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.     

Biochemical characterization of the chloroplastic β-carbonic anhydrase from Flaveria bidentis (L.) “Kuntze”

Abstract

C₃ and C₄ plant carbonic anhydrases (CAs) are zinc-enzymes that catalyze the reversible hydration of CO₂. They are sub-divided in three classes: α, β and γ, being distributed between both photosynthetic subtypes. The C₄ dicotyledon species Flaveria bidentis (L.) “Kuntze” contains a small gene family encoding three distinct β-CAs, named FbiCA1, FbiCA2 and FbiCA3. We have expressed and purified recombinant FbiCA1, which is localized in the chloroplast where it is thought to play a role in lipid biosynthesis and antioxidant activity, and biochemically characterized it by spectroscopic and inhibition experiments. FbiCA1 is a compact octameric protein that is moderately inhibited by carboxylate molecules. Surprisingly, pyruvate, but not lactate, did not inhibit FbiCA1 at concentrations up to 10?mM, suggesting that its capacity to tolerate high pyruvate concentration reflects the high concentration of pyruvate in the chloroplasts of bundle-sheath and mesophyll cells involved in C₄ photosynthesis.     

Cloning,characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea

We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO₂ hydration to bicarbonate and protons, with the following kinetic parameters: k_cat of 6.0 × 10⁵ s⁻¹ and a k_cat/K_m of 4.7 × 10⁶ M⁻¹ × s⁻¹. This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a K_I of 502 nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed K_Is in the range of 1.4–4.4 mM. Diethyldithiocarbamate was a better inhibitor (K_I of 0.58 mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with K_Is ranging between 8 and 38 μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here.     

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.     

Sulfonamide inhibition studies of the δ-carbonic anhydrase from the diatom Thalassiosira weissflogii

The δ-carbonic anhydrase (CA, EC 4.2.1.1) TweCA from the marine diatom Thalassiosira weissflogii has recently been cloned, purified and its activity/inhibition with anions investigated. Here we report the first sulfonamide/sulfamate inhibition study of a δ-class CA. Among the 40 such compounds investigated so far, 3-bromosulfanilamide, acetazolamide, ethoxzolamide, dorzolamide and brinzolamide were the most effective TweCA inhibitors detected, with K_Is of 49.6–118 nM. Many simple aromatic sulfonamides as well as dichlorophenamide, benzolamide, topiramate, zonisamide, indisulam and valdecoxib were medium potency inhibitors, (K_Is of 375–897 nM). Saccharin and hydrochlorothiazide were ineffective inhibitors of the δ-class enzyme, with K_Is of 4.27–9.20 μM. The inhibition profile of the δ-CA is very different from that of α-, β- and γ-CAs from different organisms. Although no X-ray crystal structure of this enzyme is available, we hypothesize that as for other CA classes, the sulfonamides inhibit the enzymatic activity by binding to the Zn(II) ion from the δ-CA active site.     

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.     

Anion inhibition studies of the α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae

An α-carbonic anhydrase (CA, EC 4.2.1.1) has been recently cloned and characterized in the human pathogenic bacterium Vibrio cholerae, denominated VchCA (Del Prete et al. J. Med. Chem. 2012, 55, 10742). This enzyme shows a good catalytic activity for the CO₂ hydration reaction, comparable to that of the human (h) isoform hCA I. Many inorganic anions and several small molecules were investigated as VchCA inhibitors. Inorganic anions such as cyanate, cyanide, hydrogen sulfide, hydrogen sulfite, and trithiocarbonate were effective VchCA inhibitors with inhibition constants in the range of 33–88 μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with K_Is of 7–43 μM. Halides (bromide, iodide), bicarbonate and carbonate were much less effective VchCA inhibitors, with K_Is in the range of 4.64–28.0 mM. The resistance of VchCA to bicarbonate inhibition may represent an evolutionary adaptation of this enzyme to living in an environment rich in this ion, such as the gastrointestinal tract, as bicarbonate is a virulence enhancer of this bacterium.     

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis

A carbonic anhydrase (CA, EC 4.2.1.1) denominated PgiCA, belonging to the γ-class, from the oral pathogenic bacteria Porphyromonas gingivalis, the main causative agent of periodontitis, was investigated for its inhibition profile with sulfonamides and one sulfamate. Dichlorophenamide, topiramate and many simple aromatic/heterocyclic sulfonamides were ineffective as PgiCA inhibitors whereas the best inhibition was observed with halogenosulfanilamides incorporating heavy halogens, 4-hydroxy- and 4-hydroxyalkyl-benzenesulfonamides, acetazolamide, methazolamide, zonisamide, indisulam, celecoxib, saccharin and hydrochlorothiazide (K_Is in the range of 131–380 nM). The inhibition profile of PgiCA was very different from that of CAM, hCA I and II or the β-CA from a protozoan parasite (Leishmania donovani chagasii). Identification of potent and possibly selective inhibitors of PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of this enzyme.     

