CO2 Hydration Reaction Research Articles

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

The Antarctic bacterium Colwellia psychrerythraea encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCAγ) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0×10(5) s(-1) and a k(cat)/K(m) of 4.7×10(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 γ-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.

Inhibition of carbonic anhydrase isoforms I, II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.

Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea

We have cloned, purified and characterized the γ-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: kcat of 6.0×105s−1 and a kcat/Km of 4.7×106M−1×s−1. This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a KI of 502nM. 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 KIs in the range of 1.4–4.4mM. Diethyldithiocarbamate was a better inhibitor (KI of 0.58mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with KIs ranging between 8 and 38μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here.

An efficient absorbent of amine-based amino acid-functionalized ionic liquids for CO2 capture: High capacity and regeneration ability

An efficient and high capacity absorbent of amine-based amino acid-functionalized ionic liquid (IL) ([APmim][Gly]) was designed and prepared for CO2 capture in this work. The achieved capacity of [APmim][Gly] was 1.23mol CO2/mol IL (4.31mol CO2/kg IL), which was much higher than that of the most existing dual functionalized ILs due to its low molecular weight. The largest weight loss temperature of [APmim][Gly] was found to be 370°C, at that point, the weight loss was nearly 60% of its initial weight. Besides, [APmim][Gly] also owned a high regeneration ability even after multiple regenerations. In order to clarify the reaction mechanism of CO2 absorption into [APmim][Gly] solution, 13C nuclear magnetic resonance (NMR) was carried out to analyze the species of [APmim][Gly] solution at different CO2 loadings. Different from the most reported mechanisms for CO2 capture into functionalized ILs, the reaction between [APmim][Gly] and CO2 was influenced by CO2 loadings. The reaction could divide into two stages. It was started with the reversible chemical reaction to form carbamate, and followed by the CO2 hydration reaction. At high CO2 loading, the production of carbamate was easy to be decomposed.

Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate

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 α-, β-, and γ-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°C, suggesting a possible role of the two coupled enzymes in biotechnological processes.

New natural product carbonic anhydrase inhibitors incorporating phenol moieties

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. They represent a typical example of enzyme convergent evolution, as six genetically unrelated families of such enzymes were described so far. The need to find selective CA inhibitors (CAIs) triggered the investigation of natural product libraries, which proved to be a valid source of agents with such an activity, as demonstrated for the phenols, polyamines and coumarins. Herein we report an in vitro inhibition study of human (h) CA isoforms hCAs I, II, IV, VII and XII with a panel of natural polyphenols including flavones, flavonols, flavanones, flavanols, isoflavones and depsides, some of which extracted from Quercus ilex and Salvia miltiorrhiza. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting some important isoforms over the off-target ones hCA I and II.

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis.

The Antarctic bacterium Pseudoalteromonas haloplanktis encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (PhaCAγ) has a good catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) of 1.4×10(5) s(-1) and a k(cat)/K(m) of 1.9×10(6) M(-1)×s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. Methazolamide and indisulam showed the best inhibitory properties (K(I)s of 86.7-94.7 nM). This contribution shed new light on γ-CAs inhibition profiles with a relevant class of pharmacologic agents.

Cloning, characterization and anion inhibition studies of a new γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis

A new γ-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from the Antarctic bacterium Pseudoalteromonas haloplanktis, PhaCAγ. The enzyme has a medium-low catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a kcat of 1.4×105s−1 and a kcat/Km of 1.9×106M−1s−1. An anion inhibition study of PhaCAγ 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 1.7–9.3mM). The best PhaCAγ inhibitors detected in this study were diethyldithiocarbamate (KI of 0.96mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KI in the range of 82–91μM). Since γ-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.

