Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis. (1st January 2017)

• Record Type: Journal Article
• Authors: Perfetto, Rosa; Del Prete, Sonia; Vullo, Daniela; Carginale, Vincenzo; Sansone, Giovanni; Barone, Carmela M. A.; Rossi, Mosè; Alasmary, Fatmah A. S.; Osman, Sameh M.; AlOthman, Zeid; Supuran, Claudiu T.; Capasso, Clemente
• Is Part Of: Journal of enzyme inhibition and medicinal chemistry. Volume 32:Number 1(2017)
• Page Start: 1029
• Page End: 1035
• Publication Date: 2017-01-01
• Subjects: Carbonic anhydrase -- metalloenzymes -- α-class enzyme -- hydratase activity -- mussel -- multidomain protein -- protonography -- bivalve; Enzyme inhibitors -- Periodicals; Enzyme Inhibitors -- periodicals; Biochemistry -- periodicals; 572.7
• DOI: 10.1080/14756366.2017.1353502
• Languages: English
• ISSNs: 1475-6366

Abstract

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 k cat = 4.2 × 10 5 s −1 and k cat / K m of 3.5 × 10 7 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.