Beta-carbonic Anhydrase Research Articles

Incidence of Acute Cystoid Macular Edema after Starting a Prostaglandin Analog Compared with Other Classes of Glaucoma Medications

PurposeThere is a longstanding belief that prostaglandin analogs (PGAs) may predispose patients with glaucoma to develop acute cystoid macular edema (CME). However, there is little solid evidence supporting this notion. The purpose of this study is to compare CME incidence rates among patients initiating treatment with different glaucoma medication classes. DesignDatabase study. Participants39948 patients who were newly prescribed glaucoma medications MethodsUsing data from 10 health systems contributing data to the Sight Outcomes Research Collaborative (SOURCE) Ophthalmology Data Repository, we identified all adults with glaucoma who had been newly started on a topical glaucoma medication. Patients with pre-existing documentation of macular edema were excluded. We assessed the incidence of CME among patients with glaucoma who were newly started on PGAs, topical beta blockers (BBs), alpha agonists (AAs), and carbonic anhydrase inhibitors (CAIs). Using multivariable logistic regression, and adjusting for sociodemographic factors, we assessed the odds of developing CME among patients prescribed each of the 4 glaucoma medication classes. We also performed a subset regression analysis including lens status as a co-variate. Main outcome measuresIncidence of CME within 3 months of initiating therapy with different topical glaucoma medications. ResultsAmong the 39,948 patients were newly treated with a topical glaucoma medication, 139 (0.35%) developed CME. The incidence of CME was 0.13%, 0.65%, 0.55%, 1.76% for users of PGAs, BBs, alpha agonists (AAs) and carbonic anhydrase inhibitors (CAIs), respectively. After adjusting for sociodemographic factors, users of topical BBs, AAs and CAIs had substantially higher odds of developing CME compared with PGA users (P<0.001 for all comparisons). The subset analysis also showed higher odds ratio of the non-PGA medication classes in association with CME. ConclusionsClinicians should reconsider the notion that PGAs carry a higher risk of CME versus other glaucoma medication classes. If additional studies support the findings of these analyses, clinicians may feel more comfortable prescribing PGAs to patients with glaucoma without fear they will predispose patients to CME.

Effects of Liquid Nitrogen Freezing, Immersion Freezing, and Air Freezing on Properties of Perca fluviatilis Fillets and Analysis of Potential Protein Markers Based on Label-Free Proteomics

The effects of liquid nitrogen freezing (LNF), immersion freezing (IF), and air freezing (AF) combined with thawing in running water at 20°C on the quality of Perca fluviatilis fillets were investigated using label-free proteomics technology. The ice crystal equivalent diameter (12.30±0.33 μm) and ice crystal area ratio (7.61±2.81%) of LNF were significantly lower than IF and AF (P < 0.05). The ice crystals of LNF were small and evenly distributed, and the mechanical damage to the cell structure is small. LNF samples were closer to fresh samples regarding texture. Proteomic analysis identified a total of 11 differentially abundant proteins (DAPs) associated with LNF, IF, and AF. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these DAPs were primarily catalytic proteins, metabolic enzymes, and structural proteins involved in metabolic pathways such as pyruvate metabolism, glycolysis, and arginine and proline metabolism. Among them, tubulin beta chain (A0A8C3G6F1), carbonic anhydrase (A0A2P1JP97), immunoglobulin-like and fibronectin type III domain-containing protein 1 (A0A484DE91), and tubulin are predicted to serve as potential protein markers for freezing, thawing loss, hardness, and springiness of Perca fluviatilis fillets post-thawing. These findings provide a basis for further research into the quality change induced by protein degradation of frozen Perca fluviatilis.

Beta-carbonic anhydrase gene expression levels change depending on the drought severity in both the leaves and roots of Arabidopsis thaliana

Beta-carbonic anhydrase gene expression levels change depending on the drought severity in both the leaves and roots of Arabidopsis thaliana

Gene duplications facilitate C4-CAM compatibility in common purslane.

