A new affinity gel for the purification of α-carbonic anhdrases

Abstract

The new affinity gel reported in this study was prepared using EUPERGIT C250L as a chromatographic bed material, to which etylenediamine spacer arms were attached to prevent steric hindrance between the matrix and ligand, and to facilitate effective binding of the CA-specific ligand, of the aromatic sulfonamide type for the purification of α-carbonic anhydrases (Cas; EC 4.2.1.1). Indeed, the aminoethyl moieties of the affinity gel were derivatized by reaction with 4-isothiocyanatobenzenesulfonamide, with the formation of a thiourea-based gel, having inhibitory effects against CAs. Both bovine erythrocyte carbonic anhydrase BCA and human (h) erythrocyte CA isoforms I, II (hCA I and II) have been purified from hemolysates, by using this affinity gel. The greatest purification fold and column yields for BCA and for cytosolic (hCA I?+?II) enzymes were of 181-fold (21.07%) and 184-fold (9.49%), respectively. Maximum binding was achieved at 15?°C and I?=?0.3 ionic strength for α-carbonic anhydrases.     

Anion inhibition studies of a β-carbonic anhydrase from Clostridium perfringens

A β-carbonic anhydrases (CAs, EC 4.2.1.1) was recently cloned, purified and characterized kinetically in the pathogen Clostridium perfringens. We report here the first inhibition study of this enzyme (CpeCA). CpeCA was poorly inhibited by iodide and bromide, and was inhibited with K_Is in the range of 1–10 mM by a range of anions such as (thio)cyanate, azide, bicarbonate, nitrate, nitrite, hydrogensulfite, hydrogensulfide, stannate, tellurate, pyrophosphate, divanadate, tetraborate, peroxydisulfate, sulfate, iminodisulfonate and fluorosulfonate. Better inhibitory power, with K_Is of 0.36–1.0 mM, was observed for cyanide, carbonate, selenate, selenocyanide, trithiocarbonate and diethyldithiocarbamate, whereas the best CpeCA inhibitors were sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, which had K_Is in the range of 7–75 μM. This study thus provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.     

Protonography,a technique applicable for the analysis of η-carbonic anhydrase activity

Abstract

Protonography, a sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) technique derived from zymography was recently reported by our group to be an effective, cheap and reproducible technique for evidencing catalytically active α-carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as the bovine red blood cell isoform bCA or the bacterial enzyme from Vibrio cholerae, VchCA. CA activity was also observed on the protonogram of a cellular extract of Escherichia coli, evidencing the presence of one or more β-class such enzymes. Here we show that protonography can also be applied to the recently discovered η-CA family using the Plasmodium falciparum enzyme PfCA as an example. The protonogram of PfCA clearly showed catalytically active η-CA with a specific band at 22.0?kDa, which was quite distinct from the band of the red blood cell bovine enzyme bCA, which was observed at 28.8?kDa. The different migration pattern of α- and η-CAs might be a useful tool to detect Plasmodium falciparum in infected human red blood cells by an easy, routine inexpensive technique.     

The molecular evolution of β-carbonic anhydrase in Flaveria

Limited information exists regarding molecular events that occurred during the evolution of C(4) plants from their C(3) ancestors. The enzyme β-carbonic anhydrase (CA; EC 4.2.1.1), which catalyses the reversible hydration of CO(2), is present in multiple forms in C(3) and C(4) plants, and has given insights into the molecular evolution of the C(4) pathway in the genus Flaveria. cDNAs encoding three distinct isoforms of β-CA, CA1-CA3, have been isolated and examined from Flaveria C(3) and C(4) congeners. Sequence data, expression analyses of CA orthologues, and chloroplast import assays with radiolabelled CA precursor proteins from the C(3) species F. pringlei Gandoger and the C(4) species F. bidentis (L.) Kuntze have shown that both contain chloroplastic and cytosolic forms of the enzyme, and the potential roles of these isoforms are discussed. The data also identified CA3 as the cytosolic isoform important in C(4) photosynthesis and indicate that the C(4) CA3 gene evolved as a result of gene duplication and neofunctionalization, which involved mutations in coding and non-coding regions of the ancestral C(3) CA3 gene. Comparisons of the deduced CA3 amino acid sequences from Flaveria C(3), C(4), and photosynthetic intermediate species showed that all the C(3)-C(4) intermediates investigated and F. brownii, a C(4)-like species, have a C(3)-type CA3, while F. vaginata, another C(4)-like species, contains a C(4)-type CA3. These observations correlate with the photosynthetic physiologies of the intermediates, suggesting that the molecular evolution of C(4) photosynthesis in Flaveria may have resulted from a temporally dependent, stepwise modification of protein-encoding genes and their regulatory elements.     