Anion inhibition study of the β-carbonic anhydrase (CahB1) from the cyanobacterium Coleofasciculus chthonoplastes (ex-Microcoleus chthonoplastes)

We investigated the catalytic activity and inhibition of the β-class carbonic anhydrase (CA, EC 4.2.1.1) CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO₂ hydration, with a k_cat of 2.4 × 10⁵ s⁻¹ and a k_cat/K_m of 6.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme (K_Is >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with K_Is of 29.9–48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a range of heavy metal ions-containing anions were submillimolar–millimolar inhibitors (K_Is in the range of 0.15–0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 8–75 μM, whereas acetazolamide inhibited the enzyme with a K_I of 76 nM. This is the first kinetic and inhibition study of a cyanobacterial CA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentrating mechanism which assures substrate to RubisCO for the CO₂ fixation by these organisms, a detailed kinetic/inhibition study may be essential for a better understanding of this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO₂ capture processes.     

Characterization and anions inhibition studies of an α-carbonic anhydrase from the teleost fish Dicentrarchus labrax

Carbonic anhydrase (CA; EC 4.2.1.1) was purified from the gill of the teleost fish Dicentrarchus labrax (European seabass). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 84.9-fold with a yield of 58%, and a specific activity of 838.9 U/mg proteins. It has an optimum pH at 8.0; an optimum temperature at 10°C. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate. The following anions, H?NSO??, I?, SCN?, NO??, NO??, N??, Br?, Cl?, SO?2?, and F? showed inhibitory effects on the enzyme. Sulfamic acid, iodide, and thiocyanate exhibited the strongest inhibitory action, in the micromolar range (K(i)s of 87-187 μM). NO??, NO?? and N?? were moderate inhibitors, whereas other anions showed only weak actions. All tested anions inhibited the enzyme in a competitive manner. Our findings indicate that these anions inhibit the fish enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various anions differs significantly between the fish and mammalian CAs.     

Carbonic anhydrase inhibitors. Characterization and inhibition studies of the most active β-carbonic anhydrase from Mycobacterium tuberculosis,Rv3588c

The Rv3588c gene product of Mycobacterium tuberculosis, a β-carbonic anhydrase (CA, EC 4.2.1.1) denominated here mtCA 2, shows the highest catalytic activity for CO₂ hydration (k_cat of 9.8 × 10⁵ s^?1, and k_cat/K_m of 9.3 × 10⁷ M^?1 s^?1) among the three β-CAs encoded in the genome of this pathogen. A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 2, and some diazenylbenzenesulfonamides were synthesized from sulfanilamide/metanilamide by diazotization followed by coupling with amines or phenols. Several low nanomolar mtCA 2 inhibitors have been detected among which acetazolamide, ethoxzolamide and some 4-diazenylbenzenesulfonamides (K_Is of 9–59 nM). As the Rv3588c gene was shown to be essential to the growth of M. tuberculosis, inhibition of this enzyme may be relevant for the design of antituberculosis drugs possessing a novel mechanism of action.     

Characterization and inhibition studies of an α-carbonic anhydrase from the endangered sturgeon species Acipenser gueldenstaedti

An α-carbonic anhydrase (CA, EC 4.2.1.1) was purified and characterized kinetically from erythrocytes of the sturgeon Acipenser gueldenstaedti, an endangered species. The sturgeon enzyme (AgCA) showed kinetic parameters for the CO(2) hydration reaction comparable with those of the human erythrocytes enzyme hCA II, being a highly active enzyme, whereas its esterase activity with 4-nitrophenyl acetate as substrate was lower. Sulphonamide inhibitors (acetazolamide, sulphanilamide) strongly inhibited AgCA, whereas metal ions (Ag(+), Zn(2+), Cu(2+) and Co(2+)) were weak, millimolar inhibitors. Several widely used pesticides (2,4-dichlorophenol, dithiocarbamates, parathion and carbaryl) were also assayed as inhibitors of this enzyme. The dithiocarbamates were low micromolar AgCA inhibitors (IC(50) of 16-18 μM), whereas the other pesticides inhibited the enzyme with IC(50)s in the range of 102-398 μM. The wide use of dithiocarbamate pesticides may be one of the factors enhancing the vulnerability of this sturgeon species to pollutants.     