Common purslane (Portulaca oleracea) integrates both C4 and crassulacean acid metabolism (CAM) photosynthesis pathways and is a promising model plant to explore C4-CAM plasticity. Here, we report a high-quality chromosome-level genome of nicotinamide adenine dinucleotide (NAD)-malic enzyme (ME) subtype common purslane that provides evidence for 2 rounds of whole-genome duplication (WGD) with an ancient WGD (P-β) in the common ancestor to Portulacaceae and Cactaceae around 66.30 million years ago (Mya) and another (Po-α) specific to common purslane lineage around 7.74 Mya. A larger number of gene copies encoding key enzymes/transporters involved in C4 and CAM pathways were detected in common purslane than in related species. Phylogeny, conserved functional site, and collinearity analyses revealed that the Po-α WGD produced the phosphoenolpyruvate carboxylase-encoded gene copies used for photosynthesis in common purslane, while the P-β WGD event produced 2 ancestral genes of functionally differentiated (C4- and CAM-specific) beta carbonic anhydrases involved in the C4 + CAM pathways. Additionally, cis-element enrichment analysis in the promoters showed that CAM-specific genes have recruited both evening and midnight circadian elements as well as the Abscisic acid (ABA)-independent regulatory module mediated by ethylene-response factor cis-elements. Overall, this study provides insights into the origin and evolutionary process of C4 and CAM pathways in common purslane, as well as potential targets for engineering crops by integrating C4 or CAM metabolism.

Drug utilization, medication adherence and treatment satisfaction amongst glaucoma patients receiving care at secondary health facilities

Abstract Objectives The objective of this study was to assess treatment satisfaction, medication adherence and drug utilization amongst glaucoma patients receiving care at secondary health facilities. Methods A cross-sectional study was conducted amongst glaucoma patients attending eye clinics of General Hospital Ilorin and Civil Service Hospital Ilorin in Kwara State, Nigeria from October 2020 to January 2021. Three hundred and eleven patients were enrolled in the study. Sampling was based on convenience as dictated by the inclusion and exclusion criteria. Ethical permission and informed consent were obtained before data collection. Validated instruments of Morisky Medication Adherence Scale and Treatment Satisfaction Questionnaire for Medication were used during the data collection process. Descriptive and Chi-square analyses were employed. Key findings Amongst the 311 patients studied 184(59.2%) were females with a mean age of 63.79 ± 14.89 years. Timolol eye drop was the drug of choice for monotherapy in (8.7%) of patients. Combination therapy of beta blockers and carbonic anhydrase inhibitors had the highest number (59.5%) of prescriptions. More than half (55.6%) of the patients were adherents. Participants reported being more satisfied with their anti-glaucoma therapy in the domain of global satisfaction (62.89 ± 21.85) followed by medication effectiveness (62.67 ± 20.78) and convenience (60.16 ± 15.79) while medication side effects had the least score (17.17 ± 11.09). There is a statistically significant relationship between patient satisfaction with treatment and medication adherence at P= 0.001. Conclusions Combination therapy was the most frequently utilized anti-glaucoma agent. Most of the patients adhered to their medications and were satisfied with the treatment received.

Identification and molecular characterization of the alternative spliced variants of beta carbonic anhydrase 1 (βCA1) from Arabidopsis thaliana.

Carbonic anhydrases (CAs) are ubiquitous zinc metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. Higher plants mainly contain the three evolutionarily distinct CA families αCA, βCA, and γCA, with each represented by multiple isoforms. Alternative splicing (AS) of the CA transcripts is common. However, there is little information on the spliced variants of individual CA isoforms. In this study, we focused on the characterization of spliced variants of βCA1 from Arabidopsis. The expression patterns and subcellular localization of the individual spliced variants of βCA1 were examined. The results showed that the spliced variants of βCA1 possessed different subcellular and tissue distributions and responded differently to environmental stimuli. Additionally, we addressed the physiological role of βCA1 in heat stress response and its protein-protein interaction (PPI) network. Our results showed that βCA1 was regulated by heat stresses, and βca1 mutant was hypersensitive to heat stress, indicating a role for βCA1 in heat stress response. Furthermore, PPI network analysis revealed that βCA1 interacts with multiple proteins involved in several processes, including photosynthesis, metabolism, and the stress response, and these will provide new avenues for future investigations of βCA1.

