Identification and inhibition of carbonic anhydrases from nematodes

Abstract

Carbonic anhydrases (CAs) are metalloenzymes, and classified into the evolutionarily distinct α, β, γ, δ, ζ, and η classes. α-CAs are present in many living organisms. β- and γ-CAs are expressed in most prokaryotes and eukaryotes, except for vertebrates. δ- and ζ-CAs are present in phytoplanktons, and η-CAs have been found in Plasmodium spp. Since the identification of α- and β-CAs in Caenorhabditis elegans, the nematode CAs have been considered as an emerging target in research focused on antiparasitic CA inhibitors. Despite the presence of α-CAs in both helminths and vertebrates, structural studies have revealed different kinetic and inhibition results. Moreover, lack of β-CAs in vertebrates makes this enzyme as an attractive target for inhibitory studies against helminthic infection. Some CA inhibitors, such as sulfonamides, have been evaluated against nematode CAs. This review article aims to present comprehensive information about the nematode CAs and their inhibitors as potential anthelminthic drugs.     

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.     

A matter of structure: structural comparison of fungal carbonic anhydrases

Carbonic anhydrases (CAs) are metalloenzymes that catalyze the interconversion of carbon dioxide (CO₂) and hydrogen carbonate. CAs are distributed over all the three domains of life and are divided into five distinct evolutionarily unrelated gene families (α, β, γ, δ, ζ). In the large fungal kingdom, the majority of fungi encode multiple copies of β-CAs, with some also possessing genes for α-class CAs. Hemiascomycetous and basidiomycetous yeasts encode one or two β-CAs, while most of the filamentous ascomycetes have multiple copies of genes encoding α- and β-CAs. The functions of fungal β-CAs have been investigated intensively, while the role of fungal α-CAs is mostly unknown. The β-CAs are involved in sexual development, CO₂-sensing, pathogenicity, and survival in ambient air. Only recently, researchers have begun to use functional and structural data of CAs from pathogenic and non-pathogenic organisms to develop powerful and effective drugs and inhibitors or to identify enzymes that can be utilized in industrial applications. Despite the large number of fungal CAs known, only five have been characterized structurally: the α-CA AoCA of Aspergillus oryzae, the full length β-CA Can2 from the pathogenic basidiomycete Cryptococcus neoformans, the N-terminally truncated Saccharomyces cerevisiae β-CA Nce103, and two β-CAs of Sordaria macrospora. This review focuses on the functional and structural properties of fungal CAs.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Transgenic overexpression of Leucaena β-carbonic anhydrases in tobacco does not affect carbon assimilation and overall biomass

The role of β-carbonic anhydrases (CAs) in C₃ plant carbon assimilation is not clear. In this study, the primary role of C₃ plant β-CAs in carbon assimilation was investigated for which, a chloroplastic β-CA gene (cacp) and a cytoplasmic β-CA gene (cacyt) from a C₃ tree-legume of tropics, Leucaena leucocephala (leucaena) were overexpressed in Nicotiana tabacum (tobacco). The cacp and cacyt β-CA isoforms from leucaena were overexpressed separately and also together in tobacco resulting in three types of transgenic tobacco plants (i) expressing cacp only (ii) expressing cacyt only and (iii) co-expressing both cacp and cacyt. These transgenic plants exhibited significantly higher activity of β-CAs as compared with wild-type plants. The percent increase in the CA activity of transgenic plants expressing leucaena cacyt or cacp was found to be ～51 and ～55%, respectively. The transgenic tobacco expressing both the leucaena β-CA isoforms exhibited ～63% increase in CA activity as compared with the wild-type. However, despite notable increase in the CA activity of transgenic tobacco plants, no difference was observed in their phenotype, chlorophyll content and the overall dry weight compared with that of wild-type suggesting that C₃ β-CAs are not involved in active accumulation of inorganic carbon.     

New light on bacterial carbonic anhydrases phylogeny based on the analysis of signal peptide sequences

Among protein families, carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes characterized by a common reaction mechanism in all life domains: the carbon dioxide hydration to bicarbonate and protons (CO₂+H₂O ? HCO₃^?+H⁺). Six genetically distinct CA families are known to date, the α-, β-, γ-, δ-, ζ- and η-CAs. The last CA class was recently discovered analyzing the amino acid sequences of CAs from Plasmodia. Bacteria encode for enzymes belonging to the α-, β-, and γ-CA classes and recently, phylogenetic analysis revealed an interesting relationship regarding the evolution of bacterial CA classes. This result evidenced that the three bacterial CA classes, in spite of the high level of the structural similarity, are evolutionarily distinct, but we noted that the primary structure of some β-CAs identified in the genome of Gram-negative bacteria present a pre-sequence of 18 or more amino acid residues at the N-terminal part. These observations and subsequent phylogenetic data presented here prompted us to propose that the β-CAs found in Gram-negative bacteria with a periplasmic space and characterized by the presence of a signal peptide might have a periplasmic localization and a role similar to that described previously for the α-CAs.     