Anion inhibition studies of an α-carbonic anhydrase from the living fossil Astrosclera willeyana

An α-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the living fossil sponge Astrosclera willeyana, Astrosclerin, was investigated for its inhibition profile with simple inorganic anions, complex anions and other small molecules known to interact with these zinc enzymes. Astrosclerin is a catalytically highly efficient enzyme, and is inhibited in the low micromolar range by sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, and in the submillimolar range by a variety of anions including fluoride, chloride, cyanate, thiocyanate, cyanide, hydrogen sulfide, bisulfate, stannate, perosmate, divanadate, perrhenate, perruthenate, selenocyanide, trithiocarbonate, diethyldithiocarbamate and iminodisulfonate. Less efficient Astrosclerin inhibitors were sulfate, bromide, iodide, azide, bicarbonate, carbonate, tetraborate and perchlorate (K(I)s of 5.11-30.6mM) whereas tetrafluoroborate was not at all inhibitory. Because Astrosclerin is involved in calcification processes in vivo, its anion inhibition profile may be important for future studies designed to shed light on the physiologic functions of α-CAs in marine organisms.     

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 K_I 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 K_I 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.     

Mono- and di-thiocarbamate inhibition studies of the δ-carbonic anhydrase TweCAδ from the marine diatom Thalassiosira weissflogii

The inhibition of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii, TweCAδ, was investigated using a panel of 36 mono- and di-thiocarbamates chemotypes that have recently been shown to inhibit mammalian and pathogenic CAs belonging to the α- and β-classes. TweCAδ was not significantly inhibited by most of such compounds (K_I values above 20 µM). However, some aliphatic, heterocyclic, and aromatic mono and di-thiocarbamates inhibited TweCAδ in the low micromolar range. For some compounds incorporating the piperazine ring, TweCAδ was effectively inhibited (K_Is from 129 to 791?nM). The most effective inhibitors identified in this study were 3,4-dimethoxyphenyl-ethyl-mono-thiocarbamate (K_I of 67.7?nM) and the R-enantiomer of the nipecotic acid di-thiocarbamate (K_I of 93.6?nM). Given that the activity and inhibition of this class of enzyme have received limited attention until now, this study provides new molecular probes and information for investigating the role of δ-CAs in the carbon fixation processes in diatoms, which are responsible for significant amounts of CO₂ taken from the atmosphere by these marine organisms.     

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.     

Purification and inhibition studies with anions and sulfonamides of an α-carbonic anhydrase from the Antarctic seal Leptonychotes weddellii

A high activity α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from various tissues of the Antarctic seal Leptonychotes weddellii. The new enzyme, denominated lwCA, has a catalytic activity for the physiologic CO(2) hydration to bicarbonate reaction, similar to that of the high activity human isoform hCA II, with a k(cat) of 1.1×10(6) s(-1), and a k(cat)/K(m) of 1.4×10(8) M(-1) s(-1). The enzyme was highly inhibited by cyanate, thiocyanate, cyanide, bicarbonate, carbonate, as well as sulfamide, sulfamate, phenylboronic/phenylarsonic acids (K(I)s in the range of 46-100 μM). Many clinically used sulfonamides, such as acetazolamide, methazolamide, dorzolamide, brinzolamide and benzolamide were low nanomolar inhibitors, with K(I)s in the range of 5.7-67 nM. Dichlorophenamide, zonisamide, saccharin and hydrochlorothiazide were weaker inhibitors, with K(I)s in the range of 513-5390 nM. The inhibition profile with anions and sulfonamides of the seal enzyme was rather different from those of the human isoforms hCA I and II. The high sensitivity to bicarbonate inhibition of lwCA, unlike that of the human enzymes, may reflect an evolutionary adaptation to the deep water, high CO(2) partial pressure and hypoxic conditions in which Weddell seals spend much of their life.     

Anion inhibition studies of an α-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1

The newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 encodes an α-carbonic anhydrases (CAs, EC 4.2.1.1) which is highly catalytically active and thermostable. Here we report the inhibition of this enzyme, denominated SspCA, with inorganic and complex anions and other molecules interacting with zinc proteins. SspCA was inhibited in the micromolar range by diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic and phenylarsonic acid, trithiocarbonate and selenocyanide (K(I)s of 4-70μM) and in the submillimolar one by iodide, cyanide, (thio)cyanate, hydrogen sulfide, azide, nitrate, nitrite, many complex anions incorporating heavy metal ions and iminodisulfonate (K(I)s of 0.48-0.86mM). SspCA was not substantially inhibited by bicarbonate and carbonate, hydrogensulfite and peroxidisulfate (K(I)s in the range of 21.1-84.6mM). The exceptional thermostability and lack of strong affinity for hydrogensulfide, bicarbonate, and carbonate make this enzyme an interesting candidate for biotechnological applications of enzymatic CO(2) fixation.