Β-carbonic Anhydrase Research Articles

Repurposing eugenol and cinnamaldehyde as potent antimicrobial agents: A comprehensive in-vitro and in-silico study.

Multi-drug-resistant (MDR) pathogens represent a critical global health threat, necessitating the development of novel antimicrobial agents with broad-spectrum activity and minimal toxicity. This study investigates the antimicrobial and anti-biofilm properties of 4-Allyl-2-methoxyphenol (eugenol, EU) and (E)-3-Phenylprop-2-enal (cinnamaldehyde, CN) against 19 clinically significant pathogens through a combination of in-vitro assays and in-silico analyses. EU displayed remarkable activity, particularly against Aspergillus niger (20.5±0.5mm), and strong binding affinities with key protein targets, including peptide deformylase and β-carbonic anhydrase, with binding free energies (ΔG) ranging from -12.75 to -0.60kcal/mol. CN exhibited exceptional activity against Staphylococcus epidermidis (29.6±0.4mm) and Candida albicans (36.6±0.4mm), supported by a significant binding affinity with β-carbonic anhydrase (ΔG: -5.23kcal/mol). Dissociation constants (Kd) derived from MM-GBSA analyses indicated EU's strong inhibitory potential with nano- to picomolar Kd values, directly correlating with low IC50 values. CN demonstrated moderate inhibitory activity with Kd in the micromolar range. Molecular dynamics (MD) simulations confirmed the stability of these protein-ligand complexes, revealing critical hydrophobic interactions, such as those involving PHE122, that contributed to binding stabilization. ADMET profiling further underscored the favorable pharmacokinetics and safety of both compounds. These findings establish EU and CN as promising candidates for antimicrobial therapy, with potential applications in combating MDR pathogens and biofilm-associated infections. The complementary strengths of EU and CN warrant further structural optimization and combination studies, offering new avenues in the development of next-generation antimicrobial agents.

Expression and localization of two β-carbonic anhydrases in Bienertia, a single-cell C4 plant

Expression and localization of two β-carbonic anhydrases in Bienertia, a single-cell C4 plant

Characterization of fungal carbonyl sulfide hydrolase belonging to clade D β-carbonic anhydrase.

Carbonyl sulfide hydrolase (COSase) is a unique enzyme that exhibits high activity towards carbonyl sulfide (COS) but low carbonic anhydrase (CA) activity, despite belonging to the CA family. COSase was initially identified in a sulfur-oxidizing bacterium and later discovered in the ascomycete Trichoderma harzianum strain THIF08. The COSase from T. harzianum has been recognized as a key enzyme in the assimilation of gaseous COS, and homologous genes are widely present not only in Ascomycota but also in Basidiomycota. Here, we characterized the COSases from the basidiomycete Gloeophyllum trabeum NBRC 6430 and T. harzianum to obtain detailed characteristics of fungal COSase. This study contributes to a better understanding of COS metabolism in fungi.

Functional plasticity of HCO3– uptake and CO2 fixation in Cupriavidus necator H16

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3– uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.

A comprehensive investigation of the anion inhibition profile of a β-carbonic anhydrase from Acinetobacter baumannii for crafting innovative antimicrobial treatments

This study refers to the intricate world of Acinetobacter baumannii, a resilient pathogenic bacterium notorious for its propensity at antibiotic resistance in nosocomial infections. Expanding upon previous findings that emphasised the bifunctional enzyme PaaY, revealing unexpected γ-carbonic anhydrase (CA) activity, our research focuses on a different class of CA identified within the A. baumannii genome, the β-CA, designated as 𝛽-AbauCA (also indicated as CanB), which plays a crucial role in the resistance mechanism mediated by AmpC beta-lactamase. Here, we cloned, expressed, and purified the recombinant 𝛽-AbauCA, unveiling its distinctive kinetic properties and inhibition profile with inorganic anions (classical CA inhibitors). The exploration of 𝛽-AbauCA not only enhances our understanding of the CA repertoire of A. baumannii but also establishes a foundation for targeted therapeutic interventions against this resilient pathogen, promising advancements in combating its adaptability and antibiotic resistance.

Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase.

The conversion of CO2 by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, in situ methods following the microscopic details of CO2 conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO2, water, bicarbonate, and other reactants with β-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-CoA carboxylase/reductase from Kitasatospora setae (KsCcr), two of the fastest CO2-converting enzymes in nature. Our data reveal that KsCcr possesses a so far unknown metal-independent CA-like activity. Site-directed mutagenesis of conserved active site residues combined with molecular dynamics simulations tracing CO2 distributions in the active site of KsCCr identify an 'activated' water molecule forming the hydroxyl anion that attacks CO2 and yields bicarbonate (HCO3-). Computer simulations also explain why substrate binding inhibits the anhydrase activity. Altogether, we demonstrate how in situ infrared spectroscopy combined with molecular dynamics simulations provides a simple yet powerful new approach to investigate the atomistic reaction mechanisms of different enzymes with CO2.

Design and synthesis of betulinic acid-dithiocarbamate conjugates as potential antifungal agents against Candida albicans.

Diverse betulinic acid-dithiocarbamate conjugates were designed and synthesized via a two-step reaction at room temperature. Among the fourteen dithiocarbamate analogs of betulinic acid, DTC2 demonstrated the best antifungal activity against Candida albicans, with a minimum inhibitory concentration (MIC) of 4 μg mL-1, achieving 99% fungicidal activity at the same concentration. These compounds were found to be ineffective against common Gram-negative and Gram-positive pathogens, suggesting their specificity to fungi. Furthermore, DTC2 exhibited synergistic effects with the antifungal drugs fluconazole and nystatin, resulting in a significant decrease in MIC by 64 and 16 folds, respectively, when co-administered. Notably, the molecule also hindered hyphae formation in Candida albicans, thereby reducing its pathogenicity. Furthermore, it displayed time- and concentration-dependent kill kinetics, sterilizing C. albicans within 8 hours at 8× MIC. Additionally, DTC2 exhibits greater efficacy against β-carbonic anhydrase with better docking scores and binding patterns than ethoxyzolamide, a well-known inhibitor of β-carbonic anhydrase.

Mycobacterial β-carbonic anhydrases: Molecular biology, role in the pathogenesis of tuberculosis and inhibition studies.

Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB), is still a major global health problem. According to the World Health Organization (WHO), TB still causes more deaths worldwide than any other infectious agent. Drug-sensitive TB is treatable using first-line drugs; treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB requires second- and third-line drugs. However, due to the long duration of treatment, the noncompliance of patients with different levels of resistance of Mtb to these drugs has worsened the situation. Previously developed anti-TB drugs targeted the replication machinery, protein synthesis, and cell wall biosynthesis pathways of Mtb. Therefore, novel drugs targeting alternate pathways crucial for the survival and pathogenesis of Mtb in the human host are needed. The genome of Mtb encodes three β-carbonic anhydrases (CAs) that are fundamental for pH homeostasis, hypoxia, survival, and pathogenesis. Recently, several studies have shown that the β-CAs of Mtb could be inhibited both in vitro and in vivo using small chemical molecules, suggesting that these enzymes could be novel targets for developing anti-TB compounds that are devoid of resistance by Mtb. In addition, homologs of β-CAs are absent in humans; therefore, drugs developed to target these enzymes might have minimal off-target effects. In this work, we describe the roles of β-CAs in Mtb and discuss bioinformatics and cheminformatics tools used in development and discovery of novel inhibitors of these enzymes. In addition, we summarize the in vitro and in vivo studies demonstrating that the β-CAs of Mtb are indeed druggable targets.

Bacterial β-carbonic anhydrases.

