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

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

Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (KIs of 28.5– 2954 nM), being highly effective as hCA II (KIs in the range of 0.62–12.4 nM) and XII (KIs of 0.54– 7.11 nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated

The first activation study of a δ-carbonic anhydrase: TweCAδ from the diatom Thalassiosira weissflogii is effectively activated by amines and amino acids

The activation of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAδ) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activator of TweCAδ was d-Tyr (KA of 51 nM), whereas several other amino acids and amines, such as L-His, L-Trp, d-Trp, dopamine and serotonin were submicromolar activators (KAs from 0.51 to 0.93 µM). The most ineffective activator of TweCAδ was 4-amino-l-Phe (18.9 µM), whereas d-His, l-/d-Phe, l-/d-DOPA, l-Tyr, histamine, some pyridyl-alkylamines, l-adrenaline and aminoethyl-piperazine/morpholine were moderately potent activators (KAs from 1.34 to 8.16 µM). For any δ-CA, there are no data on the crystal structure, homology modelling and the amino acid residues that are responsible for proton transfer to the active site are currently unknown making it challenging to provide a detailed rational for these findings. However, these data provide further evidence that this class of underexplored CA deserves more attention

Comparison of the amine/amino acid activation profiles of the β- and γ-carbonic anhydrases from the pathogenic bacterium Burkholderia pseudomallei

The β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, BpsCAβ, that is responsible for the tropical disease melioidosis was investigated for its activation with natural and non-natural amino acids and amines. Previously, the γ-CA from this bacterium has been investigated with the same library of 19 amines/amino acids, which show very potent activating effects on both enzymes. The most effective BpsCAβ activators were L- and D-DOPA, L- and D-Trp, L-Tyr, 4-amino-L-Phe, histamine, dopamine, serotonin, 2-pyridyl-methylamine, 1-(2-aminoethyl)-piperazine and L-adrenaline with KAs of 0.9–27 nM. Less effective activators were D-His, L- and D-Phe, D-Tyr, 2-(2-aminoethyl)pyridine and 4-(2-aminoethyl)-morpholine with KAs of 73 nM–3.42 µM. The activation of CAs from bacteria, such as BpsCAγ/β, has not been considered previously for possible biomedical applications. It would be of interest to perform studies in which bacteria are cultivated in the presence of CA activators, which may contribute to understanding processes connected with the virulence and colonization of the host by pathogenic bacteria

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s−1 and kcat/Km = 1.1 × 108 M−1 s−1) which was maintained after heating the enzyme at 80 °C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe3O4 nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst

Activation studies of the α- and β-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with amines and amino acids

The α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAα, and VchCAβ, were investigated for their activation with natural and non-natural amino acids and amines. The most effective VchCAα activators were L-tyrosine, histamine, serotonin, and 4-aminoethyl-morpholine, which had KAs in the range of 8.21–12.0 µM. The most effective VchCAβ activators were D-tyrosine, dopamine, serotonin, 2-pyridyl-methylamine, 2-aminoethylpyridine, and 2-aminoethylpiperazine, which had KAs in the submicromolar – low micromolar range (0.18–1.37 µM). The two bacterial enzymes had very different activation profiles with these compounds, between each other, and in comparison to the human isoforms hCA I and II. Some amines were selective activators of VchCAβ, including 2-pyridylmethylamine (KA of 180 nm for VchCAβ, and more than 20 µM for VchCAα and hCA I/II). The activation of CAs from bacteria, such as VchCAα/β has not been considered previously for possible biomedical applications. It would be of interest to study in more detail the extent that CA activators are implicated in the virulence and colonisation of the host by such pathogenic bacteria, which for Vibrio cholerae, is highly dependent on the bicarbonate concentration and pH in the surrounding tissue

Comparison of the Sulfonamide Inhibition Profiles of the α-Carbonic Anhydrase Isoforms (SpiCA1, SpiCA2 and SpiCA3) Encoded by the Genome of the Scleractinian Coral Stylophora pistillata

