Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec;32(1):1029-1035.
doi: 10.1080/14756366.2017.1353502.

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

Rosa Perfetto  1 , Sonia Del Prete  1   2 , Daniela Vullo  2 , Vincenzo Carginale  1 , Giovanni Sansone  3 , Carmela M A Barone  4 , Mosè Rossi  1 , Fatmah A S Alasmary  5 , Sameh M Osman  5 , Zeid AlOthman  5 , Claudiu T Supuran  2   5   6 , Clemente Capasso  1
Affiliations

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

Rosa Perfetto et al. J Enzyme Inhib Med Chem. 2017 Dec.

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.

Keywords: Carbonic anhydrase; bivalve; hydratase activity; metalloenzymes; multidomain protein; mussel; protonography; α-class enzyme.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Blast output reporting the CA library sequences. By going down the list, it is possible to see less than perfect matches, slowly degrading as the corresponding score decreases and the E-value increases. The E-value is an assessment of the statistical significance of the score. E-value close to 1 are a warning that the alignment is not reliable.
Figure 2.
Figure 2.
Alignment of α-CA sequences from Mytilus galloprovincialis (MgaCA, Accession number: ALF62133.1) Corallium rubrum (CruCA4, Accession number: KU557746), Homo sapiens I (hCAI, Accession number: NP001729) and II (hCAII, Accession number: P00918), hCA I numbering system was used. The zinc ligands (His94, 96 and 119) and the gate-keeper residues (Glu106 and Thr199) are conserved in aligned sequences; while the proton shuttle residue (His64) is preserved only in the human enzymes. The asterisk (*) indicates identity at all aligned positions; the symbol (:) relates to conserved substitutions, while (.) means that semi-conserved substitutions are observed. Multialignment was performed with the program Muscle, version 3.1.
Figure 3
Figure 3
(A and B). A. SDS-PAGE; B. Protonography. In both panels: Lane 1, Molecular weight markers; lane 2, the native MgaCA purified from the mantles of M. galloprovincialis; lane 3, the recombinant rec-MgaCA, lane 4, the commercial bovine CA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Supuran CT.Structure and function of carbonic anhydrases. Biochem J 2016;473:2023–32. - PubMed
    1. Del Prete S, Vullo D, Fisher GM, et al. . Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum – the eta-carbonic anhydrases. Bioorg Med Chem Lett 2014;24:4389–96. - PubMed
    1. Kikutani S, Nakajima K, Nagasato C, et al. . Thylakoid luminal theta-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum. Proc Natl Acad Sci U S A 2016;113:9828–33. sss - PMC - PubMed
    1. Capasso C, Supuran CT.. Bacterial, fungal and protozoan carbonic anhydrases as drug targets. Expert Opin Ther Targets 2015;19:1689–704. - PubMed
    1. Capasso C, Supuran CT.. An overview of the selectivity and efficiency of the bacterial carbonic anhydrase inhibitors. Curr Med Chem 2015;22:2130–9. - PubMed

Substances

Grants and funding

This research was financed by the grant “SMART GENERATION – Sistemi e tecnologie sostenibili per la generazione di energia – PON03PE_00157_1, OR3 – Bio-sistemi di cattura ed utilizzazione della CO2”. We also thank the Distinguished Scientist Fellowship Program (DSFP) of King Saud University, Riyadh, Saudi Arabia.

LinkOut - more resources