β-Carbonic anhydrases (β-CA; EC 4.2.1.1) are widespread zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. They have been isolated in many pathogenic and non-pathogenic bacteria where they are involved in multiple roles, often related to their growth and survival. β-CAs are structurally distant from the CAs of other classes. In the active site, located at the interface of a fundamental dimer, the zinc ion is coordinated to two cysteines and one histidine. β-CAs have been divided in two subgroups depending on the nature of the fourth ligand on the zinc ion: class I have a zinc open configuration with a hydroxide ion completing the metal coordination, which is the catalytically active species in the mechanism proposed for the β-CAs similar to the well-known of α-CAs, while in class II an Asp residue substitute the hydroxide. This latter active site configuration has been showed to be typical of an inactive form at pH below 8. An Asp-Arg dyad is thought to play a key role in the pH-induced catalytic switch regulating the opening and closing of the active site in class II β-CAs, by displacing the zinc-bound solvent molecule. An allosteric site well-suited for bicarbonate stabilizes the inactive form. This bicarbonate binding site is composed by a triad of well conserved residues, strictly connected to the coordination state of the zinc ion. Moreover, the escort site is a promiscuous site for a variety of ligands, including bicarbonate, at the dimer interface, which may be the route for bicarbonate to the allosteric site.

Antimicrobial Potency and E. coli β-Carbonic Anhydrase Inhibition Efficacy of Phenazone-Based Molecules.

In this investigation, 4-antipyrinecarboxaldhyde was reacted with methyl hydrazinecarbodithioate to afford the carbodithioate derivative 3. The as-prepared carbodithioate derivative 3 is considered to be a key molecule for the preparation of new antipyrine-1,3,4-thiadiazole-based molecules (4-9) through its reaction with the appropriate hydrazonoyl halides. Furthermore, a typical Biginelli three-component cyclocondensation reaction involving ethyl acetoacetate, 4-antipyrinecarboxaldhyde, and thiourea under the standard conditions is carried out in the presence of sulfuric acid to afford the corresponding antipyrine-pyrimidine hybrid molecule (10). The latter was submitted to react with hydrazine monohydrate to provide the corresponding hydrazide derivative (11) which, under reaction with ethyl acetoacetate in refluxing ethanol containing catalytic amount of acetic acid, afforded the corresponding derivative (12). The structure of the newly synthesized compounds was affirmed by their spectral and microanalytical data. We also screened for their antimicrobial potential (ZOI and MIC) and conducted a kinetic study. Additionally, the mechanism of biological action was assessed by a membrane leakage assay and SEM imaging technique. Moreover, the biological activities and the binding modes of these compounds were further supplemented by an in silico docking study against E. coli β-carbonic anhydrase. The amount of cellular protein released by E. coli is directly correlated to the concentration of compound 9, which was found to be 177.99 µg/mL following treatment with 1.0 mg/mL of compound 9. This finding supports compound 9's antibacterial properties and explains how the formation of holes in the E. coli cell membrane results in the release of proteins from the cytoplasm. The newly synthesized compounds represent acceptable antimicrobial activities with potential action against E. coli β-carbonic anhydrase. The docking studies and antimicrobial activity test proved that compound (9) declared a greater activity than the other synthesized compounds.

Cooperation of an external carbonic anhydrase and HCO3- transporter supports underwater photosynthesis in submerged leaves of the amphibious plant Hygrophila difformis.

