Carbonic anhydrases of anaerobic microbes

Carbonic anhydrases (CAs) catalyze the reversible hydration of carbon dioxide to bicarbonate and are abundantly distributed in prokaryotes and eukaryotes. There are five classes (α,β,γ,δ,ζ) with no significant sequence or structural identity among them, a remarkable example of convergent evolution. The β and γ classes predominate in anaerobic microbes, living without O₂, that comprise a substantial portion of the living protoplasm on Earth. Anaerobes reside in the lower intestinal tract of humans, one of many O₂-free environments on Earth, where they convert complex biomass to methane and CO₂ contributing an essential link in the global carbon cycle. Carbon dioxide is a universal metabolite of anaerobes necessitating CA for a diversity of proposed functions.     

The combined use of selective deuteration and double resonance experiments in assigning the 1H resonances of valine and tyrosine residues of dihydrofolate reductase

Selective deuteration is a general solution to the resolution problem which limits the application of double resonance experiments to the assignment of the ¹H NMR spectra of proteins. Spin-decoupling and NOE experiments have been carried out on Lactobacillus casei dihydrofolate reductase and on selectively deuterated derivatives of the enzyme containing either [γ-²H₆]Val or (α,δ₂,?₁-²H₃]His, [α,δ₁,δ₂,?₁,?₂,ζ-²H₆]Phe, [α,δ₁,?₃,ζ₂,ζ₃,η₂-²H₆]Trp and [α,?₁,?₂-²H₃]Tyr. When combined with ring-current shift calculations based on the crystal structure of the enzyme, these experiments allow us to assign ¹H resonances of Val 61, Val 115, Tyr 46 and Tyr 68.     

Carbonic anhydrase — a universal enzyme of the carbon-based life

Carbonic anhydrase (CA) is a metalloenzyme that performs interconversion between CO₂ and the bicarbonate ion (HCO₃ ^?). CAs appear among all taxonomic groups of three domains of life. Wide spreading of CAs in nature is explained by the fact that carbon, which is the major constituent of the enzyme’s substrates, is a key element of life on the Earth. Despite the diversity of CAs, they all carry out the same reaction of CO₂/HCO₃ ^? interconversion. Thus, CA obviously represents a universal enzyme of the carbon-based life. Within the classification of CAs, here we proposed the existence of an extensive family of CA-related proteins (γCA-RPs)–the inactive forms of γ-CAs, which are widespread among the Archaea, Bacteria, and, to a lesser extent, in Eukarya. This review focuses on the history of CAs discovery and integrates the most recent data on their classification, catalytic mechanisms, and physiological roles at various organisms.     

p24 Proteins from the same subfamily are functionally nonredundant

The p24 proteins function in early secretory pathway transport processes, but their exact role is unclear. In physiologically activated Xenopus melanotrope cells, a representative of each p24 subfamily (p24α₃, -β₁, -γ₃, -δ₂) is upregulated coordinately with the major melanotrope cargo, proopiomelanocortin (POMC), whereas two other p24s (p24γ₂ and -δ₁) are also expressed, but not coordinately with POMC. Using melanotrope-specific transgene expression, we here find that the roles of both p24γ₂ and p24δ₁ in the transport, glycosylation, sulphation and cleavage of POMC are different from those of their upregulated subfamily relatives (p24γ₃ and p24δ₂, respectively). Thus, even p24 proteins from the same subfamily have distinct functions in secretory cargo biosynthesis.     

The effect of pCO2 on carbon acquisition and intracellular assimilation in four marine diatoms

The effect of pCO₂ on carbon acquisition and intracellular assimilation was investigated in the three bloom-forming diatom species, Eucampia zodiacus (Ehrenberg), Skeletonema costatum (Greville) Cleve, Thalassionema nitzschioides (Grunow) Mereschkowsky and the non-bloom-forming Thalassiosira pseudonana (Hust.) Hasle and Heimdal. In vivo activities of carbonic anhydrase (CA), photosynthetic O₂ evolution, CO₂ and HCO₃⁻ uptake rates were measured by membrane-inlet mass spectrometry (MIMS) in cells acclimated to pCO₂ levels of 370 and 800 μatm. To investigate whether the cells operate a C₄-like pathway, activities of ribulose-1,5-bisphosphate carboxylase (RubisCO) and phosphoenolpyruvate carboxylase (PEPC) were measured at the mentioned pCO₂ levels and a lower pCO₂ level of 50 μatm. In the bloom-forming species, extracellular CA activities strongly increased with decreasing CO₂ supply while constantly low activities were obtained for T. pseudonana. Half-saturation concentrations (K_1/2) for photosynthetic O₂ evolution decreased with decreasing CO₂ supply in the two bloom-forming species S. costatum and T. nitzschioides, but not in T. pseudonana and E. zodiacus. With the exception of S. costatum, maximum rates (V_max) of photosynthesis remained constant in all investigated diatom species. Independent of the pCO₂ level, PEPC activities were significantly lower than those for RubisCO, averaging generally less than 3%. All examined diatom species operate highly efficient CCMs under ambient and high pCO₂, but differ strongly in the degree of regulation of individual components of the CCM such as C_i uptake kinetics and extracellular CA activities. The present data do not suggest C₄ metabolism in the investigated species.     

