence of the top library sequences was the carbonic anhydrases II from M. galloprovincialis with a “Query score” and “Identity” of 100% (Figure 1)
The mussel enzyme had a residue of lysine as substituent of the proton shuttle residue His[64] (Figure 2), characteristic of the human isozymes, explaining the relatively low catalytic activity of the native M. galloprovincialis (MgaCA), with the following kinetic parameters for the CO2 hydration reaction: kcat 1⁄4 4.1 Â 105 sÀ1 and kcat/Km of 3.6 Â 107 MÀ1 Â sÀ1 45, with respect to hCA II, which is considered as one of the most active among the a-CAs and the other CAclasses[51]
Summary
Carbonic anhydrases superfamily (CAs, EC 4.2.1.1) are metalloenzymes, which have been found in all the three domains of life (Eubacteria, Archaea, and Eukarya) and represent a very interesting example of convergent/divergent evolution phenomenon with seven known families: a-, b-, c-, d-, f-, g-, and h-CAs1–3. C-CAs are probably Fe(II) enzymes, this family is active with bound Zn(II) or Co(II) ions. The metal ion from the CA active site is coordinated by three His residues in the a-, c-, d- and, probably, h-classes; by one His, and two Cys residues in b- and f-CAs or by two His and one Gln residues in the g-class, with the fourth ligand being a water molecule/hydroxide ion acting as nucleophile in the catalytic cycle of the enzyme[1,5,6,7,29,30]. Some of the catalytically active a-CAs catalyse the hydrolysis of esters/thioesters, e.g. 4-nitrophenyl acetate (4-NpA) hydrolysis, as well as other hydrolytic reactions
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