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Aim: Among 15 human (h) carbonic anhydrase (CA; EC 4.2.1.1) isoforms, two (hCA IX and XII) play important roles in the growth and survival of tumor cells, making them therapeutic targets for cancer treatment. This study aimed to develop novel sulfonamide-based compounds as selective hCA IX and XII inhibitors. Materials & methods: A library of novel N-sulfonyl carbamimidothioates was obtained for CA inhibitory activity studies against four hCA isoforms. Results: None of the developed compounds displayed inhibitory potential against off-target isoforms hCA I and II. However, they effectively inhibited tumor-associated hCA IX and XII. Conclusion: The present study suggests potent lead compounds as selective hCA IX and XII inhibitors with anticancer activity.

Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO2/HCO3-/pH-sensing in fungi is triggered by HCO3- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the β-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.

A 58-years old woman came to the emergency department due to syncope; she had been found lying on the ground in her residence. At the admission the patient was unconscious but had preserved blood pressure 120/80 mmHg. CT brain scan was negative for hemorrhage. Admission ECG showed a 52 b/m sinus bradycardia, elevated antero-septal ST segment and prolonged QT interval. Diagnosis of antero-septal STEMI was made and a coronary angiogram performed via right femoral access showed mild atheromasia in the absence of angiographically significant lesions. Bedside echocardiogram showed severe left ventricular dysfunction (LVEF 35%) with akinesia of apical and mid-ventricular segments. The right ventricle presented a marked hypocontractility of the apical region and a preserved kinetics of the basal portion (TAPSE 22 mm). There was also a moderate mitral regurgitation and a mild tricuspid regurgitation with a sPAP of 50 mmHg (40+10). On blood sampling, high-sensitivity troponin was slightly elevated (3950 pg/ml) and marked hyponatriemia (117 mmol/l) and slight hypokalemia (3.1 mmol/l) were noted. The echocardiogram on the third day showed an improvement in global and segmental kinetics of left ventricle with an estimated EF of 45% and persistence of a mild hypocontractility of the apex of right ventricle. During her hospital stay, her cognitive state gradually improved and she remained asymptomatic without evidence of arrhythmic events on monitoring. The ECG evolved on days with normalization of the ST-T tract, negative T wave in the anterior region and prolonged QTc interval. The pre-discharge echocardiogram showed a recovery of biventricular kinetics with an estimated EF of 50%. The presence of a transient abnormality in biventricular wall motion beyond a single epicardial coronary artery perfusion territory with new electrocardiographic change met the diagnostic criteria of Tako-tsubo Syndrome defined by Mayo Clinic criteria. The electrocardiogram on the day of discharge showed a 55 b/m sinus bradycardia with a negative T wave from V1 to V4. Tako-tsubo syndrome (TTS) is an acute and reversible left ventricular dysfunction that improves spontaneously within days or weeks and is often preceded by emotional or physical stress. Biventricular involvement in TTS is typically identified on imaging studies such as echocardiography and MRI. TTS with RV involvement is associated with a higher rate of in-hospital mortality and long-term adverse clinical events than isolated LV TTS. In our clinical case, biventricular involvement was associated with a severe clinical presentation (syncope and unconsciousness). This case shows that biventricular Tako-tsubo is a clinical scenario that requires intensive care monitoring and strict cardiological evaluation.

Mammaliicoccus sciuri, previously known as Staphylococcus sciuri, is a Gram-positive bacterium involved in gene transfer phenomena that confer resistance to multiple antibiotics. These plasmid-encoded genes can be easily transferred to other pathogenic staphylococci. Because antibiotic resistance is rising, inhibiting M. sciuri proliferation may be a credible strategy for restricting antimicrobial resistance gene transfer to other pathogenic bacteria. Recently, it has been shown that blocking bacterial carbonic anhydrases (CAs, EC 4.2.1.1), metalloenzymes sustaining bacterial metabolic activities, can reduce pathogen survival and fitness. Here, the recombinant M. sciuri γ-CA (MscCAγ) has been cloned and purified, utilizing the DNA recombinant technology. Its kinetic properties for the CO2 hydration reaction, as well as the sulfonamide inhibition profile, were investigated and compared with those reported earlier for MscCAβ (previously described as SauBCA) and the two off-target human CA isoforms (hCA I and hCA II). The recombinant MscCAγ showed significant hydratase activity. Moreover, the MscCAγ sulfonamide inhibitory profile was different from that of MscCAβ, implying that a varied amino acid set typifies the catalytic pocket of the two enzymes. These differences provide additional evidence for the possibility of developing novel CA class-specific inhibitors.

Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat value of 3.0 × 105 s−1 and kcat/KM value of 3.9 × 107 M−1 s−1. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (KI values of 6.2–94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (KI values of 0.71–0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed KI values in the range of 3.1–9.3 mM.

We here report a study on the activation of the ι-class bacterial CA from Burkholderia territorii (BteCAι). This protein was recently characterised as a zinc-dependent enzyme that shows a significant catalytic activity (kcat 3.0 × 105 s−1) for the physiological reaction of CO2 hydration to bicarbonate and protons. Some amino acids and amines, among which some proteinogenic derivatives as well as histamine, dopamine and serotonin, showed efficient activating properties towards BteCAι, with activation constants in the range 3.9–13.3 µM. L-Phe, L-Asn, L-Glu, and some pyridyl-alkylamines, showed a weaker activating effect towards BteCAι, with KA values ranging between 18.4 µM and 45.6 µM. Nowadays, no information is available on active site architecture, metal ion coordination and catalytic mechanism of members of the ι-group of CAs, and this study represents another contribution towards a better understanding of this still uncharacterised class of enzymes.

A series of novel 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives were synthesized and evaluated for the inhibition of eukaryotic and human carbonic anhydrases (CAs, EC 4.2.1.1). The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two β-CAs from fungal pathogens. The best inhibition was observed against hCA II and VII as well as Candida glabrata enzyme CgNce103. hCA I and Malassezia globosa MgCA enzymes were, on the other hand, less effectively inhibited by these compounds. The inhibitory potency of these compounds against CAs was found to be dependent on the electronic and steric effects of substituent groups on the N3-position of the hydantoin ring, which included alkyl, alkenyl and substituted benzyl moieties. The interesting results against CgNce103 make the compounds of interest for investigations in vivo as potential antifungals.

Carbonic Anhydrases (CAs) are ubiquitous metalloenzymes involved in several disease conditions. There are 15 human CA (hCA) isoforms and their high homology represents a challenge for the discovery of potential drugs devoid of off-target side effects. For this reason, many synthetic and pharmacologic research efforts are underway to achieve the full pharmacological potential of CA modulators of activity. We report here a novel series of sulfanilamide derivatives containing heterocyclic carboxamide moieties which were evaluated as CA inhibitors against the physiological relevant isoforms hCA I, II, IX, and XII. Some of them showed selectivity toward isoform hCA II and hCA XII. Molecular docking was performed for some of these compounds on isoforms hCA II and XII to understand the possible interaction with the active site amino acid residues, which rationalized the reported inhibitory activity.

This study reports our continued efforts to identify inhibitors capable of targeting carbonic anhydrases (CAs) expressed in bacteria. Based on previously identified chemotypes, we designed and synthesized new analogs that were screened toward the α, β, and γ classes encoded in Vibrio cholerae (Vch). The K

Fungi are exposed to various environmental variables during their life cycle, including changes in CO2 concentration. CO2 has the potential to act as an activator of several cell signaling pathways. In fungi, the sensing of CO2 triggers cell differentiation and the biosynthesis of proteins involved in the metabolism and pathogenicity of these microorganisms. The molecular machineries involved in CO2 sensing constitute a promising target for the development of antifungals. Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial enzymes in the CO2 sensing systems of fungi, because they catalyze the reversible hydration of CO2 to proton and HCO3-. Bicarbonate in turn boots a cascade of reactions triggering fungal pathogenicity and metabolism. Accordingly, CAs affect microorganism proliferation and may represent a potential therapeutic target against fungal infection. Here, the inhibition of the unique β-CA (MpaCA) encoded in the genome of Malassezia pachydermatis, a fungus with substantial relevance in veterinary and medical sciences, was investigated using a series of conventional CA inhibitors (CAIs), namely aromatic and heterocyclic sulfonamides. This study aimed to describe novel candidates that can kill this harmful fungus by inhibiting their CA, and thus lead to effective anti-dandruff and anti-seborrheic dermatitis agents. In this context, current antifungal compounds, such as the azoles and their derivatives, have been demonstrated to induce the selection of resistant fungal strains and lose therapeutic efficacy, which might be restored by the concomitant use of alternative compounds, such as the fungal CA inhibitors.