The ubiquitous metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) are responsible for the reversible hydration of CO2 to bicarbonate (HCO3−) and protons (H+). Bicarbonate may subsequently generate carbonate used in many functional activities by marine organisms. CAs play a crucial role in several physiological processes, e.g., respiration, inorganic carbon transport, intra and extra-cellular pH regulation, and bio-mineralization. Multiple transcript variants and protein isoforms exist in the organisms. Recently, 16 α-CA isoforms have been identified in the coral Stylophora pistillata. Here, we focalized the interest on three coral isoforms: SpiCA1 and SpiCA2, localized in the coral-calcifying cells; and SpiCA3, expressed in the cytoplasm of the coral cell layers. The three recombinant enzymes were heterologously expressed and investigated for their inhibition profiles with sulfonamides and sulfamates. The three coral CA isoforms differ significantly in their susceptibility to inhibition with sulfonamides. This study provides new insights into the coral physiology and the comprehension of molecular mechanisms involved in the bio-mineralization processes, since CAs interact with bicarbonate transporters, accelerating the trans-membrane bicarbonate movement and modulating the pH at both sides of the plasma membranes

Evaluation of anti-ulcer and ulcerative colitis of Sonchus oleraceus L

Sonchus oleraceus L. was evaluated for its gastro antiulcerogenic and anti-ulcerative colitis activities Different extracts and fractions from Sonchus oleraceus aerial parts and roots were evaluated at different dose; total alcohol extracts of aerial parts SA and roots SR were evaluated doses 250 & 500 mg/kg, While Successive extracts (SAL, SRL, CSA, CSR, BSA & BSR) were evaluated at dose of 150 mg/kg. Absolute ethanol-induced ulcer model was used for evaluation of the anti-ulcerogenic activity. The root extract showed promising antiulcerogenic activity as the total alcohol extract of the root SR (500 mg/kg) produced 88.5% protection from control ulcer which is significantly more effective than the standard drug omeprazole (20 mg/kg), in addition, the butanol fraction of the root extract BSR also produced 76.66% protection from control ulcer. On the other hand, the aerial parts total extract SA showed low antiulcerogenic activity in both tested doses (250 & 500 mg/kg) as it produced 25% & 28.33% protection from control ulcer respectively. Only the butanol fraction of the aerial parts extract BSA showed promising activity 54.16%. In the acetic acid-induced ulcerative colitis model, among the investigated extracts of Sonchus oleraceus; only the total extract of the aerial parts (SA) at dose 500 mg/kg showed strong anti-ulcerative colitis activity and this activity is followed by the activity of the butanol and chloroform fractions of the aerial parts, they produced 77.28%, 57.4% & 47.68% protection from control colitis respectively. The standard drug dexamethasone produced 63.36% protection from control colitis. The total alcohol extracts SR & SA showed no alteration on liver and kidney functions and these extracts are safe up to 5000 mg/kg. Phytochemical screening of the investigated extracts revealed the presence of carbohydrates, flavonoids, tannins, unsaturated sterols, proteins and lactones which could be responsible for the activities. Keywords: Peptic ulcer, Liver function, Kidney function, Acute toxicity, Asteraceae, Phytochemical screenin

Activation studies with amines and amino acids of the β-carbonic anhydrase encoded by the Rv3273 gene from the pathogenic bacterium Mycobacterium tuberculosis

The activation of a β-class carbonic anhydrase (CAs, EC 4.2.1.1) from Mycobacterium tuberculosis, encoded by the gene Rv3273 (mtCA 3), was investigated using a panel of natural and non-natural amino acids and amines. mtCA 3 was effectively activated by D-DOPA, L-Trp, dopamine and serotonin, with KAs ranging between 8.98 and 12.1 µM. L-His and D-Tyr showed medium potency activating effects, with KAs in the range of 17.6–18.2 µM, whereas other amines and amino acids were relatively ineffective activators, with KAs in the range of 28.9–52.2 µM. As the physiological roles of the three mtCAs present in this pathogen are currently poorly understood and considering that inhibition of these enzymes has strong antibacterial effects, discovering molecules that modulate their enzymatic activity may lead to a better understanding of the factors related to the invasion and colonisation of the host during Mycobacterium tuberculosis infection