Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase from<i>Sulfurihydrogenibium yellowstonense YO3AOP1</i>

Journal of Enzyme Inhibition and Medicinal Chemistry

Aşık

et al. 2015

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co 2+ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the b-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 C and pH of 8.3: k cat of 4.8 Â 10 5 s À1 and k cat /K m of 5.6 Â 10 7 M À1 Â s À1. This activity was potently inhibited by acetazolamide which showed a K I of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO 3 biomineralization processes.

Section: Introductionmentioning

confidence: 99%

Cloning, expression and biochemical characterization of aβ-carbonic anhydrase from the soil bacteriumEnterobactersp. B13

Eminoğlu

Journal of Enzyme Inhibition and Medicinal Chemistry

Aşık

et al. 2015

“…The assay was performed at 0 C adding 0.5 mL ice-cold CO 2 -saturated water to 0.5 mL mixtures of 25 mM Tris-SO 4 buffer containing the enzyme at different concentrations. The details concerning the assay have been described by Capasso et al 16 Esterase enzyme activity Esterase activity for pNpA hydrolysis was determined at 0 C using a modification of the method proposed by Armstrong et al 30 . The reaction mixture contained 0.3 mL of freshly prepared 3 mM pNpA and 0.7 mL of 15 mM Tris sulphate buffer, pH 7.6.…”

Section: Hydratase Enzyme Activitymentioning

confidence: 99%

“…The enzyme stability was studied in the 25-80 C temperature range incubating the enzyme for 10, 30 and 60 min (Figure 4). After incubation at the indicated times, enzyme activity was determined using CO 2 as substrate as described by Capasso et al 16 . Interestingly, TweCA enzyme was stable up to 80 C when the incubation time did not exceed 30 min.…”

Section: Thermostability Studiesmentioning

confidence: 99%

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

Prete

Journal of Enzyme Inhibition and Medicinal Chemistry

Luca

et al. 2014

Self Cite

Diatom genome sequences clearly reveal the presence of different systems for HCO 3 À uptake. Carbon-concentrating mechanisms (CCM) based on HCO 3 À 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, a-, b-, g-, d-and z and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the d-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO 2 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 d-CA of T. weissflogii. The d-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 d-CAs are closer to the a-CAs than to the other classes of such enzymes.

“…The main classes of antibiotics clinically used nowadays act towards the inhibition of four classical targets: (a) cell wall biosynthesis; (b) protein biosynthesis; (c) DNA and RNA biosynthesis; (d) folate biosynthesis 1,2 . Carbonic anhydrases (CAs, EC 4.2.1.1) 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 bacteria, CAs are essential for the life cycle of the organism and that their inhibition leads to growth impairment or growth defects of the pathogen [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . CAs are ubiquitous metalloenzymes, present in prokaryotes and eukaryotes.…”

Section: Introductionmentioning

confidence: 99%

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

Prete

Journal of Enzyme Inhibition and Medicinal Chemistry

Luca

et al. 2014

Self Cite

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the a-, b-, g-, d-and z-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 b-and a g-CAs. Recently, our group purified the recombinant g-CA (named PgiCA) which was shown to possess a significant catalytic activity for the reaction that converts CO 2 to bicarbonate and protons, with a k cat of 4.1 Â 10 5 s À1 and a k cat /K m of 5.4 Â 10 7 M À1 Â s À1 . 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 b-CA, named PgiCAb. This enzyme has a good catalytic activity, with a k cat of 2.8 Â 10 5 s À1 and a k cat /K m of 1.5 Â 10 7 M À1 Â s À1 . 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.