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.     

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.     

Genetic Diversity among 3-Chloroaniline- and Aniline-Degrading Strains of the Comamonadaceae

We examined the diversity of the plasmids and of the gene tdnQ, involved in the oxidative deamination of aniline, in five bacterial strains that are able to metabolize both aniline and 3-chloroaniline (3-CA). Three strains have been described and identified previously, i.e., Comamonas testosteroni I2 and Delftia acidovorans CA28 and BN3.1. Strains LME1 and B8c were isolated in this study from linuron-treated soil and from a wastewater treatment plant, respectively, and were both identified as D. acidovorans. Both Delftia and Comamonas belong to the family Comamonadaceae. All five strains possess a large plasmid of ca. 100 kb, but the plasmids from only four strains could be transferred to a recipient strain by selection on aniline or 3-CA as a sole source of carbon and/or nitrogen. Plasmid transfer experiments and Southern hybridization revealed that the plasmid of strain I2 was responsible for total aniline but not 3-CA degradation, while the plasmids of strains LME1 and B8c were responsible only for the oxidative deamination of aniline. Several transconjugant clones that had received the plasmid from strain CA28 showed different degradative capacities: all transconjugants could use aniline as a nitrogen source, while only some of the transconjugants could deaminate 3-CA. For all four plasmids, the IS1071 insertion sequence of Tn5271 was found to be located on a 1.4-kb restriction fragment, which also hybridized with the tdnQ probe. This result suggests the involvement of this insertion sequence element in the dissemination of aniline degradation genes in the environment. By use of specific primers for the tdnQ gene from Pseudomonas putida UCC22, the diversity of the PCR-amplified fragments in the five strains was examined by denaturing gradient gel electrophoresis (DGGE). With DGGE, three different clusters of the tdnQ fragment could be distinguished. Sequencing data showed that the tdnQ sequences of I2, LME1, B8c, and CA28 were very closely related, while the tdnQ sequences of BN3.1 and P. putida UCC22 were only about 83% identical to the other sequences. Northern hybridization revealed that the tdnQ gene is transcribed only in the presence of aniline and not when only 3-CA is present.     

Bioluminescent bioreporter integrated-circuit sensing of microbial volatile organic compounds

A bioluminescent bioreporter for the detection of the microbial volatile organic compound p-cymene was constructed as a model sensor for the detection of metabolic by-products indicative of microbial growth. The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fused to a p-cymene/p-cumate-inducible promoter derived from the P. putida F1 cym operon. Exposure of strain UT93 to 0.02–850 ppm p-cymene produced self-generated bioluminescence in less than 1.5 h. Signals in response to specific volatile organic compounds (VOCs) such as m- and p-xylene and styrene, also occurred, but at two-fold lower bioluminescent levels. The bioreporter was interfaced with an integrated-circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures. This bioluminescent bioreporter integrated-circuit device was capable of detecting fungal presence within approximately 3.5 h of initial exposure to a culture of p-cymene-producing Penicillium roqueforti.     

Synthesis of sulfonamides with effective inhibitory action against Porphyromonas gingivalis γ-carbonic anhydrase

New benzenesulfonamides incorporating GABA or N-α-acetyl-l-lysine scaffolds as well as guanidine functionalities as water solubilizing moieties were obtained, using 4-aminoethyl/methyl-benzenesulfonamide and metanilamide/sulfanilamide as zinc-binding motives. The new compounds were medium potency inhibitors of the widespread cytosolic human carbonic anhydrase (CA, EC 4.2.1.1) isoforms I and II and more effective inhibitors (K_Is low nanomolar range) of the bacterial γ-CA from the oral pathogen Porphyromonas gingivalis. These sulfonamides may be useful tools for understanding the physiological role of bacterial CAs in pathogenesis of some infectious disease.     