Cloning, characterization and anion inhibition study of a β-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans

The oral pathogenic bacterium involved in human dental caries formation Streptococcus mutans, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the α- and the other one to the β-class. This last enzyme (SmuCA) has been cloned, characterized and investigated for its inhibition profile with a major class of CA inhibitors, the inorganic anions. Here we show that SmuCA has a good catalytic activity for the CO2 hydration reaction, with kcat 4.2×105s−1 and kcat/Km of 5.8×107M−1×s−1, being inhibited by cyanate, carbonate, stannate, divannadate and diethyldithiocarbamate in the submillimolar range (KIs of 0.30–0.64mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KIs of 15–46μM). The anion inhibition profile of the S. mutans enzyme is very different from other α- and β-CAs investigated earlier. Identification of effective inhibitors of this new enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.

Inhibitory effects of nitrite on the reactions of bovine carbonic anhydrase II with CO2 and bicarbonate consistent with zinc-bound nitrite

Carbonic anhydrase (CA) is a zinc enzyme that catalyzes hydration of carbon dioxide (CO2) and dehydration of bicarbonate in red blood cells, thus facilitating CO2 transport and excretion. Bovine CA II may also react with nitrite to generate nitric oxide, although nitrite is a known inhibitor of the CO2 hydration reaction. To address the potential in vivo interference of these reactions and the nature of nitrite binding to the enzyme, we here investigate the inhibitory effect of 10–30mM nitrite on Michaelis–Menten kinetics of CO2 hydration and bicarbonate dehydration by stopped-flow spectroscopy. Our data show that nitrite significantly affects the apparent dissociation constant KM for CO2 (11mM) and bicarbonate (221mM), and the turnover number kcat for the CO2 hydration (1.467×106s−1) but not for the bicarbonate dehydration (7.927×105s−1). These effects demonstrate mixed and competitive inhibition for the reaction with CO2 and bicarbonate, respectively, and are consistent with nitrite binding to the active site zinc. The high apparent dissociation constant found here for CO2, bicarbonate and nitrite (16–120mM) are all overall consistent with published data and reveal a large capacity of free enzyme available for binding each of the three substrates at their in vivo levels, with little or no significant interference among reactions. The low affinity of the enzyme for nitrite suggests that the in vivo interaction between red blood cell CA II and nitrite requires compartmentalization at the anion exchanger protein of the red cell membrane to be physiologically relevant.

Sulfonamide inhibition study of the β-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans

Streptococcus mutans, the oral pathogenic bacterium provoking dental caries formation, encodes for a β-class carbonic anhydrase (CA, EC 4.2.1.1), SmuCA. This enzyme was cloned, characterized and investigated for its inhibition profile with the major class of CA inhibitors, the primary sulfonamides. SmuCA has a good catalytic activity for the CO2 hydration reaction, with a kcat of 4.2×10(5) s(-1) and kcat/Km of 5.8×10(7) M(-1)×s(-1), and is efficiently inhibited by most sulfonamides (KIs of 246 nM-13.5 μM). The best SmuCA inhibitors were bromosulfanilamide, deacetylated acetazolamide, 4-hydroxymethylbenzenesulfonamide, a pyrimidine-substituted sulfanilamide derivative, aminobenzolamide and compounds structurally similar to it, as well as acetazolamide, methazolamide, indisulam and valdecoxib. These compounds showed inhibition constants ranging between 246 and 468 nM. Identification of effective inhibitors of this enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.

Catalysis of CO2 absorption in aqueous solution by vanadate and sulfate and their application to post combustion capture

A promising option to improve post combustion capture (PCC) is to use inorganic catalysts to accelerate the absorption process of CO2, in particular the reaction between CO2 and water to form carbonic acid. In this study, the efficiency of sulfate and vanadate on enhancing the hydration reaction of CO2 to form H2CO3 for PCC has been studied at 25°C by stopped-flow spectrophotometry. The catalytic rate constants were determined to be 14.2(2) and 277(6)M−1s−1 for sulfate and vanadate, respectively. Their application to PCC was further investigated by simulating the effect of these catalysts on the absorption process under PCC operating conditions. Vanadate was confirmed to have a greater efficiency toward CO2 hydration than sulfate and also a series of other inorganic catalysts reported earlier.

Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense.

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.

Hydration of Carbon Dioxide in Copper-Alkoxide Functionalized Metal–Organic Frameworks: A DFT Study

The adsorption of CO2 and its hydration to carbonic acid on copper alkoxide functionalized Metal–Organic Frameworks (Cu-MOFs) is studied by density functional theory calculations. The adsorption Gibbs free energies of CO2, H2O and two forms of carbonic acid on Cu-MOFs are −2.4, −14.1, −13.5, and −28.9 kcal/mol, respectively. The preferred adsorption of H2O on the Cu enhances also the stability of the Cu-MOFs against moisture. Two possible pathways are considered for the hydration reaction of CO2. In the first one, the reaction starts with the simultaneous forming of a C—O bond and the breaking of an O—H bond, leading to a HCO3 intermediate. This intermediate then undergoes a hydride-shift and is subsequently protonated to form carbonic acid. The second step is considered to be the rate-determining step of this reaction with an activation energy of 28.2 kcal/mol. In the second pathway, a three-step process is considered. It commences by dissociation of the water O—H bond and the formation of a C—O bond between the resulting OH species and the incoming CO2 molecule. Finally, the hydrogen transfers to the HCO3 intermediate to generate carbonic acid. The first step has the highest activation energy, 19.1 kcal/mol, and is therefore rate-determining. Since this is also much lower compared with the first pathway, the second pathway is preferred for the CO2 hydration on Cu-MOFs. Without a catalyst, the barrier is much higher, 53.4 kcal/mol, indicating that Cu-MOFs might be a suitable material to adsorb and hydrate CO2.

Nickel Nanoparticles for Enhancing Carbon Capture

Hydration reaction of CO2 is one of the rate limiting steps for CO2 absorption (in aqueous solutions) and aqueous CO2 mineralization. The catalytic activity of nickel nanoparticles (NiNPs) for CO2 hydration is studied at different temperatures, pH, and low CO2 partial pressures to mimic the true flue gas conditions. Results show that NiNPs can work as active catalyst for hydration of CO2 in applications such as CO2 separation and CO2 mineralization. The NiNPs display optimum activity within 20–30°C and at pH value <8. NiNPs show catalytic activity even at low CO2 partial pressures (12 vol%). In 50 wt% K2CO3 solution, an enhancement of 77% is observed in the rate of CO2 absorption with NiNPs. Commercially, CO2 saturated K2CO3 solutions are usually regenerated at 150°C; at these conditions, NiNPs show no considerable surface oxidation. They still exhibit catalytic activity for hydration reaction of CO2. CO2 absorption and mineralization (as CaCO3) in DI water are three times higher in presence of NiNPs. Calcite (CaCO3) particles precipitated in presence of NiNPs are spherical in morphology.

Anion inhibition study of the β-class carbonic anhydrase (PgiCAb) from the oral pathogen Porphyromonas gingivalis

The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the β-class (PgiCAb) and another one to the γ-class (PgiCA). This last enzyme has been characterized earlier for its inhibition profile with various classes of CA inhibitors, such as the sulfonamides and anions, whereas for PgiCAb such data were not yet reported. Here we show that PgiCAb has a good catalytic activity for the CO2 hydration reaction, with kcat 2.8×105s−1 and kcat/Km of 1.5×107M−1×s−1, being inhibited by cyanate and diethyldithiocarbamate in the submillimolar range (KIs of 0.23–0.76mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KIs of 60–78μM). The anion inhibition profile of the two P. gingivalis enzymes is very different. Identification of selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available.

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.

