Bacterial Carbonic Anhydrases Research Articles

Bacterial carbonic anhydrases: underexploited antibacterial therapeutic targets.

Bacterial carbonic anhydrases: underexploited antibacterial therapeutic targets.

Carbonic Anhydrases: New Perspectives on Protein Functional Role and Inhibition in Helicobacter pylori.

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.

Benzylaminoethylureido-Tailed Benzenesulfonamides Show Potent Inhibitory Activity against Bacterial Carbonic Anhydrases.

A series of benzylaminoethylureido-tailed benzenesulfonamides was analyzed for their inhibition potential against bacterial carbonic anhydrases (CAs) such as VhCA α, β, and γ from Vibrio cholerae, and BpsCA β and γ-CAs from Burkholderia pseudomallei. Growing drug resistance against antibiotics demands alternative targets and mechanisms of action. As CA is essential for the survival of bacteria, such enzymes have the potential for developing new antibiotics. Most of the compounds presented excellent inhibition potential against VhCA γ compared to α and β, with Ki values in the range of 82.5-191.4 nM. Several sulfonamides exhibited excellent inhibition against BpsCA β with Ki values in the range of 394-742.8 nM. Recently it has been demonstrated that sufonamide CA inhibitors are effective against vancomycin-resistant enterococci. These data show that CA inhibition of pathogenic bacteria may lead to a new class of antibiotics.

Calcium carbonate precipitation mediated by bacterial carbonic anhydrase in a karst cave: Crystal morphology and stable isotopic fractionation

Stable oxygen (δ18O) and carbon (δ13C) isotopes of speleothems are widely used as important proxies for paleoclimatic and paleoenvironment reconstructions. However, the influence of microbial activity on carbon and oxygen isotope fractionation during speleothem precipitation remains unclear. Bacterial carbonic anhydrase (CA) can promote calcium carbonate precipitation to catalyze the mutual transformation between CO2 and HCO3-. CA-producing bacteria (Lysinibacillus sp. strain LHXY2) were separated in the Xueyu Cave, Chongqing, SW China, and used in laboratory and cave in situ models to investigate their influence on the precipitation amount, mineral components, crystal morphology and carbon and oxygen isotope fractionation of CaCO3. A CA activity gradient was applied in the laboratory model by considering various CA inhibitor acetazolamide (AZ) concentrations, which showed that the CA activity could substantially enhance precipitation, alter the mineral components and morphology, and reduce the δ13C and δ18O values of the CaCO3 formed. Most importantly, the laboratory and in situ model results revealed approximately -7‰ and -1.4‰ δ13C shifts, respectively, compared to the bacteria-free model results, which indicated that microbial-driven carbon isotopic fractionation can cause great uncertainties in paleoclimate and paleoenvironment reconstructions.

Carbon Dioxide Sequestering Ability of Bacterial Carbonic Anhydrase in a Mangrove Soil Microcosm and Its Bio-mineralization Properties

In this study, we attempt to prospect potential bacterial isolates from mangrove sediments of Mangalavanam, Kerala, India, with positive carbonic anhydrase (CA) activity to sequester carbon dioxide by calcium precipitation process. Fifteen bacterial colonies (M1–M15) isolated were screened for their carbonic anhydrase enzyme production potential based on p-nitro phenol acetate assay. Based on the secondary screening, M3 and M8 were identified as the most potential for carbonic anhydrase production. The specific activity of the partially purified CA enzyme from M3 and M8 were 44 U mg−1 and 76 U mg−1 respectively. The enzyme activity increased by 1.6-fold upon precipitation by acetone (80%). The potential isolate which higher CA production, M8 was identified as Bacillus altitudinis based on 16S rDNA sequencing. Soil microcosm was established to study carbonic anhydrase production and CO2 sequestration ability of B. altitudinis M8 strain. B. altitudinis M8 strain could reduce CO2 by 75 ± 0.12% in microcosm composed of sterilized soil with bacteria (SSB) and by 97 ± 0.34% in microcosm with sterile soil with enzyme (SSE). Hence, the application of enzyme was found to be more effective in removing CO2 when compared to bacterial inoculum. To further understand the bio-mineralization ability of this microbial isolate, calcium precipitation assay was conducted. There was a reduction of 42.22 ± 0.23% of free calcium in the medium through calcite precipitation. The carbonic anhydrase-mediated calcium precipitation by B. altitudinis M8 strain could be effectively employed in the process of carbon dioxide sequestration.

