Α-carbonic Anhydrase Research Articles

Expression and Functional Study of Single Mutations of Carbonic Anhydrase 8 in Neuronal Cells.

Carbonic anhydrase 8 (CA8), an isozyme of α-carbonic anhydrases, lacks the ability to catalyze the reversible hydration of CO2 to bicarbonate and proton. Previous studies have shown that single point mutations of CA8, CA8-S100P, and CA8-G162R, are associated with novel syndromes including congenital ataxia and mild cognitive impairment. Our previous results demonstrated that overexpression of wild type (WT) CA8 promoted cell proliferation, neurite outgrowth, anti-apoptosis, invasion and migration abilities in neuronal cells. In this study, we examined the expressions and functions of CA8-S100P and CA8-G162R in neuroblastoma cells lines, compared with those of WT CA8. Our results show that the protein expressions of mutant CA8-S100P and CA8-G162R were significantly decreased in Neuro-2a and SK-N-SH cells. Interestingly, CA8-S100P demonstrated a significant increase in cell proliferation in both Neuro-2a and SK-N-SH cells. However, both CA8 mutations showed significantly decreased effects on cell protection and migration in SK-N-SH cells. Surprisingly, a significant increase of invasive ability was observed in SK-N-SH cells with overexpression of CA8-S100P as compared with those with overexpression of WT CA8 under retinoic acid (RA) treatment. In addition, we found that Neuro-2a cells with overexpression of CA8-S100P and CA8-G162R showed significantly increased neurite outgrowth. Taken together, our data suggest that the expressions of CA8-S100P and CA8-G162R in neuronal cells alter cell morphology, proliferation, mobility and viability; indicating that the homozygous point mutations of CA8 lead to not only the loss of WT CA8 function, but also the gain of novel functions leading to neuromuscular dysfunction.

Sulfonamide Inhibition Studies of an α-Carbonic Anhydrase from Schistosoma mansoni, a Platyhelminth Parasite Responsible for Schistosomiasis.

Schistosomiasis is a debilitating infection provoked by parasitic flatworms called schistosomes. The species Schistosoma mansoni is endemic in Africa, where it causes intestinal schistosomiasis. Recently, an α-carbonic anhydrase (CA, EC 4.2.1.1) was cloned and characterized from this organism and designated as SmCA. The protein is expressed in the tegument (skin) of S. mansoni at the host–parasite interface. Recombinant SmCA possesses high catalytic activity in the CO2 hydration reaction, similar to that of human CA isoform II with a kcat of 1.2 × 106 s−1 and a kcat/KM of 1.3 × 108 M−1·s−1. It has been found that schistosomes whose SmCA gene is suppressed using RNA interference are unable to establish a robust infection in mice, suggesting that the chemicals that inhibit SmCA function should have the same debilitating effect on the parasites. In this study, a collection of aromatic/heterocyclic sulfonamides were investigated as possible SmCA inhibitors. Several sulfonamides inhibited SmCA with medium to weak potency (KI values of 737.2 nM−9.25 μM), whereas some heterocyclic compounds inhibited the enzyme with KI values in the range of 124−325 nM. The α-CA from S. mansoni, SmCA, is proposed as a new anti-schistosomiasis drug target.

Characterization and High-Level Periplasmic Expression of Thermostable α-Carbonic Anhydrase from Thermosulfurimonas Dismutans in Escherichia Coli for CO2 Capture and Utilization.