Anion inhibition profiles of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae

Among the numerous metalloenzymes known to date, carbonic anhydrase (CA, EC 4.2.1.1) was the first zinc containing one, being discovered decades ago. CA is a hydro-lyase, which catalyzes the following hydration–dehydration reaction: CO₂ + H₂O ⇋ HCO₃⁻ + H⁺. Several CA classes are presently known, including the α-, β-, γ-, δ-, ζ- and η-CAs. In prokaryotes, the existence of genes encoding CAs from at least three classes (α-, β- and γ-class) suggests that these enzymes play a key role in the physiology of these organisms. In many bacteria CAs are essential for the life cycle of microbes and their inhibition leads to growth impairment or growth defects of the pathogen. CAs thus started to be investigated in detail in bacteria, fungi and protozoa with the aim to identify antiinfectives with a novel mechanism of action. Here, we investigated the catalytic activity, biochemical properties and anion inhibition profiles of the three CAs from the bacterial pathogen Vibrio cholera, VchCA, VchCAβ and VchCAγ. The three enzymes are efficient catalysts for CO₂ hydration, with k_cat values ranging between (3.4 − 8.23) × 10⁵ s⁻¹ and k_cat/K_M of (4.1 − 7.0) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. The most potent VchCAγ inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging between 44 and 91 μM.     

Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO₂, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO₂-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.     

Construction and application of an Escherichia coli bioreporter for aniline and chloroaniline detection

Aniline and chlorinated anilines (CAs) are classified as priority pollutants; therefore, an effective method for detection and monitoring is required. In this study, a green-fluorescence protein-based bioreporter for the detection of aniline and CAs was constructed in Escherichia coli DH5α, characterized and tested with soil and wastewater. The sensing capability relied on the regulatory control between a two-component regulatory protein, TodS/TodT, and the P _todX promoter of Pseudomonas putida T-57 (PpT57), since the gene expression of todS, todT, and todC2 are positively induced with 4-chloroaniline. The bioreporter system (DH5α/pPXGFP–pTODST) is markedly unique with the two co-existing plasmids. The inducibility of the fluorescence response was culture-medium- and time-dependent. Cells grown in M9G medium exhibited a low background fluorescence level and were readily induced by 4CA after 3-h exposure, reaching the maximum induction level at 9?h. When tested with benzene, toluene, ethyl-benzene and xylene, aniline and CAs, the response data were best fit by a sigmoidal dose–response relationship, from which the K _1/2 value was determined for the positive effectors. 3CA and 4CA were relatively powerful inducers, while some poly-chlorinated anilines could also induce green fluorescence protein expression. The results indicated a broader recognition range of PpT57’sTodST than previously reported for P. putida. The test results with environmental samples were reliable, indicating the potential application of this bioreporter in the ecotoxicology assessment and bioremediation of areas contaminated with aniline- and/or CAs.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Recombinant plant gamma carbonic anhydrase homotrimers bind inorganic carbon

Gamma carbonic anhydrases (γCA) are widespread in Prokaryotes. In Eukaryotes, homologous genes were found only in plant genomes. In Arabidopsis and maize, the corresponding gene products are subunits of mitochondrial Complex I.At present, only γCA homotrimers of Methanosarcina thermophila (CAM) show reversible carbon dioxide (CO₂) hydration activity. In the present work, it is shown that recombinant plant γCA2 could form homotrimers and bind . However, they are unable to catalyse the reversible hydration of CO₂.These results suggest that plant γCAs do not act as carbonic anhydrases but with a related activity possibly contributing to recycle CO₂ in the context of photorespiration.