Cloning,expression and purification of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

Abstract

The antimalarial drugs are of fundamental importance in the control of malaria, especially for the lack of efficient treatments and acquired resistance to the existing drugs. For this reason, there is a continuous work in identifying novel, less toxic and effective chemotherapies as well as new therapeutic targets against the causative agents of malaria. In this context, a superfamily of metalloenzymes named carbonic anhydrases (CAs, EC 4.2.1.1) has aroused a great interest as druggable enzymes to limit the development of Plasmodium falciparum gametocytes. CAs catalyze a common reaction in all life domains, the carbon dioxide hydration to bicarbonate and protons (CO₂?+?H₂O ? HCO₃^-?+?H⁺). P. falciparum synthesizes pyrimidines de novo starting from HCO₃^-, which is generated from CO₂ through the action of the η-CA identified in the genome of the protozoan. Here, we propose a procedure for the preparation of a wider portion of the protozoan η-CA, named PfCAdom (358 amino acid residues), with respect to the truncated form prepared by Krungkrai et al. (PfCA1, 235 amino acid residues). The results evidenced that the recombinant PfCAdom, produced as a His-tag fusion protein, was 2.7 times more active with respect the truncated form PfCA1.     

Sulfonamide inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae

The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. VchCA, the α-CA from this species was investigated earlier, whereas the β-class enzyme, VchCAβ was recently cloned, characterized kinetically and its X-ray crystal structure reported by this group. Here we report an inhibition study with sulfonamides and one sulfamate of this enzyme. The best VchCAβ inhibitors were deacetylated acetazolamide and methazolamide and hydrochlorothiazide, which showed inhibition constants of 68.2–87.0 nM. Other compounds, with medium potency against VchCAβ, (K_Is in the range of 275–463 nM), were sulfanilamide, metanilamide, sulthiame and saccharin whereas the clinically used agents such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, zonisamide and celecoxib were micromolar inhibitors (K_Is in the range of 4.51–8.57 μM). Identification of potent and possibly selective inhibitors of VchCA and VchCAβ over the human CA isoforms, may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzymes.     

Sulphonamide inhibition studies of the β-carbonic anhydrase from the bacterial pathogen Clostridium perfringens

The β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Clostridium perfringens (CpeCA) was recently characterised kinetically and for its anion inhibition profile. In the search of effective CpeCA inhibitors, possibly useful to inhibit the growth/pathogenicity of this bacterium, we report here an inhibition study of this enzyme with a panel of aromatic, heterocyclic and sugar sulphonamides/sulphamates. Some sulphonamides, such as acetazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, sulthiame and 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide were effective CpeCA inhibitors, with K_Is in the range of 37.4–71.6?nM. Zonisamide and saccharin were the least effective such inhibitors, whereas many other aromatic and heterocyclic sulphonamides were moderate – weak inhibitors with K_Is ranging between 113 and 8755?nM. Thus, this study provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.     