Carbonic anhydrases (CAs) are essential metalloenzymes in nature, catalyzing the carbon dioxide reversible hydration into bicarbonate and proton. In humans, breathing and many other critical physiological processes depend on this enzymatic activity. The CA superfamily function and inhibition in pathogenic bacteria has recently been the object of significant advances, being demonstrated to affect microbial survival/virulence. Targeting bacterial CAs may thus be a valid alternative to expand the pharmacological arsenal against the emergence of widespread antibiotic resistance. Here, we report an extensive study on the inhibition profile of the recently discovered &iota, CA class present in some bacteria, including Burkholderia territorii, namely BteCA&iota, using substituted benzene-sulfonamides and clinically licensed sulfonamide-, sulfamate- and sulfamide-type drugs. The BteCA&iota, inhibition profile showed: (i) several benzene-sulfonamides with an inhibition constant lower than 100 nM, (ii) a different behavior with respect to other &alpha, &beta, and &gamma, CAs, (iii) clinically used drugs having a micromolar affinity. This prototype study contributes to the initial recognition of compounds which efficiently and selectively inhibit a bacterial member of the &iota, CA class, for which such a selective inhibition with respect to other protein isoforms present in the host is highly desired and may contribute to the development of novel antimicrobials.

Our understanding of the function of bacterial carbonic anhydrases (CAs, EC 4.2.1.1) has increased significantly in the last years. CAs are metalloenzymes able to modulate CO2, HCO3– and H+ concentration through their crucial role in catalysis of reversible CO2 hydration (CO2 + H2O ⇄ HCO3– + H+). In all living organisms, CA activity is linked to physiological processes, such as those related to the transport and supply of CO2 or HCO3–, pH homeostasis, secretion of electrolytes, biosynthetic processes and photosynthesis. These important processes cannot be ensured by the very low rate of the non-catalyzed reaction of CO2 hydration. It has been recently shown that CAs are important biomolecules for many bacteria involved in human infections, such as Vibrio cholerae, Brucella suis, Salmonella enterica, Pseudomonas aeruginosa, and Helicobacter pylori. In these species, CA activity promotes microorganism growth and adaptation in the host, or modulates bacterial toxin production and virulence. In this review, recent literature in this research field and some of the above-mentioned issues are discussed, namely: (i) the implication of CAs from bacterial pathogens in determining the microorganism growth and virulence; (ii) the druggability of these enzymes using classical CA inhibitors (CAIs) of the sulfonamide-type as examples; (iii) the role played by Helicobacter pylori CAs in the acid tolerance/adaptation of the microbe within the human abdomen; (iv) the role of CAs played in the outer membrane vesicles spawned by H. pylori in its planktonic and biofilm phenotypes; (v) the possibility of using H. pylori CAIs in combination with probiotic strains as a novel anti-ulcer treatment approach. The latter approach may represent an innovative and successful strategy to fight gastric infections in the era of increasing resistance of pathogenic bacteria to classical antibiotics.

Proteins are relevant antimicrobial drug targets, and among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central metabolism, or are necessary for the pathogen vitality. Genomic exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant ?-CA (CynT2) identified in the genome of the Gram-negative bacterium Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO2. Surprisingly, this enzyme has not been investigated for its inhibition with any class of CA inhibitors. Here, we show that CynT2 was strongly inhibited by some substituted benzene-sulfonamides and the clinically used inhibitor sulpiride (KIs in the range of 82&ndash, 97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure&ndash, activity relationship for this class of enzyme inhibitors.