2-Aminobenzoxazole-appended coumarins as potent and selective inhibitors of tumour-associated carbonic anhydrases

We have carried out the design, synthesis, and evaluation of a small library of 2-aminobenzoxazole-appended coumarins as novel inhibitors of tumour-related CAs IX and XII. Substituents on C-3 and/or C-4 positions of the coumarin scaffold, and on the benzoxazole moiety, together with the length of the linker connecting both units were modified to obtain useful structure-activity relationships. CA inhibition studies revealed a good selectivity towards tumour-associated CAs IX and XII (K_i within the mid-nanomolar range in most of the cases) in comparison with CAs I, II, IV, and VII (K_i > 10 µM); CA IX was found to be slightly more sensitive towards structural changes. Docking calculations suggested that the coumarin scaffold might act as a prodrug, binding to the CAs in its hydrolysed form, which is in turn obtained due to the esterase activity of CAs. An increase of the tether length and of the substituents steric hindrance was found to be detrimental to in vitro antiproliferative activities. Incorporation of a chlorine atom on C-3 of the coumarin moiety achieved the strongest antiproliferative agent, with activities within the low micromolar range for the panel of tumour cell lines tested.     

Intracellular carbonic anhydrase activities in Dunaliella tertiolecta (Butcher) and Chlamydomonas reinhardtii (Dangeard) in relation to inorganic carbon concentration during growth: further evidence for the existence of two distinct carbonic anhydrases associated with the chloroplasts

Using mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ intracellular carbonic anhydrase (CA) activity was investigated in the unicellular green algae Dunaliella tertiolecta and Chlamydomonas reinhardtii which were either grown on air enriched with 5% CO₂ (high-C_i cells) or on air (low-C_i cells). In D. tertiolecta high- and low-C_i cells had detectable levels of internal CA activity when measured under in-vivo conditions and this activity could be split up into three distinct forms. One CA was not associated with the chloroplasts, while two isozymes were found to be located within the plastids. The activities of all intracellular CAs were always about twofold higher in low than in high-C_i cells of D. tertiolecta and the chloroplastic enzymes were completely induced within 4 h of adaptation to air. One of the chloroplastic CAs was found to be soluble the other was insoluble. In addition to the physical differences, MgSO₄ in vitro caused a more than twofold stimulation of the soluble activity while the insoluble form of CA remained rather unaffected. In C. reinhardtii, MgSO₄ increased the soluble CA activity by 346% and the concentration of MgSO₄ required for half-maximum stimulation was between 10 and 15 mM. Again, the insoluble CA activity was not affected by MgSO₄. Furthermore, the soluble isoenzyme was considerably more sensitive to ethoxyzolamide, a potent inhibitor of CA, than the insoluble enzyme. The concentration of inhibitor causing 50% inhibition of soluble CA activity was 110 and 85 μM ethoxyzolamide for D. tertiolecta and C. reinhardtii, respectively. From these data we conclude that the two chloroplast-associated CAs are distinct enzymes.     

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.     

Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum—The η-carbonic anhydrases

The genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) thought to belong to the α-class, PfCA. Here we demonstrate the kinetic properties of PfCA for the CO₂ hydration reaction, as well as an inhibition study of this enzyme with inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, and phenylboronic/arsonic acids, detecting several low micromolar inhibitors. A closer examination of the sequence of this and the CAs from other Plasmodium spp., as well as a phylogenetic analysis, revealed that these protozoa encode for a yet undisclosed, new genetic family of CAs termed the η-CA class. The main features of the η-CAs are described in this report.     

Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.     

New family of biosensors for monitoring BTX in aquatic and edaphic environments

Benzene, toluene, ethylbenzene and xylenes (BTEX) contamination is a serious threat to public health and the environment, and therefore, there is an urgent need to detect its presence in nature. The use of whole‐cell reporters is an efficient, easy‐to‐use and low‐cost approach to detect and follow contaminants outside specialized laboratories; this is especially important in oil spills that are frequent in marine environments. The aim of this study is the construction of a bioreporter system and its comparison and validation for the specific detection of monocyclic aromatic hydrocarbons in different host bacteria and environmental samples. Our bioreporter system is based on the two component regulatory system TodS–TodT of P. putida DOT‐T1E, and the P_todX promoter fused to the GFP protein as the reporter protein. For the construction of different biosensors, this bioreporter was transferred into three different bacterial strains isolated from three different environments, and their performance was measured. Validation of the biosensors on water samples spiked with petrol, diesel and crude oil on contaminated waters from oil spills and on contaminated soils demonstrated that they can be used in mapping and monitoring some BTEX compounds (specifically benzene, toluene and two xylene isomers). Validation of biosensors is an important issue for the integration of these devices into pollution‐control programmes.     

Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353

One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V_max) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V_max during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V_max during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1°C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at ~35°C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.     

