Abstract
The carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons. In the last years, and especially, to the rapid emergence of the bacterial antibiotic resistance that is occurring worldwide, the understanding of the function of bacterial CAs has increased significantly. Recently, a new CA-class (ι-CA) was discovered in the marine diatom T. pseudonana. It has been reported that bacterial genomes may contain genes with relevant homology to the diatom ι-class CA. Still, the catalytic activity of the enzyme encoded by the gene was not investigated. Thus, herein, for the first time, we cloned, expressed, and purified the recombinant bacterial ι-CA (acronym BteCAι) identified in the genome of Burkholderia territorii. The recombinant BteCAι resulted in a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat of 3.0 × 105 s −1 and kcat/KM of 3.9 × 107 M −1 s −1, and is also sensitive to inhibition by the sulphonamide acetazolamide. Furthermore, with the aid of the protonography, it has been demonstrated that BteCAι can be present as a dimer. This result is corroborated by the construction of a molecular model of BteCAι, which showed that the enzyme is formed by two equivalent monomers having a structure similar to a butterfly.
Highlights
The metalloenzyme carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons (CO2 þ H2O À HCO3– þ Hþ)[1]
The analysis of BteCAi primary structure evidenced a secretory signal peptide consisting of 25 amino acid residues at the Nterminal part of the protein
The phylogenetic analysis carried out using bacterial a, b, c, and i-CAs evidenced that the new i-CAs were closer to the c-classes than the other bacterial CAs
Summary
The metalloenzyme carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons (CO2 þ H2O À HCO3– þ Hþ)[1]. Since the CA hydratase/dehydratase activity contributes to numerous physiological functions involving dissolved inorganic carbon, CAs are crucial biomolecules in many physiological and pathological conditions in all types of organisms[6]. CAs encoded in the genome of Helicobacter pylori, are essential for the acid acclimatisation of the pathogen within the stomach[13–15]; Vibrio cholerae uses CAs as a system to colonise the host since CAs are involved in the production of sodium bicarbonate, which induces cholera toxin expression[16]; Brucella suis needed functional CAs for growing[17–20]; b-CA from Salmonella enterica is highly expressed during the bacterial infection[21]. Starting from 1920, when was observed for the first time the CA activity in hemolyzed blood[23], the knowledge of the enzymes responsible for that activity, i.e., the CA, has extensively been improved
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