HCO3- can be a major carbon resource for photosynthesis in underwater environments. Here we investigate the underlying mechanism of uptake and membrane transport of HCO3- in submerged leaves of Hygrophila difformis, a heterophyllous amphibious plant. To characterize these mechanisms, we evaluated the sensitivity of underwater photosynthesis to an external carbonic anhydrase (CA) inhibitor and an anion exchanger protein inhibitor, and we attempted to identify components of the mechanism of HCO3- utilization. We evaluated the effects of the external CA inhibitor and anion exchanger protein inhibitor on the NaHCO3 response of photosynthetic O2 evolution in submerged leaves of H. difformis. Furthermore, we performed a comparative transcriptomic analysis between terrestrial and submerged leaves. Photosynthesis in the submerged leaves was decreased by both the external CA inhibitor and anion exchanger protein inhibitor, but no additive effect was observed. Among upregulated genes in submerged leaves, two α-CAs, Hdα-CA1 and Hdα-CA2, and one β-carbonic anhydrase, Hdβ-CA1, were detected. Based on their putative amino acid sequences, the α-CAs are predicted to be localized in the apoplastic region. Recombinant Hdα-CA1 and Hdβ-CA1 showed dominant CO2 hydration activity over HCO3- dehydration activity. We propose that the use of HCO3- for photosynthesis in submerged leaves of H. difformis is driven by the cooperation between an external CA, Hdα-CA1, and an unidentified HCO3- transporter.

Green synthesis, antibacterial and antifungal evaluation of new thiazolidine-2,4-dione derivatives: molecular dynamic simulation, POM study and identification of antitumor pharmacophore sites

In this study, a series of thiazolidine-2,4-dione derivatives 3a–i were synthesized and evaluated for antibacterial activity against Gram-positive and Gram-negative strains of Bacillus licheniformis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Newly prepared thiazolidine (TZD) derivatives were further screened separately for in vitro antifungal activity against cultures of fungal species, namely, Aspergillus niger, Alternaria brassicicola, Chaetomium murorum, Fusarium oxysporum, Lycopodium sp. and Penicillium notatum. The electron-donating substituents (–OH and –OCH3) and electron-withdrawing substituents (–Cl and –NO2) on the attached arylidene moieties of five-membered heterocyclic ring enhanced the broad spectrum of antimicrobial and antifungal activities. The molecular docking study has revealed that compound 3h strongly interacts with the catalytic residues of the active site of the β-carbonic anhydrase (P. aeruginosa) and has the best docking score. In silico pharmacokinetics studies showed the drug-likeness and non-toxic nature of the synthesized compounds, which indicates the combined antibacterial, antiviral and antitumor pharmacophore sites of the targeted drug. This work demonstrates that potential TZD derivatives bind to different types of bacterial and fungal pathogens for circumventing their activities and opens avenues for the development of newer drug candidates that can target bacterial and fungal pathogens. Communicated by Ramaswamy H. Sarma

Expression of a periplasmic β-carbonic anhydrase (CA) gene is positively correlated with HCO3- utilization by the gametophytes of Saccharina japonica (Phaeophyceae, Ochrophyta)

Expression of a periplasmic β-carbonic anhydrase (CA) gene is positively correlated with HCO3- utilization by the gametophytes of Saccharina japonica (Phaeophyceae, Ochrophyta)

The pivotal ripening gene SlDML2 participates in regulating disease resistance in tomato.

Fruit ripening and disease resistance are two essential biological processes for quality formation and maintenance. DNA methylation, in the form of 5-methylcytosine (5mC), has been elucidated to modulate fruit ripening, but its role in regulating fruit disease resistance remains poorly understood. In this study, we show that mutation of SlDML2, the DNA demethylase gene essential for fruit ripening, affects multiple developmental processes of tomato besides fruit ripening, including seed germination, leaf length and width and flower branching. Intriguingly, loss of SlDML2 function decreased the resistance of tomato fruits against the necrotrophic fungal pathogen Botrytis cinerea. Comparative transcriptomic analysis revealed an obvious transcriptome reprogramming caused by SlDML2 mutation during B. cinerea invasion. Among the thousands of differentially expressed genes, SlβCA3 encoding a β-carbonic anhydrase and SlFAD3 encoding a ω-3 fatty acid desaturase were demonstrated to be transcriptionally activated by SlDML2-mediated DNA demethylation and positively regulate tomato resistance to B. cinerea probably in the same genetic pathway with SlDML2. We further show that the pericarp tissue surrounding B. cinerea infection exhibited a delay in ripening with singnificant decrease in expression of ripening genes that are targeted by SlDML2 and increase in expression of SlβCA3 and SlFAD3. Taken together, our results uncover an essential layer of gene regulation mediated by DNA methylation upon B. cinerea infection and raise the possible that the DNA demethylase gene SlDML2, as a multifunctional gene, participates in modulating the trade-off between fruit ripening and disease resistance.