The interconversion of CO2 and HCO3&minus, is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO2 and HCO3&minus, In the genome of Escherichia coli, a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a &beta, CA, and CynS gene, encoding for the cyanase. CynT (&beta, CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second &beta, CA (CynT2 or Can or yadF), as well as a &gamma, and &iota, CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO2 hydratase reaction with the following parameters: kcat = 5.3 &times, 105 s&minus, 1 and kcat/KM = of 4.1 &times, 107 M&minus, 1 s&minus, 1. Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (KI = 2.5 to 84 &micro, M). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.

Carbonic anhydrase IX is a promising target for the search for new antitumor compounds with improved properties. Using the molecular hybridization approach, on the basis of structures of a selective carbonic anhydrase IX inhibitor 3 and an activator of apoptosis 2 (1), a series of 1-substituted isatin-5-sulfonamides 5a–5u were designed and synthesized. The study of the inhibitory activity of isatin-5-sulfonamides showed the ability to inhibit I, II, IX, XII isoforms at nano- and micromolar concentrations. Docking of compounds 5e and 5k into the active site of II and IX carbonic anhydrase isoforms showed the coordination of sulfonamidate anions with zinc cations, as well as a number of additional hydrophobic interactions. The trifluoromethylthio derivative 5r suppressed the growth of tumor cells at low micromolar concentrations, maintaining activity on resistant lines and under hypoxic conditions. Immunoblotting of MCF-7 cells treated with the 5r revealed its antiestrogenic activity and ability to activate apoptosis in tumor cells.

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.

Carbonic anhydrases (CA, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyze the conversion of carbon dioxide (CO2) to bicarbonate (HCO3) and protons (H+). In prokaryotes, the existence of genes encoding for alpha-, beta- and gamma-classes suggests that these enzymes play an important role in the prokaryotic physiology. It has been demonstrated, in fact, that their inhibition in vivo leads to growth impairment or growth defects of the microorganism. Ultimately, we started to investigate the biochemical properties and the inhibitory profiles of the alpha- and beta-CAs identified in the genome of Vibrio cholerae, which is the causative agent of cholera. The genome of this pathogen encodes for CAs belonging to alpha, beta and gamma classes. Here, we report a sulfonamide inhibition study of the gamma-CA (named VchCA gamma) comparing it with data obtained for the alpha- and beta-CA enzymes. VchCA gamma activity (k(cat) = 7.39 x 10(5) s(-1)) was significantly higher than the other gamma-CAs. The inhibition study with a panel of sulfonamides and one sulfamate led to the detection of a large number of nanomolar VchCA gamma inhibitors, including simple aromatic/heterocyclic sulfonamides (compounds 2-9, 11, 13-15, 24) as well as EZA, DZA, BRZ, BZA, TPM, ZNS, SLP, IND (K(I)s in the range of 66.2-95.3 nM). As it was proven that bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit this virulence in vivo, we propose that VchCA, VchCA beta and VchCA gamma may be a target for antibiotic development, exploiting a mechanism of action rarely considered up until now, i.e., interference with bicarbonate supply as a virulence factor. (C) 2016 Elsevier Ltd. All rights reserved.

We have cloned, purified and characterized the gamma-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 x 10(5) s(-1) and a k(cat)/K-m of 4.7 x 10(6) M-1 x s(-1). This activitywas 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(I)s 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(I)s ranging between 8 and 38 mu M. The present study may shed some more light regarding the role that gamma-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here. (c) 2016 Elsevier Ltd. All rights reserved.

The Antarctic bacterium Colwellia psychrerythraea encodes for a gamma-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCA gamma) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0 x 10(5) s(-1) and a k(cat)/K-m of 4.7 x 10(6) M-1 s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. The best inhibitor was metanilamide (K-I of 83.5 nM) followed by indisulam, valdecoxib, celecoxib, sulthiame and hydrochlorothiazide (K(I)s ranging between 343 and 491 nM). Acetazolamide, methazolamide as well as other aromatic/heterocyclic derivatives showed inhibition constants between 502 and 7660 nM. The present study may shed some more light regarding the role that gamma-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here, by allowing the identification of inhibitors which may be useful as pharmacologic tools. (C) 2016 Elsevier Ltd. All rights reserved.

