Alpha Carbonic Anhydrase from Nitratiruptor tergarcus Engineered for Increased Activity and Thermostability.

The development of carbon capture and storage technologies has resulted in a rising interest in the use of carbonic anhydrases (CAs) for CO2 fixation at elevated temperatures. In this study, we chose to rationally engineer the α-CA (NtCA) from the thermophilic bacterium Nitratiruptor tergarcus, which has been previously suggested to be thermostable by in silico studies. Using a combination of analyses with the DEEPDDG software and available structural knowledge, we selected residues in three regions, namely, the catalytic pocket, the dimeric interface and the surface, in order to increase thermostability and CO2 hydration activity. A total of 13 specific mutations, affecting seven amino acids, were assessed. Single, double and quadruple mutants were produced in Escherichia coli and analyzed. The best-performing mutations that led to improvements in both activity and stability were D168K, a surface mutation, and R210L, a mutation in the dimeric interface. Apart from these, most mutants showed improved thermostability, with mutants R210K and N88K_R210L showing substantial improvements in activity, up to 11-fold. Molecular dynamics simulations, focusing particularly on residue fluctuations, conformational changes and hydrogen bond analysis, elucidated the structural changes imposed by the mutations. Successful engineering of NtCA provided valuable lessons for further engineering of α-CAs.

In vitro and In silico assessment of antischistosomal activities of ethanolic extract of Cornulacamonacantha.

Schistosomiasis is the second most prevailing parasitic disease worldwide. Although praziquantel is considered an effective drug in the treatment against schistosomiasis to some extent, there is an emerging drug resistance that widely recorded. Therefore, there is an urgent need to develop effective and safe anti-schistosomal drugs. In this study, Cornulaca monacantha (C. monacantha), a sub-saharan plant, was extracted using aqueous ethanol and characterized by High-Performance Liquid Chromatography (HPLC). Major constituents of the extract are belonging to flavonoids, tannins and phenolic glycosides. Worms' viability and surface morphology of Schistosoma mansoni (S. mansoni) adult worms treated with the extract were assessed using in vitro viability assay, Scanning Electron Microscopy (SEM), and histological examination. The extract (80-350 µg/ml) reduced viability percentage of worms by 40-60% and caused degeneration of both oral and ventral suckers, tegumental, sub-tegumental and muscular damage. Molecular docking approach was utilized to assess the binding affinities of the extracted compounds with S. mansoni alpha-carbonic anhydrase (SmCA), an essential tegument protein. Pharmacokinetic analysis using SwissADME showed that 7 compounds have high drug similarity. This study confirms the in vitro schistomicidal activity of C. monacantha extract against S. mansoni adult worms and suggests potential SmCA inhibition.

The production and biochemical characterization of α-carbonic anhydrase from Lactobacillus rhamnosus GG.

We report the production and biochemical characterization of an α-carbonic anhydrase (LrhCA) from gram-positive probiotic bacteria Lactobacillus rhamnosus GG. CAs form a family of metalloenzymes that catalyze hydration of CO2/interconversion between CO2 and water to bicarbonate ions and protons. They are divided into eight independent gene families (α, ß, γ, δ, ζ, η, θ, and ι). Interestingly, many pathogens have been identified with only ß- and/or γ-CAs, which can be targeted with CA-specific inhibitors (CAIs) acting as anti-pathogen drugs. Since it is important to study the potential off-target effects of CAIs for both the human body and its commensal bacteria, we took L. rhamnosus GG as our study subject. To date, only a single α-CA has been identified in L. rhamnosus GG, which was successfully produced and biochemically characterized. LrhCA showed moderate catalytic activity with the following kinetic parameters: kcat of 9.86 × 105 s-1 and kcat/KM of 1.41 × 107 s-1 M-1. Moderate inhibition was established with 11 of the 39 studied sulfonamides. The best inhibitors were 5-((4-aminophenyl)sulfonamido)-1,3,4-thiadiazole-2-sulfonamide, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide, and benzolamide with Ki values of 319 nM, 378 nM, and 387 nM, respectively. The other compounds showed weaker inhibitory effects. The Ki of acetazolamide, a classical CAI, was 733 nM. In vitro experiments with acetazolamide showed that it had no significant effect on cell growth in L. rhamnosus GG culture. Several sulfonamides, including acetazolamide, are in use as clinical drugs, making their inhibition data highly relevant to avoid any adverse off-target effects towards the human body and its probiotic organisms. KEY POINTS: • The α-carbonic anhydrase from Lactobacillus rhamnosus GG (LrhCA) is 24.3 kDa. • LrhCA has significant catalytic activity with a kcat of 9.9 × 105 s-1. • Acetazolamide resulted in a marginal inhibitory effect on cell growth.

Alpha-Carbonic Anhydrases from Hydrothermal Vent Sources as Potential Carbon Dioxide Sequestration Agents: In Silico Sequence, Structure and Dynamics Analyses.

With the increase in CO2 emissions worldwide and its dire effects, there is a need to reduce CO2 concentrations in the atmosphere. Alpha-carbonic anhydrases (α-CAs) have been identified as suitable sequestration agents. This study reports the sequence and structural analysis of 15 α-CAs from bacteria, originating from hydrothermal vent systems. Structural analysis of the multimers enabled the identification of hotspot and interface residues. Molecular dynamics simulations of the homo-multimers were performed at 300 K, 363 K, 393 K and 423 K to unearth potentially thermostable α-CAs. Average betweenness centrality (BC) calculations confirmed the relevance of some hotspot and interface residues. The key residues responsible for dimer thermostability were identified by comparing fluctuating interfaces with stable ones, and were part of conserved motifs. Crucial long-lived hydrogen bond networks were observed around residues with high BC values. Dynamic cross correlation fortified the relevance of oligomerization of these proteins, thus the importance of simulating them in their multimeric forms. A consensus of the simulation analyses used in this study suggested high thermostability for the α-CA from Nitratiruptor tergarcus. Overall, our novel findings enhance the potential of biotechnology applications through the discovery of alternative thermostable CO2 sequestration agents and their potential protein design.