Biochemical and structural characterization of beta-carbonic anhydrase from the parasite Trichomonas vaginalis

Trichomonas vaginalis is a unicellular parasite and responsible for one of the most common sexually transmittable infections worldwide, trichomoniasis. Carbonic anhydrases (CAs) are enzymes found in all lifeforms and are known to play a vital role in many biochemical processes in organisms including the maintenance of acid–base homeostasis. To date, eight evolutionarily divergent but functionally convergent forms of CAs (α, β, γ, δ, ζ, η, θ, and ι) have been discovered. The human genome contains only α-CAs, whereas many clinically significant pathogens express only β-CAs and/or γ-CAs. The characterization of pathogenic β- and γ-CAs provides important knowledge for targeting these biomolecules to develop novel anti-invectives against trichomoniasis. Here, we report the recombinant production and characterization of the second β-CA of T. vaginalis (TvaCA2). Light scattering analysis revealed that TvaCA2 is a dimeric protein, which was further supported with in silico modeling, suggesting similar structures between TvaCA2 and the first β-CA of T. vaginalis (TvaCA1). TvaCA2 exhibited moderate catalytic activity with the following kinetic parameters: kcat of 3.8 × 105 s−1 and kcat/KM of 4.4 × 107 M−1 s−1. Enzyme activity inhibition was studied with a set of clinically used sulfonamides and sulfonamide derivates. Twenty-seven out of the 39 compounds resulted in inhibition with a nanomolar range. These initial results encourage for future work entailing the design of more potent inhibitors against TvaCA2, which may provide new assets to fight trichomoniasis.Key messages• Protozoan parasite Trichomonas vaginalis has two β-carbonic anhydrases (TvaCA1/2).• TvaCA1/TvaCA2 represents promising targets for antitrichomonal drug development.• TvaCA2 is a dimer of 20.3 kDa and possesses moderate catalytic activity.• The most efficient inhibitor was clinical drug acetazolamide with KI of 222.9 nM.• The 39 tested sulfonamides form the basis for the design of more potent inhibitors.

Application of beta and gamma carbonic anhydrase sequences as tools for identification of bacterial contamination in the whole genome sequence of inbred Wuzhishan minipig (Sus scrofa) annotated in databases.

Sus scrofa or pig was domesticated thousands of years ago. Through various indigenous breeds, different phenotypes were produced such as Chinese inbred miniature minipig or Wuzhishan pig (WZSP), which is broadly used in the life and medical sciences. The whole genome of WZSP was sequenced in 2012. Through a bioinformatics study of pig carbonic anhydrase (CA) sequences, we detected some β- and γ-class CAs among the WZSP CAs annotated in databases, while β- or γ-CAs had not previously been described in vertebrates. This finding urged us to analyze the quality of whole genome sequence of WZSP for the possible bacterial contamination. In this study, we used bioinformatics methods and web tools such as UniProt, European Bioinformatics Institute, National Center for Biotechnology Information, Ensembl Genome Browser, Ensembl Bacteria, RSCB PDB and Pseudomonas Genome Database. Our analysis defined that pig has 12 classical α-CAs and 3 CA-related proteins. Meanwhile, it was approved that the detected CAs in WZSP are categorized in the β- and γ-CA families, which belong to Pseudomonas spp. and Acinetobacter spp. The protein structure study revealed that the identified β-CA sequence from WZSP belongs to Pseudomonas aeruginosa with PDB ID: 5JJ8, and the identified γ-CA sequence from WZSP belongs to P. aeruginosa with PDB ID: 3PMO. Bioinformatics and computational methods accompanied with bacterial-specific markers, such as 16S rRNA and β- and γ-class CA sequences, can be used to identify bacterial contamination in mammalian DNA samples.

Lower Eyelid Horizontal Tightening in Prostaglandin Associated Periorbitopathy.