The first activation studies of the η-carbonic anhydrase from the malaria parasite Plasmodium falciparum with amines and amino acids

The first activation study of a η-class carbonic anhydrase (CAs, EC 4.2.1.1) is reported. A panel of 24 natural and non-natural amino acids and amines was used to explore the activation profile of Plasmodium falciparum CA (PfACA). The most effective activators belonged to the amino acid chemotype, with d-Glu, l-Asp, l-/d-Phe and l-/d-DOPA possessing activation constant in the range of 82 nM–0.75 µM, whereas l-/d-His, l-Tyr, 4-amino-l-Phe and l-Gln were slightly less effective (K_A in the range of 1.00–2.51 µM. The only amine with submicromolar activating properties was 1-(2-aminoethyl-piperazine) with a K_A of 0.71 µM, whereas histamine, dopamine and serotonin showed K_A ranging between 7.18 and 9.97 µM. As CA activators have scarcely been investigated for their interaction with protozoan CAs, this study may be relevant for an improved understanding of the role of this enzyme in the life cycle of the malaria producing organisms belonging to the genus Plasmodium.     

Advances in understanding the physiological role and locations of carbonic anhydrases in C3 plant cells

The review describes the structures of plant carbonic anhydrases (CAs), enzymes catalyzing the interconversion of inorganic carbon forms and belonging to different families, as well as the interaction of inhibitors and activators of CA activity with the active sites of CAs in representatives of these families. We outline the data that shed light on the location of CAs in green cells of C3 plants, algae and angiosperms, with the emphasis on the recently obtained data. The proven and proposed functions of CAs in these organisms are listed. The possibility of the involvement of several chloroplast CAs in acceleration of the conversion of bicarbonate to CO₂ and in supply of CO₂ for fixation by Rubisco is particularly considered. Special attention is paid to CAs in various parts of thylakoids and to discussion about current knowledge of their possible physiological roles. The review states that, despite the significant progress in application of the mutants with suppressed CAs synthesis, the approach based on the use of the inhibitors of CA activity in some cases remains quite effective. Combination of these two approaches, namely determining the effect of CA activity inhibitors in plants with certain knocked-out CA genes, turns out to be very useful for understanding the functions of other CAs.

     

Discovery of 2,4-thiazolidinedione-tethered coumarins as novel selective inhibitors for carbonic anhydrase IX and XII isoforms

Different 2,4-thiazolidinedione-tethered coumarins 5a–b, 10a–n and 11a–d were synthesised and evaluated for their inhibitory action against the cancer-associated hCAs IX and XII, as well as the physiologically dominant hCAs I and II to explore their selectivity. Un-substituted phenyl-bearing coumarins 10a, 10 h, and 2-thienyl/furyl-bearing coumarins 11a–c exhibited the best hCA IX (K_Is between 0.48 and 0.93 µM) and hCA XII (K_Is between 0.44 and 1.1 µM) inhibitory actions. Interestingly, none of the coumarins had any inhibitory effect on the off-target hCA I and II isoforms. The sub-micromolar compounds from the biochemical assay, coumarins 10a, 10 h and 11a–c, were assessed in an in vitro antiproliferative assay, and then the most potent antiproliferative agent 11a was tested to explore its impact on the cell cycle phases and apoptosis in MCF-7 breast cancer cells to provide more insights into the anticancer activity of these compounds.     

Synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one’s aryl Schiff base derivatives and investigation of carbonic anhydrase and cholinesterase (AChE,BuChE) inhibitory properties

Carbonic anhydrase enzymes (EC 4.2.1.1, CAs) are metalloenzyme families that catalyze the rapid conversion of H₂O and CO₂ to HCO₃^– and H⁺. CAs are found in different tissues where they participate in various significant biochemical processes such as ion transport, carbon dioxide respiration, ureagenesis, lipogenesis, bone resorption, electrolyte secretion, acid-base balance, and gluconeogenesis. In such processes, many CAs are significant therapeutic targets because of their inhibitory potentials especially in the treatment of some diseases such as edema, glaucoma, obesity, cancer, epilepsy, and osteoporosis. Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) inhibitors are also valuable compounds for different therapeutic applications including Alzheimer’s disease. In this work, we report a fast and effective synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one’s aryl Schiff base derivatives and also their CA and cholinesterases inhibitory properties. Our findings showed that these Schiff base derivatives, with triazole ring, found as strong CA and cholinesterases inhibitors.