Carbonic anhydrase (CA) is a diffusion-controlled enzyme that rapidly catalyzes carbon dioxide (CO2) hydration. CA has been considered as a powerful and green catalyst for bioinspired CO2 capture and utilization (CCU). For successful industrial applications, it is necessary to expand the pool of thermostable CAs to meet the stability requirement under various operational conditions. In addition, high-level expression of thermostable CA is desirable for the economical production of the enzyme. In this study, a thermostable CA (tdCA) of Thermosulfurimonas dismutans isolated from a deep-sea hydrothermal vent was expressed in Escherichia coli and characterized in terms of expression level, solubility, activity and stability. tdCA showed higher solubility, activity, and stability compared to those of CA from Thermovibrio ammonificans, one of the most thermostable CAs, under low-salt aqueous conditions. tdCA was engineered for high-level expression by the introduction of a point mutation and periplasmic expression via the Sec-dependent pathway. The combined strategy resulted in a variant showing at least an 8.3-fold higher expression level compared to that of wild-type tdCA. The E. coli cells with the periplasmic tdCA variant were also investigated as an ultra-efficient whole-cell biocatalyst. The engineered bacterium displayed an 11.9-fold higher activity compared to that of the recently reported system with a halophilic CA. Collectively these results demonstrate that the highly expressed periplasmic tdCA variant, either in an isolated form or within a whole-cell platform, is a promising biocatalyst with high activity and stability for CCU applications.

The role of carbonic anhydrase α-CA4 in the adaptive reactions of photosynthetic apparatus: the study with α-CA4 knockout plants

The role of α-carbonic anhydrase 4 (α-CA4) in photosynthetic machinery functioning in thylakoid membranes was studied, using Arabidopsis thaliana wild type plants (WT) and the plants with knockout of At4g20990 gene encoding α-CA4 (αCA4-mut) grown both in low light (LL, 80 μmol quantam-2 s-1) or in high light (HL, 400 μmol quantam-2 s-1). It was found that a content of PsbS protein, one of determinants of non-photochemical quenching of chlorophyll fluorescence, increased in mutants by 30% and 100% compared with WT plants in LL and in HL, respectively. Violaxanthin cycle pigments content and violaxanthin deepoxidase activity in HL were also higher in αCA4-mut than in WT plants. The content of PSII core protein, D1, when adapting to HL, decreased in WT plants and remained unchanged in mutants. This indicates, that the decrease in the content of Lhcb1 and Lhcb2 proteins in HL (Rudenko et al. Protoplasma 55(1):69-78, 2018) in WT plants resulted from decrease of both Photosystem II (PSII) complex content and content of these proteins in this complex, whereas in αCA4-mut plants from the latter process only. The absence of α-CA4 did not affect the rate of electron transport through Photosystem I (PSI) in thylakoids of mutant vs. WT, but led to 50-80% increase in the rate of electron transport from H2O to QA, evidencing the location of α-CA4 close to PSII. The latter difference may raise the question about its causal connection with the difference in the D1 protein content change during adapting to increased illumination in the presence and the absence of α-CA4.

Mechanism of Action of Non-Synonymous Single Nucleotide Variations Associated with α-Carbonic Anhydrase II Deficiency.

Human carbonic anhydrase II (CA-II) is a Zinc (Zn) metalloenzyme responsible for maintenance of acid-base balance within the body through the reversible hydration of CO to produce protons (H) and bicarbonate (BCT). Due to its importance, alterations to the amino acid sequence of the protein as a result of single nucleotide variations (nsSNVs) have detrimental effects on homeostasis. Six pathogenic CA-II nsSNVs, K18E, K18Q, H107Y, P236H, P236R and N252D were identified, and variant protein models calculated using homology modeling. The effect of each nsSNV was analyzed using motif analysis, molecular dynamics (MD) simulations, principal component (PCA) and dynamic residue network (DRN) analysis. Motif analysis identified 11 functionally important motifs in CA-II. RMSD data indicated subtle SNV effects, while PCA analysis revealed that the presence of BCT results in greater conformational sampling and free energy in proteins. DRN analysis showed variant allosteric effects, and the average betweenness centrality (BC) calculations identified Glu117 as the most important residue for communication in CA-II. The presence of BCT was associated with a reduction to Glu117 usage in all variants, suggesting implications for Zn dissociation from the CA-II active site. In addition, reductions to Glu117 usage are associated with increases in the usage of the primary and secondary Zn ligands; His94, His96, His119 and Asn243 highlighting potential compensatory mechanisms to maintain Zn within the active site. Compared to traditional MD simulation investigation, DRN analysis provided greater insights into SNV mechanism of action, indicating its importance for the study of missense mutation effects in proteins and, in broader terms, precision medicine related research.