To determine the effect of chronic topical use of prostaglandin analogs for glaucoma on lower eyelid tension. A prospective case-control study was performed. Lower eyelid tension was measured in a group of glaucoma patients (73 eyelids) using prostaglandin analogs and a paired control group (87 eyelids). Seven glaucoma patients with unilateral use of prostaglandin analogs had the lower eyelid tension of the exposed side compared with the contralateral eyelid. Eyelid tension was digitally measured in photographs in neutral position and after manual downward traction in the center of the eyelid (modified distraction test). Duration of drug exposition, age, use of other topical medication and other prostaglandin-associated periorbitopathy signs as long eyelashes, eyelid hyperemia, eyelid pigmentation, and deepening of upper eyelid sulcus were recorded for analysis. Mean lower eyelid tension in prostaglandin group was significantly higher than in control group: distraction 5.26 mm (SD 1.52) versus 6.80 mm (SD 1.29) (p < 0.001). On the unilateral prostaglandin use intragroup comparison, mean lower eyelid distraction in prostaglandin side was 4.24 mm (SD 1.80) and in control side was 6.79 mm (SD 1.63) (p < 0.012). Beta blocker and carbonic anhydrase inhibitor concomitant use was associated with lower eyelid tension measures in prostaglandin users. Presence of long eyelashes was associated with higher eyelid tension. All other measured variables did not demonstrate interaction with eyelid tension. Chronic topical use of prostaglandin analogs for glaucoma is associated with lower eyelid tightening.

Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (β-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world’s leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, β, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only β- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The β-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of β-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.

Expression profiles of 12 drought responsive genes in two European (deciduous) oak species during a two-year drought experiment with consecutive drought periods

Based on an earlier comparative transcriptomics study (Madritsch et al., 2019, in revision) 12 representative genes were selected that were differentially expressed in response to drought stress in two major European oak species, pedunculate oak (Quercus robur) and pubescent oak (Q. pubescens). RT-qPCR technique was used to validate the quantification data from the transcriptomics study and to generate detailed gene expression profiles over the course of a two-year drought experiment with repeated long-term drought events comprising eight sampling dates. The 12 genes were selected based on quantification data and on their functional annotation and belong to different pathways and phytohormone signaling networks. PP2C27 (Protein phosphatase 2C 27), RD22 (Response to desiccation 22) and STP13 (Sugar transport protein 13) are key molecules of the abscisic acid (ABA) signaling pathway that plays a major role in the modulation of the molecular drought stress response. AOS1 (Allene oxide synthase 1) is the first enzyme in the biosynthesis of jasmonates (JAs), ERF4 (Ethylen responsive transcription factor 4) is part of the ethylene-activated pathway and CDR1 (Constitutive disease resistance 1) is part of the salicylic acid (SA) dependent pathway. Two genes related to the phenylpropanoid pathway were included, PAL (Phenylalanine ammoniase-lyase) catalyzes the first step of the pathway and UGT73C6 (UDP-glycosyltransferase 73C6) plays a role in the biosynthesis of quercetins and other flavonoids, these contribute to tolerance against oxidative stress. NRX2 (Nucleoredoxin 2) also plays a role in protection against oxidative stress. Also, two genes that are related to photosynthesis, BCA2 (Beta carbonic anhydrase 2) and GPT2 (Glucose-6-phosphate/phosphate translocator 2) and the nutrient transporter BOR2 (Boron transporter 2) were included. Most genes revealed clearly different expression profiles over the course of the experiment, indicating an adjustment process during the long-term repeated drought periods. Despite being part of the same signaling network, the ABA-related genes showed divers expression profiles, reflecting the complexity of the drought response and the modulation of the response over the course of the experiment in the two Quercus species. The expression profiles for AOS1, ERF4 and PAL indicate that they were upregulated by JA pathway in Q. pubescens independently from drought stress treatment, contradicting an involvement of JA pathway to the drought stress response. BCA2, NRX2, PP2C27 showed a contrary regulation in the two species that might be linked to their different drought tolerance while BOR2 and UGT73C6 were shown to be drought responsive for the first time.