The Carbonic Anhydrase βCA1 Functions in PopW-Mediated Plant Defense Responses in Tomato.

β-Carbonic anhydrase (βCA) is very important for plant growth and development, but its function in immunity has also been examined. In this study, we found that the expression level of Solanum lycopersicum βCA1 (SlβCA1) was significantly upregulated in plants treated with Xanthomonas euvesicatoria 85-10. The protein was localized in the nucleus, cell membrane and chloroplast. Using tomato plants silenced with SlβCA1, we demonstrated that SlβCA1 plays an active role in plant disease resistance. Moreover, we found that the elicitor PopW upregulated the expression of SlβCA1, while the microbe-associated molecular pattern response induced by PopW was inhibited in TRV-SlβCA1. The interaction between PopW and SlβCA1 was confirmed. Here, we found that SlβCA1 was positively regulated during PopW-induced resistance to Xanthomonas euvesicatoria 85-10. These data indicate the importance of SlβCA1 in plant basic immunity and its recognition by the Harpin protein PopW as a new target for elicitor recognition.

CanB, a Druggable Cellular Target in Mycobacterium tuberculosis.

Treatment against tuberculosis can lead to the selection of drug-resistant Mycobacterium tuberculosis strains. To tackle this serious threat, new targets from M. tuberculosis are needed to develop novel effective drugs. In this work, we aimed to provide a possible workflow to validate new targets and inhibitors by combining genetic, in silico, and enzymological approaches. CanB is one of the three M. tuberculosis β-carbonic anhydrases that catalyze the reversible reaction of CO2 hydration to form HCO3- and H+. To this end, we precisely demonstrated that CanB is essential for the survival of the pathogen in vitro by constructing conditional mutants. In addition, to search for CanB inhibitors, conditional canB mutants were also constructed using the Pip-ON system. By molecular docking and minimum inhibitory concentration assays, we selected three molecules that inhibit the growth in vitro of M. tuberculosis wild-type strain and canB conditional mutants, thus implementing a target-to-drug approach. The lead compound also showed a bactericidal activity by the time-killing assay. We further studied the interactions of these molecules with CanB using enzymatic assays and differential scanning fluorimetry thermal shift analysis. In conclusion, the compounds identified by the in silico screening proved to have a high affinity as CanB ligands endowed with antitubercular activity.

Cloning, characterization, and inhibition of the novel β-carbonic anhydrase from parasitic blood fluke, Schistosoma mansoni

Schistosoma mansoni is an intestinal parasite with one β-class carbonic anhydrase, SmaBCA. We report the sequence enhancing, production, catalytic activity, and inhibition results of the recombinant SmaBCA. It showed significant catalytic activity on CO2 hydration in vitro with k cat 1.38 × 105 s−1 and k cat/Km 2.33 × 107 M−1 s−1. Several sulphonamide inhibitors, from which many are clinically used, showed submicromolar or nanomolar inhibitory effects on SmaBCA. The most efficient inhibitor with a KI of 43.8 nM was 4-(2-amino-pyrimidine-4-yl)-benzenesulfonamide. Other effective inhibitors with KI s in the range of 79.4–95.9 nM were benzolamide, brinzolamide, topiramate, dorzolamide, saccharin, epacadostat, celecoxib, and famotidine. The other tested compounds showed at least micromolar range inhibition against SmaBCA. Our results introduce SmaBCA as a novel target for drug development against schistosomiasis, a highly prevalent parasitic disease.