Crystal structure and chemical inhibition of essential schistosome host-interactive virulence factor carbonic anhydrase SmCA

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Here we identify an α-carbonic anhydrase (SmCA) that is expressed at the schistosome surface as determined by activity assays and immunofluorescence/immunogold localization. Suppressing SmCA expression by RNAi significantly impairs the ability of larval parasites to infect mice, validating SmCA as a rational drug target. Purified, recombinant SmCA possesses extremely rapid CO2 hydration kinetics (kcat: 1.2 × 106 s-1; kcat/Km: 1.3 × 108 M-1s-1). The enzyme’s crystal structure was determined at 1.75 Å resolution and a collection of sulfonamides and anions were tested for their ability to impede rSmCA action. Several compounds (phenylarsonic acid, phenylbaronic acid, sulfamide) exhibited favorable Kis for SmCA versus two human isoforms. Such selective rSmCA inhibitors could form the basis of urgently needed new drugs that block essential schistosome metabolism, blunt parasite virulence and debilitate these important global pathogens.

Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis)

The α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α–CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α–CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1.

Bacterial α-type carbonic anhydrase (α-CA) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable α-CA from Persephonella marina EX-H1 (pmCA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pmCA homodimer in which each monomer consists of a 10-stranded β-sheet in the center. The catalytic zinc ion is coordinated by three highly conserved histidine residues with an exchangeable fourth ligand (a water molecule, a bicarbonate anion, or the sulfonamide group of acetazolamide). Together with an intramolecular disulfide bond, extensive interfacial networks of hydrogen bonds, ionic and hydrophobic interactions stabilize the dimeric structure and are likely responsible for the high thermal stability. We also identified novel binding sites for calcium ions at the crystallographic interface, which serve as molecular glue linking negatively charged and otherwise repulsive surfaces. Furthermore, this large negatively charged patch appears to further increase the thermostability at alkaline pH range via favorable charge-charge interactions between pmCA and solvent molecules. These findings may assist development of novel α-CAs with improved thermal and/or alkaline stability for applications such as CO2 capture and sequestration.

Antiulcer activity of Cyperus alternifolius in relation to its UPLC-MS metabolite fingerprint: A mechanistic study

BackgroundGastric ulcer is one of the main prevalent gastrointestinal multi-etiological disorders with many associated complications and adverse effects. Our aim was to develop safer antiulcer therapies based on methanol or ethyl acetate extracts of tubers and aerial parts from Cyperus alternifolius. MethodsGastric ulceration was experimentally generated by administration of single oral doses of indomethacin (30 mg/kg) to fasted rats. The animals received methanol or ethyl acetate extracts of C. alternifolius tuber and methanol or ethyl acetate extracts of aerial parts at two dose levels (50 or 100 mg/kg). Ranitidine (50 mg/kg) was used as standard anti-ulcer drug. After 4 h, the ulcer number and the total ulcer score were determined and TNF-α was assessed. Also, pathological and histochemical examination for gastric mucosa were performed.The metabolome heterogeneity of the different extracts was explored using (UPLC-MS) aided by supervised pattern recognition, i.e., orthogonal partial least squares discriminate analysis (OPLS-DA). A second OPLS-DA model was employed to link the UPLC-MS derived metabolome of the different extracts to their antiulcer activity to identify activity mediating metabolites. ResultsThe extracts significantly reduced ulcer number, total ulcer score and TNF-α content in the stomach. Methanol or ethyl acetate extracts of tubers were most effective even more than ranitidine. In parallel, the histopathological examination showed an improvement of damaged mucosa. A high PAS reaction was observed in the treated groups indicating a relieve of the mucosal layer. A mechanistic clue of the C. alternifolius antiulcer potential was provided by the identification of its bioactive compounds using OPLS-DA. Both methanol extracts of tubers and aerial parts were more enriched in phenolic acids. The ethyl acetate extract of the aerial part was more abundant in two aldehydes. A mechanism of action was postulated based on their reported actions viz. α-carbonic anhydrase inhibition, anti-inflammatory and analgesic activity by its antioxidant activity and downregulation of several inflammatory mediators. ConclusionThis is the first study to report on the antiulcer activity of C. alternifolius tubers with identification of the key bioactive compounds and the mode of action. Future phytochemical and biological evaluation of the identified bioactive compounds are needed to confirm the plant tubers as safer alternative or adjunct therapy compared to conventional antiulcer drugs.