Characterization of marine bacterial carbonic anhydrase and their CO2 sequestration abilities based on a soil microcosm

The novel technology of biological carbon sequestration using microbial enzymes have numerous advantages over conventional sequestration strategies. In the present study, extracellular carbonic anhydrase (CA) producing bacteria were isolated from water samples in the Arabian Sea, India. A potential isolate, Bacillus safensis isolate AS-75 was identified based on 16S rDNA sequence analysis. The culture conditions suitable for CA production were 32 °C incubation temperature with 4% NaCl and 10 mM Zn supplementation. Experimental optimization of culture conditions enhanced enzyme activity to 265 U mL−1. CA specific gene was characterized and based on the analysis, the CA of B. safensis isolate AS-75 was a leucine (11.3%) with α-helices as the dominant component in its secondary structure. Based on soil microcosm studies, CA could sequester CO2 by 95.4% ± 0.11% in sterilized soil with enzyme microcosm. Hence, the application of enzyme was found to be more effective in removing CO2.

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1.

Bacterial α-type carbonic anhydrase (α-CA) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable α-CA from Persephonella marina EX-H1 (pmCA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pmCA homodimer in which each monomer consists of a 10-stranded β-sheet in the center. The catalytic zinc ion is coordinated by three highly conserved histidine residues with an exchangeable fourth ligand (a water molecule, a bicarbonate anion, or the sulfonamide group of acetazolamide). Together with an intramolecular disulfide bond, extensive interfacial networks of hydrogen bonds, ionic and hydrophobic interactions stabilize the dimeric structure and are likely responsible for the high thermal stability. We also identified novel binding sites for calcium ions at the crystallographic interface, which serve as molecular glue linking negatively charged and otherwise repulsive surfaces. Furthermore, this large negatively charged patch appears to further increase the thermostability at alkaline pH range via favorable charge-charge interactions between pmCA and solvent molecules. These findings may assist development of novel α-CAs with improved thermal and/or alkaline stability for applications such as CO2 capture and sequestration.

Click-tailed benzenesulfonamides as potent bacterial carbonic anhydrase inhibitors for targeting Mycobacterium tuberculosis and Vibrio cholerae

A series of 1,2,3-triazole-bearing benzenesulfonamides was assessed for the inhibition of carbonic anhydrases (CA, EC 4.2.1.1) from bacteria Vibrio cholerae (VchCAα and VchCAβ) and Mycobacterium tuberculosis (β-mtCA3). Growing resistance phenomena against existing antimicrobial drugs are globally spreading and highlight a urgent need of agents endowed with alternative mechanisms of action. Two global WHO strategies aim to reduce cholera deaths by 90% and eradicate the tuberculosis epidemic by 2030. The derivatives here reported represent interesting leads towards the optimization of new antibiotic agents showing excellent inhibitory efficiency and selectivity for the target CAs over the human (h) off-target isoform hCA I. In detail, the first subset of derivatives potently inhibits VchCAα in a low nanomolar range (KIs between 0.72 and 22.6 nM). Compounds of a second subset, differing from the first one for the position of the spacer between benzenesulfonamide and triazole, preferentially inhibit VchCAβ (KIs in the range 54.8–102.4 nM) and β-mtCA3 (KIs in the range 28.2–192.5 nM) even more than the clinically used AAZ, used as the standard.