A new procedure for the cloning,expression and purification of the β-carbonic anhydrase from the pathogenic yeast Malassezia globosa,an anti-dandruff drug target

Malassezia yeasts are almost exclusively the single eukaryotic members of the fungal flora of the skin. Malassezia globosa and Malassezia restricta are found on the skin of practically all humans. Malassezia globosa is highly implicated in the pathogenesis of dandruff and its genome encodes for only one carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the β-class (MgCA). It has been indeed demonstrated that in many pathogenic microorganisms, CAs are essential for their life cycle and their inhibition can lead to growth impairment and defects. In the previous work, the recombinant MgCA was investigated for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target. MgCA was cloned as GST-fusion protein, but the yield was rather low and the protein was often found in inclusion bodies. Here, we propose an alternative procedure consisting in cloning the recombinant MgCA as His-Tag fusion protein. This procedure resulted in a good method to express and purify the active recombinant MgCA, and the protein recovery was better with respect to that used for preparing MG-CA (β-CA cloned as GST-fusion protein).     

Inhibition of the β-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa with monothiocarbamates

A series of monothiocarbamates (MTCs) was investigated for the inhibition of the β-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa, MgCA. These MTCs incorporate various scaffolds, among which aliphatic amine with 1–4 carbons atom in their molecule, morpholine, piperazine, as well as phenethylamine and benzylamine derivatives. All the reported MTCs displayed a better efficacy in inhibiting MgCA compared to the clinically used sulphonamide drug acetazolamide (K_I of 74?μM), with K_Is spanning between 1.85 and 18.9?μM. The homology model of the enzyme previously reported by us was used to rationalize the results by docking some of these MTCs within the fungal CA active site. This study might be useful to enrich the knowledge of the MgCA inhibition profile, eliciting novel ideas pertaining the design of modulators with potential efficacy in combatting dandruff or other fungal infections.     

Antileishmanial activity of sulphonamide nanoemulsions targeting the β-carbonic anhydrase from Leishmania species

The β-carbonic anhydrase (CA, EC 4.2.1.1) from Leishmania spp. (LdcCA) is effectively inhibited by aromatic/heterocyclic sulphonamides, in the low nanomolar range, but no in vitro antileishmanial activity was detected for such compounds. We formulated some of these sulphonamides as nanoemulsions (NEs) in clove oil, and tested them in vitro against Leishmania infantum MHOM/BR/1974/PP75 and Leishmania amazonensis IFLA/BR/1967/PH8 strains. Interesting inhibitory concentrations IC₅₀ were observed for some of the sulphonamides NEs, with IC₅₀ as low as 3.90?µM (NE-3F) and 2.24?µM (NE-5B) for L. amazonensis and 3.47?µM (NE-5B) for L. infantum. Some of the investigated NEs displayed toxicity for macrophages beyond the parasites. For the same nonoemulsions, a selective index (SI) greater than for Amphotericin B. Haemolytic assay using human red blood cells indicate that the NEs were less cytotoxic than amphotericin B, a widely used antifungal agent. NEs demonstrated to be an excellent strategy for increasing the penetration of these hydrophilic drugs through membranes, with a huge increase of efficacy over the sulphonamide CA inhibitor (CAI) alone.     

A rapid method for the purification of β-lactamase from Bacillus cereus by affinity chromatography

A procedure for the purification of β-lactamase from Bacillus cereus in a single chromatographic step is described. The enzyme is isolated from the crude culture supernatant by affinity chromatography. An inhibitor, methicillin, was immobilised by covalent attachment to the insoluble column gel, Sepharose. The enzyme was adsorbed to the column ligand from the crude supernatant and was subsequently released by increasing the ionic strength of the eluting buffer. In this way the enzyme was selectively isolated from other proteins in the crude supernatant. About 98% of the original β-lactamase activity was recovered in the purified enzyme fraction.     

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 k_cat?=??4.2?×?10⁵?s^?1 and k_cat/K_m 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.     