Laying the Foundation for Crassulacean Acid Metabolism (CAM) Biodesign: Expression of the C4 Metabolism Cycle Genes of CAM in Arabidopsis.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to hotter and drier climates. Introducing the CAM photosynthetic machinery into C3 (or C4) photosynthesis plants (CAM Biodesign) represents a potentially breakthrough strategy for improving WUE while maintaining high productivity. To optimize the success of CAM Biodesign approaches, the functional analysis of individual C4 metabolism cycle genes is necessary to identify the essential genes for robust CAM pathway introduction. Here, we isolated and analyzed the subcellular localizations of 13 enzymes and regulatory proteins of the C4 metabolism cycle of CAM from the common ice plant in stably transformed Arabidopsis thaliana. Six components of the carboxylation module were analyzed including beta-carbonic anhydrase (McBCA2), phosphoenolpyruvate carboxylase (McPEPC1), phosphoenolpyruvate carboxylase kinase (McPPCK1), NAD-dependent malate dehydrogenase (McNAD-MDH1, McNAD-MDH2), and NADP-dependent malate dehydrogenase (McNADP-MDH1). In addition, seven components of the decarboxylation module were analyzed including NAD-dependent malic enzyme (McNAD-ME1, McNAD-ME2), NADP-dependent malic enzyme (McNADP-ME1, NADP-ME2), pyruvate, orthophosphate dikinase (McPPDK), pyruvate, orthophosphate dikinase-regulatory protein (McPPDK-RP), and phosphoenolpyruvate carboxykinase (McPEPCK). Ectopic overexpression of most C4-metabolism cycle components resulted in increased rosette diameter, leaf area, and leaf fresh weight of A. thaliana except for McNADP-MDH1, McPPDK-RP, and McPEPCK. Overexpression of most carboxylation module components resulted in increased stomatal conductance and dawn/dusk titratable acidity (TA) as an indirect measure of organic acid (mainly malate) accumulation in A. thaliana. In contrast, overexpression of the decarboxylating malic enzymes reduced stomatal conductance and TA. This comprehensive study provides fundamental insights into the relative functional contributions of each of the individual components of the core C4-metabolism cycle of CAM and represents a critical first step in laying the foundation for CAM Biodesign.

Cloning, Expression and Characterization of Two Beta Carbonic Anhydrases from a Newly Isolated CO2 Fixer, Serratia marcescens Wy064.

Bacterial strains from karst landform soil were enriched via chemostat culture in the presence of sodium bicarbonate. Two chemolithotrophic strains were isolated and identified as Serratia marcescens Wy064 and Bacillus sp. Wy065. Both strains could grow using sodium bicarbonate as the sole carbon source. Furthermore, the supplement of the medium with three electron donors (Na2S, NaNO2, and Na2S2O3) improved the growth of both strains. The activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) could be detected in the crude enzyme of strain Wy064, implying that the strain Wy064 might employ Calvin cycle to fix CO2. S. marcescens genome mining revealed four potential CA genes designated CA1-CA4. The proteins encoded by genes CA1-3 were cloned and expressed in Escherichia coli. The purified recombinant enzymes of CA1 and CA3 exhibited CO2 hydration activities, whereas enzyme CA2 was expressed in inclusion bodies. A CO2 hydration assay demonstrated that the specific activity of CA3 was significantly higher than that of CA1. The maximum CO2 hydration activities for CA1 and CA3 were observed at pH 7.5 and 40°C. The activities of CA1 and CA3 were significantly enhanced by several metal ions, especially Zn2+, which resulted in 21.1-fold and 26.1-fold increases of CO2 hydration activities, respectively. The apparent K m and V max for CO2 as substrate were 27mM and 179 WAU/mg for CA1, and 14mM and 247 WAU/mg for CA3, respectively. Structure modeling combined with sequence analysis indicated that CA1 and CA3 should belong to the Type II β-CA.

Non-linear Physiology and Gene Expression Responses of Harmful Alga Heterosigma akashiwo to Rising CO2

Heterosigma akashiwo is a raphidophyte known for forming ichthyotoxic blooms. In order to predict the potential impacts of rising CO2 on H. akashiwo it is necessary to understand the factors influencing growth rates over a range of CO2 concentrations. Here we examined the physiology and gene expression response of H. akashiwo to concentrations from 200 to 1000ppm CO2. Growth rate data were combined from this and previous studies and fit with a CO2 limitation-inhibition model that revealed an apparent growth optimum around 600-800ppm CO2. Physiological changes included a significant increase in C:N ratio at ∼800ppm CO2 and a significant decrease in hydrogen peroxide concentration at ∼1000ppm. Whole transcriptome sequencing of H. akashiwo revealed sharp distinctions in metabolic pathway gene expression between ∼600 and ∼800ppm CO2. Hierarchical clustering by co-expression identified groups of genes with significant correlations to CO2 and growth rate. Genes with significant differential expression with CO2 included carbon concentrating mechanism genes such as beta-carbonic anhydrases and a bicarbonate transporter, which may underpin shifts in physiology. Genes involved in cell motility were significantly changed by both elevated CO2 and growth rate, suggesting that future ocean conditions could modify swimming behavior in this species.