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.

α-Carbonic anhydrases are strongly activated by spinaceamine derivatives

A series of 4-substituted-spinaceamine (4,5,6,7-tetrahydro-imidazolo[4,5-c]pyridine) were prepared from histamine and aromatic aldehydes Schiff bases, and investigated as activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the new derivatives, and the nature of the moiety in position 4 of the bicyclic system was the factor influencing activation properties against all isoforms. For hCA I, these compounds showed KAs in the range of 2.52-21.5 µM, the most effective activator being 4-(2-hydroxyphenyl)-spinaceamine. For hCA II the activation constants ranged between 0.60 and 17.2 µM, with 4-(2,3,5,6-tetrafluorophenyl)- spinaceamine the best activator. Affinity for hCA IV was in the range of 0.52-63.8 µM, and the same compound as for hCA II was the most effective activator. The most sensitive isoform for activation was the brain-associated hCA VII, for which KAs in the range of 82 nM-4.26 µM were observed. Effective hCA VII activators were the (2-bromophenyl)-, 2,3,5,6-tetrafluorophenyl- and furyl-substituted spineaceamines (KAs of 82-95 nM). As CA activators may have pharmacologic applications in various fields, this work provides interesting derivatives for further studies.

Isolation and Expression Analysis of Three Types of α-Carbonic Anhydrases from the Antarctic Alga Chlamydomonas sp. ICE-L under Different Light Stress Treatments.

Carbonic anhydrases (CAs) are a class of zinc-containing metalloenzymes that can reversibly catalyse the hydration reaction of carbon dioxide. Antarctic algae are the most critical component of the Antarctic ecosystem; algae can enter the carbon cycle food chain by fixing carbon dioxide from the air. In this study, the complete open reading frames (ORFs) of CA1 (GenBank ID KY826431), CA2 (GenBank ID KY826432), and CA3 (GenBank ID KY826433), encoding CAs in the Antarctic ice microalga Chlamydomonas. sp. ICE-L, were successfully cloned using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the expression patterns of CAs under blue light, under UV light, and in the dark were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The CA1, CA2, and CA3 ORFs encode proteins of 376, 430, and 419 amino acids, respectively. Phylogenetic analysis revealed that all amino acid sequences showed high homology with those of C. sp. ICE-L. There are six types of algal CAs; we hypothesised that the CAs studied here are most likely α-CAs. Expression analysis showed that the transcription level of the CAs was influenced by both UV light and blue light. These findings provide additional insight into the molecular mechanisms of CAs and will accelerate the development of CAs for applications in agriculture and environmental governance.

Mantle transcriptome sequencing of Mytilus spp. and identification of putative biomineralization genes.