Biochemical characterization of recombinant β-carbonic anhydrase (PgiCAb) identified in the genome of the oral pathogenic bacterium Porphyromonas gingivalis

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β-, γ-, δ- and ζ-CAs are ubiquitous metalloenzymes present in prokaryotes and eukaryotes. CAs started to be investigated in detail only recently in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many such organisms they are essential for the life cycle of the organism. CA inhibition leads to growth impairment or growth defects in several pathogenic bacteria. The microbiota of the human oral mucosa consists of a myriad of bacterial species, Porphyromonas gingivalis being one of them and the major pathogen responsible for the development of chronic periodontitis. The genome of P. gingivalis encodes for a β- and a γ-CAs. Recently, our group purified the recombinant γ-CA (named PgiCA) which was shown to possess a significant catalytic activity for the reaction that converts CO₂ to bicarbonate and protons, with a k_cat of 4.1?×?10^5?s^?1 and a k_cat/K_m of 5.4?×?10^7?M^?1?×?s^?1. We have also investigated its inhibition profile with a range of inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate. Here, we describe the cloning, purification and kinetic parameters of the other class of CA identified in the genome of P. gingivalis, the β-CA, named PgiCAb. This enzyme has a good catalytic activity, with a k_cat of 2.8?×?10^5?s^?1 and a k_cat/K_m of 1.5?×?10^7?M^?1?×?s^?1. PgiCAb was also inhibited by the clinically used sulfonamide acetazolamide, with an inhibition constant of 214?nM. The role of CAs as possible virulence factors of P. gingivalis is poorly understood at the moment but their good catalytic activity and the fact that they might be inhibited by a large number of compounds, which may pave the way for finding inhibitors with antibacterial activity that may elucidate these phenomena and lead to novel antibiotics.     

Gene encoding γ-carbonic anhydrase is cotranscribed with <Emphasis Type="Italic">argC</Emphasis> and induced in response to stationary phase and high CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7

Background  

Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO₂ to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (γ-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one β-CA and two γ-CAs.     

Biochemical characterization of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis,PgiCA

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. CAs are present in many pathogenic species and are involved in the bicarbonate metabolism/biosynthetic reactions involving this ion. Ubiquity of these enzymes suggests a pivotal role in microbial virulence and pathogenicity. Porphyromonas gingivalis is an anaerobic bacterium, which colonizes the oral cavity, being involved in the pathogenesis of periodontitis, an inflammatory disease leading to tooth loss. Recently, we reported an anion inhibitory study on the γ-CA (denominated PgiCA) identified in the genome of this Gram-negative bacterium. In this paper we continue our research on PgiCA, and describe the biochemical characterization of the recombinant protein, its thermal stability, the oligomeric state and the enzyme kinetics. PgiCA is a polypeptide chain formed of 192 amino acids and displays an identity of 30–33% when compared with the prototypical γ-CAs, CAM or CAMH (from Methanosarcina thermophila) or CcmM (from Thermosynechococcus elongatus). A subunit molecular mass of 21?kDa was estimated by SDS-PAGE, while HPLC size exclusion chromatography under native conditions gave an estimated molecular mass of 65?kDa suggesting that the recombinant enzyme self-associate in a homotrimer, as all other γ-CAs studied so far. Enzyme kinetic analysis showed that PgiCA is 62 times more effective as a catalyst compared to CAM, the only other γ-CA characterized in detail kinetically. All these features represent an interesting attractive for the drug design of inhibitors/activators of this new enzyme.     

Inhibition of β-carbonic anhydrases with ureido-substituted benzenesulfonamides

A series of sulfonamides was prepared by reaction of sulfanilamide with aryl/alkyl isocyanates. The ureido-substituted benzenesulfonamides showed a very interesting profile for the inhibition of several carbonic anhydrases (CAs, EC 4.2.1.1) such as the human hCA II and three β-CAs from pathogenic fungal or bacterial species. The Candida albicans enzyme was inhibited with potencies in the range of 3.4-3970 nM, whereas the Mycobacterium tuberculosis enzymes Rv1284 and Rv3273 were inhibited with K_is in the range of 4.8-6500 nM and of 6.4-6850 nM, respectively. The structure-activity relationship for this class of inhibitors is rather complex, but the main features associated with effective inhibition of both α- and β-CAs investigated here have been delineated. The nature of the moiety substituting the second ureido nitrogen is the determining factor in controlling the inhibitory power, probably due to the flexibility of the ureido linker and the possibility of this moiety to orientate in different subpockets of the active site cavities of these enzymes.     