Herbarium genomics retraces the origins of C4-specific carbonic anhydrase in Andropogoneae (Poaceae)

ABSTRACTNatural history collections are traditionally used for taxonomic research, but their relevance can extend into studies of molecular evolution as illustrated here using the example of C4 photosynthesis. This complex trait boosts growth in open and warm conditions and evolved numerous times, providing multiple comparisons among C3 and C4 relatives. Previous genomic studies relied on comparisons of model species which are often separated by long-time divergence. The large C4 group Andropogoneae (Poaceae) was usually compared to relatively distant C3 taxa. Early-diverging C4 Andropogoneae and closely related C3 taxa are known but are difficult to collect and hence have not been sequenced yet. We show here that genome scans generated from herbarium samples can be used to retrace the evolution of βca genes, which encode beta carbonic anhydrase, an enzyme of the C4 pathway. Genomic fragments encompassing βca genes were assembled for specimens of phylogenetic importance, and their comparison to model grasses made it possible to distinguish changes that happened during the transition to C4 from those that preceded or followed it. Tandem gene duplications occurred during the early grass diversification, and the duplicate that retained the ancestral alternative exon conferring chloroplast localization was later co-opted for C4 photosynthesis. Early transition to C4 photosynthesis apparently relied on an increase of the cytosolic alternative transcript without drastic changes in the βca gene, which occurred later during the diversification of C4 groups. These insights into the genomic rearrangements linked to the evolution of an adaptive trait highlight the potential of natural history collections for genomic analyses.

Designing of phenol-based β-carbonic anhydrase1 inhibitors through QSAR, molecular docking, and MD simulation approach.

Tuberculosis (Tb) is an airborne infectious disease caused by Mycobacterium tuberculosis. Beta-carbonic anhydrase 1 (β-CA1) has emerged as one of the potential targets for new antitubercular drug development. In this work, three-dimensional quantitative structure-activity relationships (3D-QSAR), molecular docking, and molecular dynamics (MD) simulation approaches were performed on a series of natural and synthetic phenol-based β-CA1 inhibitors. The developed 3D-QSAR model (r2 = 0.94, q2 = 0.86, and pred_r2 = 0.74) indicated that the steric and electrostatic factors are important parameters to modulate the bioactivity of phenolic compounds. Based on this indication, we designed 72 new phenolic inhibitors, out of which two compounds (D25 and D50) effectively stabilized β-CA1 receptor and, thus, are potential candidates for new generation antitubercular drug discovery program.

Anion inhibition studies of a beta carbonic anhydrase from the malaria mosquito Anopheles gambiae

An anion inhibition study of the β-class carbonic anhydrase, AgaCA, from the malaria mosquito Anopheles gambiae is reported. A series of simple as well as complex inorganic anions, and small molecules known to interact with CAs were included in the study. Bromide, iodide, bisulphite, perchlorate, perrhenate, perruthenate, and peroxydisulphate were ineffective AgaCA inhibitors, with KIs > 200 mM. Fluoride, chloride, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrite, nitrate, sulphate, stannate, selenate, tellurate, diphosphate, divanadate, tetraborate, selenocyanide, and trithiocarbonate showed KIs in the range of 1.80–9.46 mM, whereas N,N-diethyldithiocarbamate was a submillimolar AgaCA inhibitor (KI of 0.65 mM). The most effective AgaCA inhibitors were sulphamide, sulphamic acid, phenylboronic acid and phenylarsonic acid, with inhibition constants in the range of 21–84 µM. The control of insect vectors responsible of the transmission of many protozoan diseases is rather difficult nowadays, and finding agents which can interfere with these processes, as the enzyme inhibitors investigated here, may arrest the spread of these diseases worldwide.