Anion inhibition studies of two α-carbonic anhydrases from Lotus japonicus, LjCAA1 and LjCAA2

The model organism for the investigation of symbiotic nitrogen fixation in legumes Lotus japonicus encodes two carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-class, LjCAA1 and LjCAA2. Here we report the kinetic characterization and inhibition of these two CAs with inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, and phenylboronic/arsonic acids. LjCAA1 showed a high catalytic activity for the CO2 hydration reaction, with a kcat of 7.4∗105s−1 and a kcat/Km of 9.6∗107M−1s−1 and was inhibited in the low micromolar range by N,N-diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic/arsonic acid (KIs of 4–62μM). LjCAA2 showed a moderate catalytic activity for the physiologic reaction, with a kcat of 4.0∗105s−1 and a kcat/Km of 4.9∗107M−1s−1. The same anions mentioned above for the inhibition of LjCAA1 showed the best activity against LjCAA2 (KIs of 7–29μM). Nitrate and nitrite, anions involved in nitrogen fixation, showed lower affinity for the two enzymes, with inhibition constants in the range of 3.7–7.0mM. Halides and sulfate also behaved in a distinct manner towards the two enzymes investigated here. As LjCAA1/2 participate in the pH regulation processes and CO2 metabolism within the nitrogen-fixing nodules of the plant, our studies may shed some light regarding these complex biochemical processes.

Surface Display of Small Peptides on Escherichia coli for Enhanced Calcite Precipitation Rates.

Mineralization has emerged as a promising strategy for long-term carbon sequestration. These processes involve carbon dioxide hydration followed by mineral precipitation. We have explored the production of whole-cell biocatalysts engineered with carbonic anhydrase (CA) activity to accelerate the CO₂ hydration reaction. In this study, short polypeptides were displayed on the surface of E. coli cells and whole-cell biocatalysts containing periplasmically expressed CAs in an attempt to enhance calcite mineral formation. It was found that cells coexpressing recombinant periplasmic CA and surface-displayed GPA peptide (PEVPEGAFDTAI) outperformed other peptide-expressing biocatalysts evaluated in terms of the amount of precipitate formed, as well as the overall formation rate of solids. Cells expressing the Cab CA isoform (BLR-pCab) and Cam isoform (BLR-pCam) with the surface-displayed GPA peptide exhibited 36 and 59% improvements in precipitation amounts, as well as 18 and 60% improvements in overall formation rates, respectively, over similar biocatalysts without GPA expression. The biocatalyst with the best performance was BLR-pCam/GPA, which generated 0.15 g of CaCO₃, while BLR cells generated only 0.08 g of CaCO₃ under the same small batch reaction conditions. The BLR-pCam/GPA cells also exhibited the fastest formation rates, achieving the maximum change in solution turbidity after only 2.2 min, as opposed to 6.3 min for BLR cells. These results demonstrate that synthetic biology approaches can be used to create novel biocatalysts with the ability to enhance both catalysis and precipitation activities.

Cloning, Characterization, and Inhibition Studies of a β-Carbonic Anhydrase from Leishmania donovani chagasi, the Protozoan Parasite Responsible for Leishmaniasis

Leishmaniasis is an infection provoked by protozoans belonging to the genus Leishmania. Among the many species and subsepecies of such protozoa, Leishmania donovani chagasi causes visceral leishmaniasis. A β-carbonic anhydrase (CA, EC 4.2.1.1) was cloned and characterized from this organism, denominated here LdcCA. LdcCA possesses effective catalytic activity for the CO2 hydration reaction, with kcat of 9.35 × 10(5) s(-1) and kcat/KM of 5.9 × 10(7) M(-1) s(-1). A large number of aromatic/heterocyclic sulfonamides and 5-mercapto-1,3,4-thiadiazoles were investigated as LdcCA inhibitors. The sulfonamides were medium potency to weak inhibitors (KI values of 50.2 nM-9.25 μM), whereas some heterocyclic thiols inhibited the enzyme with KIs in the range of 13.4-152 nM. Some of the investigated thiols efficiently inhibited the in vivo growth of Leishmania chagasi and Leishmania amazonensis promastigotes, by impairing the flagellar pocket and movement of the parasites and causing their death. The β-CA from Leishmania spp. is proposed here as a new antileishmanial drug target.