Sonia Del Prete

Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum--the η-carbonic anhydrases.

The genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) thought to belong to the α-class, PfCA. Here we demonstrate the kinetic properties of PfCA for the CO2 hydration reaction, as well as an inhibition study of this enzyme with inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, and phenylboronic/arsonic acids, detecting several low micromolar inhibitors. A closer examination of the sequence of this and the CAs from other Plasmodium spp., as well as a phylogenetic analysis, revealed that these protozoa encode for a yet undisclosed, new genetic family of CAs termed the η-CA class. The main features of the η-CAs are described in this report.

Biochemical properties of a new α-carbonic anhydrase from the human pathogenic bacterium, Vibrio cholerae

Abstract Vibrio cholerae, a Gram-negative bacterium, is the causative agent of cholera and colonizes the upper small intestine where sodium bicarbonate is present at a high concentration. Sodium bicarbonate is a potential inducer of virulence gene expression. Bacteria can increase cytosolic bicarbonate levels through the existence of transporter family proteins or through the action of metalloenzymes, called carbonic anhydrases (CAs, EC 4.2.1.1). Vibrio cholerae, lacking of transporter proteins in its genome, utilizes the CA system to accumulate bicarbonate into the cell suggesting a pivotal role of this metalloenzymes in the microbial virulence. Here, we report for the first time the characterization of the α-CA of V. cholerae (VchCA), which has been identified by translated genome inspection. The α-CA encoding gene was cloned and expressed in Escherichia coli and the recombinant protein purified to homogeneity. This investigation aimed to study the biochemical properties of VchCA and to provide preliminary insights in the field of this pathogen virulence. VchCA has a low esterase activity with 4-nitrophenyl acetate as substrate, and a high activity for the hydration of CO2 to bicarbonate.

DNA cloning, characterization, and inhibition studies of an α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

We have cloned, purified, and characterized an α-carbonic anhydrase (CA, EC 4.2.1.1) from the human pathogenic bacterium Vibrio cholerae, VchCA. The new enzyme has significant catalytic activity, and an inhibition study with sulfonamides and sulfamates led to the detection of a large number of low nanomolar inhibitors, among which are methazolamide, acetazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, and indisulam (KI values in the range 0.69-8.1 nM). As bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit the in vivo virulence, we propose that VchCA may be a target for antibiotic development, exploiting a mechanism of action rarely considered until now.

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 kcat of 4.1 × 105 s−1 and a kcat/Km of 5.4 × 107 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 kcat of 2.8 × 105 s−1 and a kcat/Km of 1.5 × 107 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.

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.

Protonography, a new technique for the analysis of carbonic anhydrase activity

Abstract All proteolytic enzymes, which are able to renature and reacquire the proteolytic activity on a copolymerized substrate, can be analyzed by zymography upon removal of sodium dodecyl sulfate (SDS). Protonography, the new technique described in this study, unlike zymography, allows the detection of a different protein, not a protease, i.e. of the carbonic anhydrase (CA, EC 4.2.1.1) activity on a SDS polyacrylamide gel electrophoresis gel. CAs are zinc-containing enzymes that catalyze the reversible conversion of carbon dioxide to bicarbonate and protons. Hydrogen ions produced during the catalyzed reaction are responsible for the change of color that appears on the gel around the CA band. For this reason, we named the new technique “protonography”. The following four salient features characterize this new technique: (a) on the basis of molecular weight markers, recombinant or native CAs with different molecular weights can be detected and quantified rapidly on a single gel; (b) the hydratase activity can be reversibly inhibited by SDS during electrophoresis and recovered by incubating the gel in aqueous Triton X-100; (c) it is possible to separate active oligomeric forms of CAs on the gel enabling their activities to be determined independently of one another. This feature is not possible when using solution assays; and (d) it can be a useful tool to establish if a putative or a newly identified CA in a genome is expressed and enzymatically active. This article outlines the general principles employed in protonography, providing an easy procedure to implement it in laboratories working with CAs. It also presents an overview of its development and current research applications through specific examples.

A highly catalytically active γ-carbonic anhydrase from the pathogenic anaerobe Porphyromonas gingivalis and its inhibition profile with anions and small molecules

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the γ-class are present in archaea, bacteria and plants but, except the Methanosarcina thermophila enzymes CAM and CAMH, they were poorly characterized so far. Here we report a new such enzyme (PgiCA), the γ-CA from the oral cavity pathogenic bacterium Porphyromonas gingivalis, the main causative agent of periodontitis. PgiCA showed a good catalytic activity for the CO2 hydration reaction, comparable to that of the human (h) isoform hCA I. Inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate were effective PgiCA inhibitors with inhibition constants in the range of 41-97 μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, and phenylboronic acid, with KIs of 4.0-9.8 μM. The role of this enzyme as a possible virulence factor of P. gingivalis is poorly understood at the moment but its good catalytic activity and the possibility to be inhibited by a large number of compounds may lead to interesting developments in the field.

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 (KIs in the range of 131-380nM). 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.

Cloning, characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii

We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO₂ hydration, its physiological reaction, with a kcat of 1.3 × 10(5)s(-1) and a k(cat)/K(M) of 3.3 × 10(7)M(-1)s(-1). A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (KIs >200 mM) whereas other halides and pseudohalides were submillimolar-millimolar inhibitors (K(I)s in the range of 0.93-8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K(I)s in the range of 9-90 μM, whereas acetazolamide inhibited the enzyme with a K(I) of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO₂ fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO₂ capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms.

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 KIs of 49.6-118nM. Many simple aromatic sulfonamides as well as dichlorophenamide, benzolamide, topiramate, zonisamide, indisulam and valdecoxib were medium potency inhibitors, (KIs of 375-897nM). Saccharin and hydrochlorothiazide were ineffective inhibitors of the δ-class enzyme, with KIs of 4.27-9.20μM. 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.