Inhibition of α-, β-, γ-, δ-, ζ- and η-class carbonic anhydrases from bacteria, fungi, algae, diatoms and protozoans with famotidine

Famotidine, an antiulcer drug belonging to the H2 antagonists class of pharmacological agents, was recently shown to potently inhibit human (h) and bacterial carbonic anhydrases (CAs, EC 4.2.1.1). We investigated the inhibitory effects of famotidine against all classes of CAs from the pathogenic bacteria Vibrio cholerae, Burkholderia pseudomallei and Mycobacterium tuberculosis Rv3273 β-CA, as well as the CAs from the nonpathogenic bacteria/cyanobacteria Sulfurihydrogenibium yellowstonensis, S. azorense, Pseudoalteromonas haloplanktis, Colwellia psychrerythraea and Nostoc commune. The δ- and ζ-CAs from the diatom Thalassiosira weissflogii, the fungal enzymes from Cryptococcus neoformans, Candida glabrata and Malassezia globosa, as well as the protozoan enzymes from Trypanosoma cruzi and Plasmodium falciparum, were also investigated. Anopheles gambiae β-CA was also investigated. All these enzymes were effectively inhibited by famotidine, with affinities between the low nanomolar to the micromolar range. The best inhibition was observed against C. glabrata β-CA and TweCAζ, with KIs ranging between 13.6 and 22.1 nM.

Seeking new approach for therapeutic treatment of cholera disease via inhibition of bacterial carbonic anhydrases: experimental and theoretical studies for sixteen benzenesulfonamide derivatives

A series of sixteen benzenesulfonamide derivatives has been synthesised and tested as inhibitors of Vibrio cholerae carbonic anhydrase (CA) enzymes, belonging to α-CA, β-CA, and γ-CA classes (VchCAα, VchCAβ, and VchCAγ). The determined Ki values were compared to those of selected human CA isoforms (hCA I and hCA II). Structure-affinity relationship analysis highlighted that all tested compounds proved to be active inhibitors of VchCAα at nanomolar concentration. The VchCAβ activity was lower to respect inhibitory efficacy toward VchCAα, whereas, these benzenesulfonamide derivatives failed to inhibit VchCAγ. Interestingly, compound 7e combined the best activity toward VchCAα and VchCAβ. In order to obtain a model for binding mode of our inhibitors toward bacterial CAs, we carried out docking simulations by using the available crystal structures of VchCAβ.

Inhibition of bacterial α-, β- and γ-class carbonic anhydrases with selenazoles incorporating benzenesulfonamide moieties

A series of benzenesulfonamides incorporating selenazoles with diverse substitution patterns were investigated as inhibitors of six bacterial carbonic anhydrases (CAs, EC 4.2.1.1) from bacterial pathogens, such as Helicobacter pylori (hpCAα was the investigated enzyme), Vibrio cholerae (all the three CAs from this pathogen were considered, VchCAα, VchCAβ and VchCAγ) and Burkholderia pseudomallei (with its two CAs, BpsCAβ and BpsCAγ). All these sulfonamides were effective CA inhibitors, with potencies in the low micromolar or submicromolar range, making them attractive as lead compounds for designing antibacterials with a novel mechanism of action, which could counteract the extensive resistance problem observed with many clinically used antibiotics.

An Overview of the Bacterial Carbonic Anhydrases.

Bacteria encode carbonic anhydrases (CAs, EC 4.2.1.1) belonging to three different genetic families, the α-, β-, and γ-classes. By equilibrating CO2 and bicarbonate, these metalloenzymes interfere with pH regulation and other crucial physiological processes of these organisms. The detailed investigations of many such enzymes from pathogenic and non-pathogenic bacteria afford the opportunity to design both novel therapeutic agents, as well as biomimetic processes, for example, for CO2 capture. Investigation of bacterial CA inhibitors and activators may be relevant for finding antibiotics with a new mechanism of action.

Role of bacterial carbonic anhydrase during CO2 capture in the CO2-H2O-carbonate system

Atmospheric CO2 are closely connected to climate change and global carbon cycle. Karst processes in CO2-H2O-carbonate system can absorb atmospheric CO2. Carbonic anhydrase (CA) can efficiently catalyze the interconversion between CO2 and HCO3−, which may help accelerate karst processes. To explore the influence of bacterial CA on CO2 capture capacity in CO2-H2O-carbonate system, a flow dissolution experimental device was designed to simulate CO2 absorption by rainwater infiltration or surface water flushing. Results showed that, when bacterial CA was added into the experimental system, it increased not only the rate but also the quantity of CO2 absorption, and the CO2 captured by limestone or dolomite dissolution increased by at least 18.9% and 22.1%, respectively. However, excessively high concentration of CO2 was not conducive to carbonate rock dissolution by bacterial CA. In accordance with the fixed carbon caused by global carbonate dissolution, the contribution of soil CA to the CO2 sinks may reach 0.567–1.938×1014gC/a. In summary, the addition of microbial CA could increase the CO2 capture in the form of DIC in CO2-H2O-carbonate system, thereby increasing the carbon sink potential of karst systems. The results help establish a promising approach for reducing CO2 emission by microbial CA.