In molluscs, the shell secreted by mantle tissue during the biomineralization process is the first barrier against predators and mechanical damage. Changing environmental conditions, such as ocean acidification, influence shell strength and thus protection of the soft body within. Mussels are marine bivalves with important commercial and ecological value worldwide. Despite this importance, the proteins involved in the biomineralization and pigmentation processes in Mytilus spp. remain unclear, as does taxonomy of Mytilus taxa, though there have been many molecular studies. To further understanding in these areas, this study aimed to characterize and compare mantle transcriptomes of four mussel taxa using next generation sequencing. Mussels representing four taxa, were collected from several localities and RNA from mantle tissue was extracted. RNA sequences obtained were assembled, annotated and potential molecular markers, including simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were identified. Candidate contigs putatively related to biomineralization and pigmentation processes were then selected and several transcripts were chosen for phylogenetic analyses from the Bivalvia class. Transcriptome comparisons between Mytilus taxa, including gene ontology (GO) enrichment analysis and orthologues identification were performed. Of assembled contigs, 46.57%, 37.28% and 17.53% were annotated using NCBI NR, GO and Kyoto Encyclopedia of Genes and Genomes databases, respectively. Potential SSRs (483) and SNPs (1,497) were identified. Results presented a total of 1,292 contigs putatively involved in biomineralization and melanogenesis. Phylogenetic analyses of α-carbonic anhydrase, chitinase and tyrosinase revealed complex evolutionary history and diversity of these genes, which may be a result of duplication events or adaptation to different environments in mussels and other bivalves. Enrichment analyses revealed GO terms associated with pH and thermal response in Mytilus edulis from the North Sea and M. galloprovincialis from the Mediterranean Sea. The phylogenetic analysis within the genus Mytilus revealed M. californianus and M. coruscus to be genetically more distant from the other taxa: M. trossulus, M. edulis, M. chilensis and M. galloprovincialis. This work represents the first mantle transcriptome comparison between Mytilus taxa and provides contigs putatively involved in biomineralization.

Activation of human α-carbonic anhydrase isoforms I, II, IV and VII with bis-histamine schiff bases and bis-spinaceamine substituted derivatives

A series of histamine bis-Schiff bases and bis-spinaceamine derivatives were synthesised and investigated as activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the new derivatives, with activation constants in the range of 4.73–10.2 µM for hCA I, 6.15–42.1 µM for hCA II, 2.37–32.7 µM for hCA IV and 32 nM–18.7 µM for hCA VII, respectively. The nature of the spacer between the two histamine/spinaceamine units of these molecules was the main contributor to the diverse activating efficacy, with a very different fine tuning for the diverse isoforms. As CA activators recently emerged as interesting agents for enhancing cognition, in the management of CA deficiencies, or for therapy memory and artificial tissues engineering, our compounds may be considered as candidates for such applications.

Thermostability enhancement of the α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-self-labelling-protein-tag system (ASLtag)

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.

Bioinspired Metal-Organic Framework for Trace CO2 Capture.

A Zn benzotriazolate metal-organic framework (MOF), [Zn(ZnO2CCH3)4(bibta)3] (1, bibta2- = 5,5'-bibenzotriazolate), has been subjected to a mild CH3CO2-/HCO3- ligand exchange procedure followed by thermal activation to generate nucleophilic Zn-OH groups that resemble the active site of α-carbonic anhydrase. The postsynthetically modified MOF, [Zn(ZnOH)4(bibta)3] (2*), exhibits excellent performance for trace CO2 capture and can be regenerated at mild temperatures. IR spectroscopic data and density functional theory (DFT) calculations reveal that intercluster hydrogen bonding interactions augment a Zn-OH/Zn-O2COH fixation mechanism.