Immobilized metal affinity chromatography in open-loop simulated moving bed technology: Purification of a heat stable histidine tagged β-glucosidase

Open-loop simulated moving bed (SMB) has been used for immobilized metal affinity chromatographic (IMAC) purification of his-tagged β-glucosidase expressed in E. coli. A simplified approach based on an optimized single column protocol is used to design the open-loop SMB. A set of columns in the SMB represent one step in the chromatographic cycle i.e. there will be one set each of columns for load, wash, elution etc within the SMB. Only the wash and elution are operated with columns in sequence. The β-glucosidase was purified to almost single band purity with a purification factor of 15 and a recovery of 91%. SMB-performance showed reduced buffer consumption, higher purification fold, a better yield and higher productivity.     

Inhibition studies of a β-carbonic anhydrase from Brucella suis with a series of water soluble glycosyl sulfanilamides

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified characterized kinetically and for inhibition with a series of water soluble glycosylated sulfanilamides. bsCA 1 has appreciable activity as catalyst for the hydration of CO₂ to bicarbonate, with a k_cat of 6.4 × 10⁵ s^?1 and k_cat/K_m of 3.9 × 10⁷ M^?1 s^?1. All types of inhibitory activities have been detected, with K_Is in the range of 8.9–110 nM. The best bsCA 1 inhibitor were the galactose and ribose sulfanilamides, with inhibition constants of 8.9–9.2 nM. Small structural changes in the sugar moiety led to dramatic differences of enzyme inhibitory activity for this series of compounds. One of the tested glycosylsulfonamides and acetazolamide significantly inhibited the growth of the bacteria in cell cultures.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids

The protein encoded by the NCE103 gene of Candida glabrata, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K_As of 21.2–37 μM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.1–16.7 μM). The best activators were l-/d-Tyr, with activation constants of 7.1–9.5 μM. This study may bring a better understanding of the catalytic/activation mechanisms of β-CAs from pathogenic fungi.     

Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae β-carbonic anhydrase

Dithiocarbamates (DTCs) prepared from primary or secondary amines, which incorporated amino/hydroxyl-alkyl, mono-/bicyclic aliphatic/heterocyclic rings based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, were investigated for the inhibition of α- and β-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, such as the human (h) isoform hCA I and II, as well as the Saccharomyces cerevisiae β-CA, scCA. The yeast and its β-CA were shown earlier to be useful models of pathogenic fungal infections. The DTCs investigated here were medium potency hCA I inhibitors (K_Is of 66.5–910?nM), were more effective as hCA II inhibitors (K_Is of 8.9–107?nM) and some of them showed excellent, low nanomolar activity against the yeast enzyme, with inhibition constants ranging between 6.4 and 259?nM. The detailed structure activity relationship for inhibition of the yeast and human enzymes is discussed. Several of the investigated DTCs showed excellent selectivity ratios for inhibiting the yeast over the human cytosolic CA isoforms.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such as l-/d-His, Phe, DOPA, Trp (K_As of 82–90 μM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.2–21.3 μM). The best activator was l-adrenaline, with an activation constant of 0.95 μM. This study may help to better understand the catalytic/activation mechanisms of the β-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.     

Carbonic anhydrase inhibitors. Inhibition and homology modeling studies of the fungal β-carbonic anhydrase from Candida albicans with sulfonamides

The β-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Candida albicans (Nce103) is involved in a CO₂ sensing pathway critical for the pathogen life cycle and amenable to drug design studies. Herein we report an inhibition study of Nce103 with a library of sulfonamides and one sulfamate, showing that Nce103, similarly to the related enzyme from Cryptococcus neoformans Can2, is inhibited by these compounds with K_Is in the range of 132 nM–7.6 μM. The best Nce103 inhibitors were acetazolamide, methazolamide, bromosulfanilamide, and 4-hydroxymethylbenzenesulfonamide (K_Is < 500 nM). A homology model was generated for Nce103 based on the crystal structure of Can2. The model shows that compounds with zinc-binding groups incorporating less polar moieties and compact scaffolds generate stronger Nce103 inhibitors, whereas highly polar zinc-binding groups and bulkier compounds appear more promising for the specific inhibition of Can2. Such compounds may be useful for the design of antifungal agents possessing a new mechanism of action.