Cloning, Over-Expression, and Purification of Carbonic Anhydrase from an Extremophilic Bacterium: An Introduction to Advanced Molecular Biology

The photosynthetic bioreactor research program is a training platform appropriate for introducing advanced molecular biology techniques to undergraduate students and advanced high school biology students. For this advanced molecular biology training exercise, the enzyme carbonic anhydrase was cloned, over-expressed, purified, and functionally characterized. Carbonic anhydrases are industrially important enzymes with potential use in carbon sequestration and biofuel production. Alpha and beta carbonic anhydrases from Photobacterium profundum, a psychrophilic, halotolerant bacterium, were characterized in this study. Carbonic anhydrases that can withstand high salinity and are active at low temperatures can be transformed into oleaginous marine microalgae to enhance biofuel production. Our research program started with a three-day boot camp with lectures in relevant topics of molecular biology, microbiology, and research methods. After the boot camp, the lab phase of the project involved training students to perform polymerase chain reaction, DNA gel electrophoresis, DNA ligation, and bacterial transformation. In the final phase of the project, students were trained in recombinant protein over-expression and protein purification techniques. Here we report successful cloning and over-expression by high school students of two novel carbonic anhydrases from a psychrohalophile with application in biofuel production.

Resurrecting ancestral genes in bacteria to interpret ancient biosignatures.

Two datasets, the geologic record and the genetic content of extant organisms, provide complementary insights into the history of how key molecular components have shaped or driven global environmental and macroevolutionary trends. Changes in global physico-chemical modes over time are thought to be a consistent feature of this relationship between Earth and life, as life is thought to have been optimizing protein functions for the entirety of its approximately 3.8 billion years of history on the Earth. Organismal survival depends on how well critical genetic and metabolic components can adapt to their environments, reflecting an ability to optimize efficiently to changing conditions. The geologic record provides an array of biologically independent indicators of macroscale atmospheric and oceanic composition, but provides little in the way of the exact behaviour of the molecular components that influenced the compositions of these reservoirs. By reconstructing sequences of proteins that might have been present in ancient organisms, we can downselect to a subset of possible sequences that may have been optimized to these ancient environmental conditions. How can one use modern life to reconstruct ancestral behaviours? Configurations of ancient sequences can be inferred from the diversity of extant sequences, and then resurrected in the laboratory to ascertain their biochemical attributes. One way to augment sequence-based, single-gene methods to obtain a richer and more reliable picture of the deep past, is to resurrect inferred ancestral protein sequences in living organisms, where their phenotypes can be exposed in a complex molecular-systems context, and then to link consequences of those phenotypes to biosignatures that were preserved in the independent historical repository of the geological record. As a first step beyond single-molecule reconstruction to the study of functional molecular systems, we present here the ancestral sequence reconstruction of the beta-carbonic anhydrase protein. We assess how carbonic anhydrase proteins meet our selection criteria for reconstructing ancient biosignatures in the laboratory, which we term palaeophenotype reconstruction.This article is part of the themed issue ‘Reconceptualizing the origins of life’.

Horizontal transfer of β-carbonic anhydrase genes from prokaryotes to protozoans, insects, and nematodes.

BackgroundHorizontal gene transfer (HGT) is a movement of genetic information occurring outside of normal mating activities. It is especially common between prokaryotic endosymbionts and their protozoan, insect, and nematode hosts. Although beta carbonic anhydrase (β-CA) plays a crucial role in metabolic functions of many living organisms, the origin of β-CA genes in eukaryotic species remains unclear.MethodsThis study was conducted using phylogenetics, prediction of subcellular localization, and identification of β-CA, transposase, integrase, and resolvase genes on the MGEs of bacteria. We also structurally analyzed β-CAs from protozoans, insects, and nematodes and their putative prokaryotic common ancestors, by homology modelling.ResultsOur investigations of a number of target genomes revealed that genes coding for transposase, integrase, resolvase, and conjugation complex proteins have been integrated with β-CA gene sequences on mobile genetic elements (MGEs) which have facilitated the mobility of β-CA genes from bacteria to protozoan, insect, and nematode species. The prokaryotic origin of protozoan, insect, and nematode β-CA enzymes is supported by phylogenetic analyses, prediction of subcellular localization, and homology modelling.ConclusionMGEs form a complete set of enzymatic tools, which are relevant to HGT of β-CA gene sequences from prokaryotes to protozoans, insects, and nematodes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1415-7) contains supplementary material, which is available to authorized users.