Extracellular CahB1 from Sodalinema gerasimenkoae IPPAS B-353 Acts as a Functional Carboxysomal β-Carbonic Anhydrase in Synechocystis sp. PCC6803

Cyanobacteria mostly rely on the active uptake of hydrated CO2 (i.e., bicarbonate ions) from the surrounding media to fuel their inorganic carbon assimilation. The dehydration of bicarbonate in close vicinity of RuBisCO is achieved through the activity of carboxysomal carbonic anhydrase (CA) enzymes. Simultaneously, many cyanobacterial genomes encode extracellular α- and β-class CAs (EcaA, EcaB) whose exact physiological role remains largely unknown. To date, the CahB1 enzyme of Sodalinema gerasimenkoae (formerly Microcoleus/Coleofasciculus chthonoplastes) remains the sole described active extracellular β-CA in cyanobacteria, but its molecular features strongly suggest it to be a carboxysomal rather than a secreted protein. Upon expression of CahB1 in Synechocystis sp. PCC6803, we found that its expression complemented the loss of endogenous CcaA. Moreover, CahB1 was found to localize to a carboxysome-harboring and CA-active cell fraction. Our data suggest that CahB1 retains all crucial properties of a cellular carboxysomal CA and that the secretion mechanism and/or the machinations of the Sodalinema gerasimenkoae carboxysome are different from those of Synechocystis.

CanB is a metabolic mediator of antibiotic resistance in Neisseria gonorrhoeae.

The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments and antibiotics. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations. We hypothesized that the requirement for supplemental CO2, present in approximately half of isolates, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO2 dependence is attributable to a single substitution in a β-carbonic anhydrase, CanB. CanB19E is necessary and sufficient for growth in the absence of CO2, and the hypomorphic CanB19G variant confers CO2 dependence. Furthermore, ciprofloxacin resistance is correlated with CanB19G in clinical isolates, and the presence of CanB19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has affected the acquisition of fluoroquinolone resistance.

Multigene manipulation of photosynthetic carbon metabolism enhances the photosynthetic capacity and biomass yield of cucumber under low-CO2 environment.

Solar greenhouses are important in the vegetable production and widely used for the counter-season production in the world. However, the CO2 consumed by crops for photosynthesis after sunrise is not supplemented and becomes chronically deficient due to the airtight structure of solar greenhouses. Vegetable crops cannot effectively utilize light resources under low-CO2 environment, and this incapability results in reduced photosynthetic efficiency and crop yield. We used cucumber as a model plant and generated several sets of transgenic cucumber plants overexpressing individual genes, including β-carbonic anhydrase 1 (CsβCA1), β-carbonic anhydrase 4 (CsβCA4), and sedoheptulose-1,7-bisphosphatase (CsSBP); fructose-1,6-bisphosphate aldolase (CsFBA), and CsβCA1 co-expressing plants; CsβCA4, CsSBP, and CsFBA co-expressing plants (14SF). The results showed that the overexpression of CsβCA1, CsβCA4, and 14SF exhibited higher photosynthetic and biomass yield in transgenic cucumber plants under low-CO2 environment. Further enhancements in photosynthesis and biomass yield were observed in 14SF transgenic plants under low-CO2 environment. The net photosynthesis biomass yield and photosynthetic rate increased by 49% and 79% compared with those of the WT. However, the transgenic cucumbers of overexpressing CsFBA and CsSBP showed insignificant differences in photosynthesis and biomass yield compared with the WT under low-CO2.environment. Photosynthesis, fluorescence parameters, and enzymatic measurements indicated that CsβCA1, CsβCA4, CsSBP, and CsFBA had cumulative effects in photosynthetic carbon assimilation under low-CO2 environment. Co-expression of this four genes (CsβCA1, CsβCA4, CsSBP, and CsFBA) can increase the carboxylation activity of RuBisCO and promote the regeneration of RuBP. As a result, the 14SF transgenic plants showed a higher net photosynthetic rate and biomass yield even under low-CO2environment.These findings demonstrate the possibility of cultivating crops with high photosynthetic efficiency by manipulating genes involved in the photosynthetic carbon assimilation metabolic pathway.