Recent advances in the discovery of zinc-binding motifs for the development of carbonic anhydrase inhibitors

Abstract

In addition to the sulfonamides and their isosteres, recently novel carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) which act by binding to the metal ion from the active site were discovered. Based on the X-ray crystal structure of the CA II–trithiocarbonate adduct, dithiocarbamates, xanthates and thioxanthates were shown to potently inhibit α- and β-CAs. The hydroxamates constitute another class of recently studied CAIs both against mammalian and protozoan enzymes. Another chemotype for which CA inhibitory properties were recently reported is the salicylaldoxime scaffold. X-ray crystal structures were reported for CA II complexed with dithiocarbamates and hydroxamates, whereas the xanthates and salicylaldoximes were investigated by kinetic measurements and docking studies. The dithiocarbamates and the xanthates showed potent antiglaucoma activity in animal models of the disease whereas some hydroxamates inhibited the growth of Trypanosoma cruzii probably by inhibiting the protozoan CA.     

Benzenesulfonamides incorporating nitrogenous bases show effective inhibition of β-carbonic anhydrases from the pathogenic fungi Cryptococcus neoformans,Candida glabrata and Malassezia globosa

There is an urgent need for new chemotherapic agents to treat human fungal infections due to emerging and spreading globally resistance mechanisms. Among the new targets that have been recently investigated for the development of antifungal drugs there are the metallo-enzymes Carbonic Anhydrases (CAs, EC 4.2.1.1). The inhibition of the β-CAs identified in many pathogenic fungi leads to an impairment of parasite growth and virulence, which in turn leads to a significant anti-infective effect. Based on antifungal nucleoside antibiotics, the inhibition of the β-CAs from the resistance-showing fungi Candida glabrata (CgNce103), Cryptococcus neoformans (Can2) and Malasszia globosa (MgCA) with a series of benzenesulfonamides bearing nitrogenous bases, such as uracil and adenine, is here reported. Many such compounds display low nanomolar (<100 nM) inhibitory potency against Can2 and CgNce103, whereas the activity of MgCA is considerably less affected (inhibition constants in the range 138.8–5601.5 nM). The β-CAs inhibitory data were compared with those against α-class human ubiquitous isoforms. Interesting selective inhibitory activities for the target fungal CAs over hCA I and II were reported, which make nitrogenous base benzenesulfonamides interesting tools and leads for further investigations in search of new antifungal with innovative mechanisms 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.     

Purification and enzymatic characterization of trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from Rhodococcus opacus and enzymatic formation of α, β-unsaturated ketones

ABSTRACT

Trans-o-hydroxybenzylidenepyruvate (tHBPA) hydratase-aldolase (RnoE) catalyzes the conversion of tHBPA to 2-hydroxybenzaldehyde and pyruvate. We purified RnoE from Rhodococcus opacus and characterized its enzymatic properties. It exhibited maximum enzyme activity at 60°C and catalyzed the reverse reaction, converting various aromatic benzaldehydes and pyruvate to benzylidenepyruvate, indicating that this enzyme can be adapted for the enzymatic synthesis of α, β-unsaturated ketones.     

Ketoreductase activity for reduction of substituted-β-tetralones utilizing aqueous-organic systems and β-cyclodextrin derivatives

Abstract

Ketoreductases (KREDs) were employed for enantioselective reduction of 7-hydroxy-2-tetralone 1a and adduct 7-methoxy-2-tetralonbisulfite 2a to their corresponding (S)-/(R)-alcohols. In addition, the effect of additives such as organic solvents and β-cyclodextrin derivatives on the enzyme reductions was investigated. The changes in enzyme activity as a function of additives were correlated to structural alterations of the KREDs using circular dichroism and fluorescence spectrophotometric measurements. The effects of both the organic solvents and β-cyclodextrin derivatives on substrate solubility and equilibrium binding constants (log K) of β-cyclodextrin-substrate complexes were determined.     

Structure and catalytic mechanism of the β-carbonic anhydrases

The β-carbonic anhydrases (β-CAs) are a diverse but structurally related group of zinc-metalloenzymes found in eubacteria, plant chloroplasts, red and green algae, and in the Archaea. The enzyme catalyzes the rapid interconversion of CO₂ and H₂O to HCO₃⁻ and H⁺, and is believed to be associated with metabolic enzymes that consume or produce CO₂ or HCO₃⁻. For many organisms, β-CA is essential for growth at atmospheric concentrations of CO₂. Of the five evolutionarily distinct classes of carbonic anhydrase, β-CA is the only one known to exhibit allosterism. Here we review the structure and catalytic mechanism of β-CA, including the structural basis for allosteric regulation.     

Plasmodium falciparum Carbonic Anhydrase is a Possible Target for Malaria Chemotherapy

Plasmodium falciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the α class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the α-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having α-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.