Activation studies with amines and amino acids of the α-carbonic anhydrase from the pathogenic protozoan Trypanosoma cruzi

The activation of a α-class carbonic anhydrase (CAs, EC 4.2.1.1) from Trypanosoma cruzi (TcCA) was investigated with the best known classes of activators, the amino acids and aromatic/heterocyclic amines. The best TcCA activators were l-/d-DOPA and 4-amino-l-phenylalanine, which had activation constants in the range of 0.38–0.83 µM. Low micromolar activators were also l-/d-Trp, l-/d-Tyr, l-Gln, histamine and serotonin (KAs of 1.79–4.92 µM), whereas l-/d-His, l-/d-Phe and l-Asp were less effective activators (KAs of 6.39–18.7 µM). Amines such as dopamine, pyridyl-alkylamines, aminoethyl-piperazine or l-adrenaline, were devoid of activating effects on TcCA. Since the role of autacoids as many of these compounds investigated here is not known for the life cycle of T. cruzi, our work provides new tools for further investigations of factors connected with this protozoan pathogen infection.

Activation Profile Analysis of CruCA4, an α-Carbonic Anhydrase Involved in Skeleton Formation of the Mediterranean Red Coral, Corallium rubrum.

CruCA4, a coral α-carbonic anhydrase (CA, EC 4.2.1.1) involved in the biomineralization process of the Mediterranean red coral, Corallium rubrum, was investigated for its activation with a panel of amino acids and amines. Most compounds showed considerable activating properties, with a rather well defined structure–activity relationship. The most effective CruCA4 activators were d-His, 4-H2N-l-Phe, Histamine, Dopamine, Serotonin, 1-(2-Aminoethyl)-piperazine, and l-Adrenaline, with activation constants in the range of 8–98 nM. Other amines and amino acids, such as d-DOPA, l-Tyr, 2-Pyridyl-methylamine, 2-(2-Aminoethyl) pyridine and 4-(2-Aminoethyl)-morpholine, were submicromolar CruCA4 activators, with KA ranging between 0.15 and 0.93 µM. Since it has been shown that CA activators may facilitate the initial phases of in-bone mineralization, our study may be relevant for finding modulators of enzyme activity, which can enhance the formation of the red coral skeleton.

Thermo-alkali-stable α-carbonic anhydrase of Bacillus halodurans: heterologous expression in Pichia pastoris and applicability in carbon sequestration.

Recombinant α-carbonic anhydrase of the polyextremophilic bacterium Bacillus halodurans TSLV1 (rBhCA) has been produced extracellularly in active form in Pichia pastoris under methanol inducible (AOX1) as well as constitutive (GAP) promoters. A marked improvement in rBhCA production was achieved by developing a P. pastoris recombinant that produces rBhCA constitutively as compared to that under inducible promoter. The purified rBhCA from P. pastoris is a glycosylated protein that displays a higher molecular mass (79.5kDa) than that produced from E. coli recombinant (75kDa); the former has a Tm of 75°C, which is slightly higher than that of the latter (72°C). The former rBhCA exhibits higher thermostability than the latter. The former sequestered CO2 efficiently similar to that of the native BhCA and the latter. This is the first report on the production of recombinant carbonic anhydrase extracellularly in P. pastoris.

Protonography and anion inhibition profile of the α-carbonic anhydrase (CruCA4) identified in the Mediterranean red coral Corallium rubrum

CruCA4 is a secreted isoform of the α-carbonic anhydrase (CA, EC 4.2.1.1) family, which has been identified in the octocoral Corallium rubrum. This enzyme is involved in the calcification process leading to the formation of the coral calcium carbonate skeleton. We report here experiments performed on the recombinant CruCA4 with the technique of protonography that can be used to detect in a simple way the enzyme activity. We have also investigated the inhibition profile of CruCA4 with one major class of CA inhibitors, the inorganic anions. A range of weak and moderate inhibitors have been identified having KI in the range of 1–100 mM, among which the halides, pseudohalides, bicarbonate, sulfate, nitrate, nitrite, and many complex inorganic anions. Stronger inhibitors were sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethylditiocarbamate, which showed a better affinity for this enzyme, with KI in the range of 75 μM–0.60 mM. All these anions/small molecules probably coordinate to the Zn(II) ion within the CA active site as enzyme inhibition mechanism.