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A β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Francisella tularensis (FtuβCA) was cloned and purified, and the anion inhibition profile was investigated. Based on the measured kinetic parameters for the enzyme catalyzed CO 2 hydration reaction (k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 ), FtuβCA is a highly effective enzyme. The activity of FtuβCA was not inhibited by a range of anions that do not typically coordinate Zn(II) effectively, including perchlorate, tetrafluoroborate, and hexafluorophosphate. Surprisingly, some anions which generally complex well with many cations, including Zn(II), also did not effectively inhibit FtuβCA, e.g., fluoride, cyanide, azide, nitrite, bisulphite, sulfate, tellurate, perrhenate, perrhuthenate, and peroxydisulfate. However, the most effective inhibitors were in the range of 90–94 µM (sulfamide, sulfamic acid, phenylarsonic and phenylboronic acid). N,N -Diethyldithiocarbamate (K I of 0.31 mM) was a moderately potent inhibitor. As Francisella tularensis is the causative agent of tularemia, the discovery of compounds that can interfere with the life cycle of this pathogen may result in novel opportunities to fight antibiotic drug resistance. [ABSTRACT FROM AUTHOR]

A new β-class carbonic anhydrase (CA, EC 4.2.1.1) has been cloned, purified and characterized in the genome of the pathogenic bacterium Francisella tularensis responsible of the febrile illness tularemia. This enzyme, FtuβCA, showed a k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 for the CO 2 hydration, physiological reaction, being one of the most effective β-CAs known to date, with a catalytic activity only 1.68-times lower than that of the human(h) isoform hCA II. A panel of 39 simple aromatic and heterocyclic sulfonamides, as well as clinically used drugs incorporating sulfonamide/sulfamate zinc-binding groups, was used to investigate the inhibition profile of FtuβCA with these classes of derivatives. The enzyme generally showed a weaker affinity for these inhibitors compared to other α- and β-CAs investigated earlier, with only acetazolamide and its deacetylated precursor having inhibition constant <1 µM. Indeed, the two compounds acetazolamide AAZ and its deacetylated precursor 13 (K I s of 655–770 nM), as well as metanilamide and methazolamide (K I s of 2.53–2.92 µM), were the best FtuβCA inhibitors detected so far. As the physiological role of bacterial β-CAs is poorly understood for the virulence/life cycle of these pathogens, the present study may constitute a starting point for the design of effective pathogenic bacteria CA inhibitors with potential use as antiinfectives. [ABSTRACT FROM AUTHOR]

We report the cloning, purification and characterization of BpsβCA, a β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei , the etiological agent of melioidosis, and compare its activity and inhibition with those of the γ-CA from the same organism, BpsγCA, recently investigated by our groups. BpsβCA showed a significant catalytic activity for the physiologic, CO 2 hydration reaction, with the following kinetic parameters, k cat of 1.6 × 10 5 s −1 and k cat /K m of 3.4 × 10 7 M −1 × s −1 . The inhibition of BpsβCA with a group of anions and small molecules was also investigated. The best inhibitors were sulfamide, sulfamic acid and phenylarsonic acid, which showed K I s in the range of 83–92 µM, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate and hexafluorophosphate showed K I s > 100 mM. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32–8.6 mM. As little is known of the life cycle and virulence of this bacterium, this type of study may bring information of interest for the development of novel strategies to fight bacterial infection and drug resistance to commonly used antibiotics. [ABSTRACT FROM AUTHOR]

We have cloned, purified, and characterized a β-carbonic anhydrase (CA, EC 4.2.1.1), BpsCAβ, from the pathogenic bacterium Burkholderia pseudomallei, responsible for the tropical disease melioidosis. The enzyme showed high catalytic activity for the physiologic CO2 hydration reaction to bicarbonate and protons, with the following kinetic parameters: kcat of 1.6 × 105 s-1 and kβcat/KM of 3.4 × 107 M-1±s-1. An inhibition study with a panel of 38 sulfonamides and one sulfamate-including 15 compounds that are used clinically-revealed an interesting structure-activity relationship for the interaction of this enzyme with these inhibitors. Many simple sulfonamides and clinically used agents such as topiramate, sulpiride, celecoxib, valdecoxib, and sulthiame were ineffective BpsCAβinhibitors (KI > 50 μM). Other drugs, such as ethoxzolamide, dorzolamide, brinzolamide, zonisamide, indisulam, and hydrochlorothiazide were moderately potent micromolar inhibitors. The best inhibition was observed with benzene-1,3-disulfonamides-benzolamide and its analogs acetazolamide and methazolamide-which showed KI in the range of 185-745 nM. The inhibition profile of BpsCAβis very different from that of the γ-class enzyme from the same pathogen, BpsCAγ. Thus, identifying compounds that would effectively interact with both enzymes is relatively challenging. However, benzolamide was one of the best inhibitors of both of these CAs with KI of 653 and 185 nM, respectively, making it an interesting lead compound for the design of more effective agents, which may be useful tools for understanding the pathogenicity of this bacterium. [ABSTRACT FROM AUTHOR]

Abstract: 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 >200mM) whereas selenate and selenocyanide were ineffective inhibitors too, with K Is of 29.9–48.61mM. 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 0.15–0.90mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K Is in the range of 8–75μM, whereas acetazolamide inhibited the enzyme with a K I of 76nM. 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. [Copyright &y& Elsevier]