Inhibition of Bacterial Carbonic Anhydrases as a Novel Approach to Escape Drug Resistance.

Clinically used antibiotics act through one of these four mechanisms: cell wall biosynthesis inhibition, inhibition of protein biosynthesis, interference with DNA and RNA synthesis and the folate pathway. The metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) widespread in microorganisms and present as three genetically distinct families may be considered for the design of antiinfective agents with a different mechanism of action compared to the clinically used antibiotics. CAs are crucial for the life cycle of the pathogen, interfering with pH regulation and biosynthetic processes in which CO2 or bicarbonate are substrates. CA inhibition was shown to lead to debilitation or growth defects of several pathogenic bacteria. CAs catalyzes the interconversion between carbon dioxide to bicarbonate, leading to the formation of protons, and thus affecting pH homeostasis. Several classes of CA inhibitors (CAIs) are known to date, among which the metal complexing anions, the unsubstituted sulfonamides, the dithiocarbamates, etc., which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere. Effective inhibitors for many bacterial CAs belonging to the α-, β-, and γ-CA classes were detected, some of which inhibited bacterial growth in vivo. Few of the inhibitors investigated so far were also selective for the bacterial over the human CA isoforms, which may pose problems for their wide clinical applications. Structure-based drug design campaigns might lead to the achievement of the desired selectivity/ potency for preferentially inhibiting bacterial but not the host CAs.

Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration.

Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

New light on bacterial carbonic anhydrases phylogeny based on the analysis of signal peptide sequences

Among protein families, carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes characterized by a common reaction mechanism in all life domains: the carbon dioxide hydration to bicarbonate and protons (CO2+H2O ⇔ HCO3−+H+). Six genetically distinct CA families are known to date, the α-, β-, γ-, δ-, ζ- and η-CAs. The last CA class was recently discovered analyzing the amino acid sequences of CAs from Plasmodia. Bacteria encode for enzymes belonging to the α-, β-, and γ-CA classes and recently, phylogenetic analysis revealed an interesting relationship regarding the evolution of bacterial CA classes. This result evidenced that the three bacterial CA classes, in spite of the high level of the structural similarity, are evolutionarily distinct, but we noted that the primary structure of some β-CAs identified in the genome of Gram-negative bacteria present a pre-sequence of 18 or more amino acid residues at the N-terminal part. These observations and subsequent phylogenetic data presented here prompted us to propose that the β-CAs found in Gram-negative bacteria with a periplasmic space and characterized by the presence of a signal peptide might have a periplasmic localization and a role similar to that described previously for the α-CAs.

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.

Inhibition studies of bacterial, fungal and protozoan β-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties

A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three β-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis.The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high β-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs.

An Overview of the Selectivity and Efficiency of the Bacterial Carbonic Anhydrase Inhibitors.

The possibility to develop new antibacterial agents raised much interest recently. 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. Recently, carbonic anhydrases (CAs, EC 4.2.1.1) started to be investigated in detail 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. CAs catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. Several classes of CA inhibitors (CAIs) are known to date: the metal complexing anions and the unsubstituted sulfonamides, which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere, generating trigonal- bipyramidal species are the classical, most frequently investigated ones. In many cases effective inhibitors were detected, some of which also inhibited the bacterial growth in vivo. However, very few of the detected inhibitors were also selective for the bacterial over the human, off target isoforms such as hCA II. Using structure-based drug design processes, we estimate that it will be possible to achieve the desired selectivity for inhibiting preferentially the bacterial but not the host CA isoforms.

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA), an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II) as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO2 substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.