Structured summary

MINT-7266044: gamma CA2 (uniprotkb:Q9C6B3) and gamma CA2 (uniprotkb:Q9C6B3) physically interact (MI:0914) by dynamic light scattering (MI:0038)MINT-7266036: gamma CA2 (uniprotkb:Q9C6B3) and gamma CA2 (uniprotkb:Q9C6B3) physically interact (MI:0914) by molecular sieving (MI:0071)     

A matter of structure: structural comparison of fungal carbonic anhydrases

Carbonic anhydrases (CAs) are metalloenzymes that catalyze the interconversion of carbon dioxide (CO₂) and hydrogen carbonate. CAs are distributed over all the three domains of life and are divided into five distinct evolutionarily unrelated gene families (α, β, γ, δ, ζ). In the large fungal kingdom, the majority of fungi encode multiple copies of β-CAs, with some also possessing genes for α-class CAs. Hemiascomycetous and basidiomycetous yeasts encode one or two β-CAs, while most of the filamentous ascomycetes have multiple copies of genes encoding α- and β-CAs. The functions of fungal β-CAs have been investigated intensively, while the role of fungal α-CAs is mostly unknown. The β-CAs are involved in sexual development, CO₂-sensing, pathogenicity, and survival in ambient air. Only recently, researchers have begun to use functional and structural data of CAs from pathogenic and non-pathogenic organisms to develop powerful and effective drugs and inhibitors or to identify enzymes that can be utilized in industrial applications. Despite the large number of fungal CAs known, only five have been characterized structurally: the α-CA AoCA of Aspergillus oryzae, the full length β-CA Can2 from the pathogenic basidiomycete Cryptococcus neoformans, the N-terminally truncated Saccharomyces cerevisiae β-CA Nce103, and two β-CAs of Sordaria macrospora. This review focuses on the functional and structural properties of fungal CAs.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

Carbonic anhydrase inhibitors

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread enzymes in all organisms, catalyzing CO₂ hydration to bicarbonate and protons. Their inhibition is exploited clinically for decades for various classes of diuretics and systemically acting antiglaucoma agents. In the last years novel applications of CA inhibitors (CAIs) emerged, such as topically acting antiglaucoma, anticonvulsants, antiobesity, antipain, and antitumor agents/diagnostic tools. Such CAIs target diverse isozymes of the 13 catalytically active α-CA isoforms present in mammals. CAs belonging to the α-, β-, γ-, δ-, and ζ-families are found in many organisms all over the phylogenetic tree, and their inhibition was studied ultimately for some pathogenic protozoa (Plasmodium falciparum), fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae), and bacteria (Helicobacter pylori, Mycobacterium tuberculosis, and Brucella suis). Novel interesting chemotypes, in addition to the sulfonamide and sulfamate CAIs, such as coumarins, phenols, and fullerenes, were also reported recently, together with their mechanism of inhibition. This class of enzyme inhibitors shows promise for designing interesting pharmacological agents and understanding in detail protein–drug interactions at molecular level.     

Isolation of sea urchin embryo histone H2Aα1 and immunological identification of other stage-specific H2A proteins

H2A_α1, the principal H2A histone synthesized prior to the blastula stage of the sea urchin, was isolated free of other putative H2A subtypes and other histones. Its amino acid composition provides confirmation that H2A_α1 is the H2A protein encoded in the histone gene cluster carried by pCO2. An antibody prepared against this protein cross-reacts strongly with CS2A (a putative H2A synthesized only during the cleavage stage) as well as with H2A_β, H2A_γ, and H2A_δ (putative H2As synthesized principally after the blastula stage) but not with non-H2A core histones or other nuclear proteins. The data support the view that CS2A, H2A_α1, H2A_β, H2A_γ, and H2A_δ are all H2A proteins.     

Emerging roles for carbonic anhydrase in mesophyll conductance and photosynthesis

Carbonic anhydrase (CA) is an abundant protein in most photosynthesizing organisms and higher plants. This review paper considers the physiological importance of the more abundant CA isoforms in photosynthesis, through their effects on CO₂ diffusion and other processes in photosynthetic organisms. In plants, CA has multiple isoforms in three different families (α, β and γ) and is mainly known to catalyze the CO₂ equilibrium. This reversible conversion has a clear role in photosynthesis, primarily through sustaining the CO₂ concentration at the site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco). Despite showing the same major reaction mechanism, the three main CA families are evolutionarily distinct. For different CA isoforms, cellular localization and total gene expression as a function of developmental stage are predicted to determine the role of each family in relation to the net assimilation rate. Reaction–diffusion modeling and observational evidence support a role for CA activity in reducing resistance to CO₂ diffusion inside mesophyll cells by facilitating CO₂ transfer in both gas and liquid phases. In addition, physical and/or biochemical interactions between CAs and other membrane‐bound compartments, for example aquaporins, are suggested to trigger a CO₂‐sensing response by stomatal movement. In response to environmental stresses, changes in the expression level of CAs and/or stimulated deactivation of CAs may correspond with lower photosynthetic capacity. We suggest that further studies should focus on the dynamics of the relationship between the activity of CAs (with different subcellular localization, abundance and gene expression) and limitations due to CO₂ diffusivity through the mesophyll and supply of CO₂ to photosynthetic reactions.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.