With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to alpha-, beta-, and gamma-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 degrees C, suggesting a possible role of the two coupled enzymes in biotechnological processes. (c) 2015 Elsevier Ltd. All rights reserved.

A new ?-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from the Antarctic bacterium Pseudoalteromonas haloplanktis, PhaCA?. The enzyme has a medium-low catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a kcat of 1.4 × 105 s-1 and a kcat/Km of 1.9 × 106 M-1 s-1. An anion inhibition study of PhaCA? with inorganic anions and small molecule inhibitors is also reported. Many anions present in sea water, such as chloride, fluoride, sulfate, iodide, but also others such as azide, perchlorate and tetrafluoroborate did not inhibit this enzyme. Pseudohalides such as cyanate, thiocyanate, cyanide, selenocyanide, and also bicarbonate, nitrate, nitrite and many complex inorganic anions showed inhibition in the millimolar range (KI in the range of 1.7-9.3 mM). The best PhaCA? inhibitors detected in this study were diethyldithiocarbamate (KI of 0.96 mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KI in the range of 82-91 ?M). Since ?-CAs are poorly understood at this moment, being present in carboxysomes and thus involved in photosynthesis, this study may be relevant for a better understanding of these processes in Antarctic bacteria/cyanobacteria.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the CO2 hydration/dehydration reversible reaction: CO2 + H2O ? (Formula presented.) + H+. Living organisms encode for at least six distinct genetic families of such catalyst, the ?-, ?-, ?-, ?-, ?- and ?-CAs. The main function of the CAs is to quickly process the CO2 derived by metabolic processes in order to regulate acid-base homeostasis, connected to the production of protons (H+) and bicarbonate. Few data are available in the literature on Antarctic CAs and most of the scientific information regards CAs isolated from mammals or prokaryotes (as well as other mesophilic sources). It is of great interest to study the biochemical behavior of such catalysts identified in organism living in the Antarctic sea where temperatures average -1.9 °C all year round. The enzymes isolated from Antarctic organisms represent a useful tool to study the relations among structure, stability and function of proteins in organisms adapted to living at constantly low temperatures. In the present paper, we report in detail the cloning, purification, and physico-chemical properties of NcoCA, a ?-CA isolated from the Antarctic cyanobacterium Nostoc commune. This enzyme showed a higher catalytic efficiency at lower temperatures compared to mesophilic counterparts belonging to ?-, ?-, ?-classes, as well as a limited stability at moderate temperatures.

Two thermostable α-carbonic anhydrases (α-CAs) isolated from thermophilic Sulfurihydrogenibium spp., namely SspCA (from S. yellowstonensis) and SazCA (from S. azorense), were shown in a previous work to possess interesting complementary properties. SspCA was shown to have an exceptional thermal stability, whereas SazCA demonstrated to be the most active α-CA known to date for the CO2 hydration reaction. Here we report the crystallographic structure of SazCA and the identification of the structural features responsible for its high catalytic activity, by comparing it with SspCA structure. These data are of relevance for the design of engineered proteins showing higher stability and catalytic activity than other α-CAs known to date.

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 ?-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 ?-class such enzymes. Here we show that protonography can also be applied to the recently discovered ?-CA family using the Plasmodium falciparum enzyme PfCA as an example. The protonogram of PfCA clearly showed catalytically active ?-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 ?- and ?-CAs might be a useful tool to detect Plasmodium falciparum in infected human red blood cells by an easy, routine inexpensive technique.

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, alpha-, beta-, gamma-, delta-and zeta and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the delta-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 delta-CA of T. weissflogii. The delta-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 delta-CAs are closer to the a-CAs than to the other classes of such enzymes.

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 gamma-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 gamma-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 gamma-CAs studied so far. Enzyme kinetic analysis showed that PgiCA is 62 times more effective as a catalyst compared to CAM, the only other gamma-CA characterized in detail kinetically. All these features represent an interesting attractive for the drug design of inhibitors/activators of this new enzyme.

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.