Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure

We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3-. This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.     

The roles of carbonic anhydrases in photosynthetic CO2 concentrating mechanisms

Cyanobacteria, algae, aquatic angiosperms and higher plants have all developed their own unique versions of photosynthetic CO₂ concentrating mechanisms (CCMs) to aid Rubisco in efficient CO₂ capture. An important aspect of all CCMs is the critical roles that the specialised location and function that various carbonic anhydrase enzymes play in the overall process, participating the interconversion of CO₂ and HCO₃ ⁻ species both inside and outside the cell. This review examines what we currently understand about the nature of the carbonic anhydrase enzymes, their localisation and roles in the various CCMs that have been studied in detail. This revised version was published online in August 2006 with corrections to the Cover Date.     

Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1.

Genomic complementation of the high-CO2-requiring mutant ca-1-12-1C of Chlamydomonas reinhardtii was achieved by transformation with DNA pools from an indexed cosmid library of wild-type genomic DNA. Transformation of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO2 levels. Transformations with cosmid DNA from each of the rows and files of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a gene encoding a recently identified C. reinhardtii chloroplast carbonic anhydrase (CAH3). Transformations with chimeric constructs combining different portions of the wild-type and mutant genes indicated the presence of a mutation in the 5'-half of the gene. Comparison of mutant and wild-type gene sequences in this region revealed a G-to-A substitution in the mutant gene, which produced a nonsense codon. The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and efficient means for isolating genes associated with defects affecting photosynthesis and other physiological processes in this eukaryotic green alga.     

Catalytic mechanism of alpha-class carbonic anhydrases: CO2 hydration and proton transfer

Carbonic anhydrases were first identified in red blood cells and have been thus traditionally addressed in a hematological context. However, recently there has been a shift of research interest to therapeutic areas, notably in solid cancers, relegating the impact of carbonic anhydrase function and pathological dysfunction in blood related physiology to secondary importance. This review addresses this paradigm and emphasizes the potential impact of recent studies on blood related carbonic anhydrase isotype expression and modulation in diverse areas such as physiology and pathology, biosensing, their use as biomarkers, and in the development of synthetic blood. A special emphasis is placed on reviewing new dynamic and quantitative studies that allow for the efficient tracking and quantitation of various carbonic anhydrase isozymes within the blood and more generally within the human body, that give new perspectives on the biochemical and physiological role of blood associated carbonic anhydrase in health and pathology.     

Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite

Nitrite (ONO(-)) exerts nitric oxide (NO)-related biological actions and its concentration in the circulation may be of particular importance. Nitrite is excreted in the urine. Hence, the kidney may play an important role in nitrite/NO homeostasis in the vasculature. We investigated a possible involvement of renal carbonic anhydrases (CAs) in endogenous nitrite reabsorption in the proximal tubule. The potent CA inhibitor acetazolamide was administered orally to six healthy volunteers (5 mg/kg) and nitrite was measured in spot urine samples before and after administration. Acetazolamide increased abruptly nitrite excretion in the urine, strongly suggesting that renal CAs are involved in nitrite reabsorption in healthy humans. Additional in vitro experiments support our hypothesis that nitrite reacts with CO(2), analogous to the reaction of peroxynitrite (ONOO(-)) with CO(2), to form acid-labile nitrito carbonate [ONOC(O)O(-)]. We assume that this reaction is catalyzed by CAs and that nitrito carbonate represents the nitrite form that is actively transported into the kidney. The significance of nitrite reabsorption in the kidney and the underlying mechanisms, notably a direct involvement of CAs in the reaction between nitrite and CO(2), remain to be elucidated.     

Very high CO2 partially restores photosynthesis in sunflower at low water potentials

We re-examined the question of whether the stomata limit photosynthesis in dehydrated sunflower (Helianthus annuus L.) plants having low leaf water potentials. A gas-exchange apparatus was modified to operate at external CO₂ partial pressures as high as 3000 Pa (3%), which were much higher than previously achieved. This allowed photosynthesis and stomatal behavior to be monitored simultaneously at very high CO₂ in the same leaf. The data were compared with those from leaves treated with abscisic acid (ABA) where effects on photosynthesis are entirely stomatal. Photosynthesis was inhibited at low water potential and was only slightly enhanced by increasing the external CO₂ partial pressure from 34 Pa (normal air) to 300 Pa. Photosynthesis in ABA-treated leaves was similarly inhibited but recovered fully at 300 Pa. In both cases, the stomata closed to the same extent as judged from the average conductance of the leaves. Because the ABA effect resulted from diffusion limitation for CO₂ caused by stomatal closure, the contrasting data show that most of the dehydration effect was nonstomatal at low water potentials. When CO₂ partial pressures were raised further to 3000 Pa, photosynthesis increased somewhat at low water potentials but not in ABA-treated leaves. This indicates that some nonstomatal component of photosynthesis responded differently in leaves at low water potential and leaves treated with ABA. Because this component was only partially restored by very high CO₂, it was likely to be metabolic and was an important source of photosynthetic inhibition.Abbreviations and Symbol ABA abscisic acid - Chl chlorophyll - p_a external partial pressure of CO₂ - P_i intercellular partial pressure of CO₂ - _w water potential This work was supported by grant DE-FG02-87ER13776 from the Department of Energy and a grant from E.I. DuPont de Nemours and Company.     

Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis

Sudden exposure of plants to high light (HL) leads to metabolic and physiological disruption of the photosynthetic cells. Changes in ROS content, adjustment of photosynthetic processes and the antioxidant pools and, ultimately, gene induction are essential components for a successful acclimation to the new light conditions. The influence of salicylic acid (SA) on plant growth, short-term acclimation to HL, and on the redox homeostasis of Arabidopsis thaliana leaves was assessed here. The dwarf phenotype displayed by mutants with high SA content (cpr1-1, cpr5-1, cpr6-1, and dnd1-1) was less pronounced when these plants were grown in HL, suggesting that the inhibitory effect of SA on growth was partly overcome at higher light intensities. Moreover, higher SA content affected energy conversion processes in low light, but did not impair short-term acclimation to HL. On the other hand, mutants with low foliar SA content (NahG and sid2-2) were impaired in acclimation to transient exposure to HL and thus predisposed to oxidative stress. Low and high SA levels were strictly correlated to a lower and higher foliar H(2)O(2) content, respectively. Furthermore high SA was also associated with higher GSH contents, suggesting a tight correlation between SA, H(2)O(2) and GSH contents in plants. These observations implied an essential role of SA in the acclimation processes and in regulating the redox homeostasis of the cell. Implications for the role of SA in pathogen defence signalling are also discussed.     

Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO₂ (high inorganic carbon, C_i) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-C_i grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO₂ supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-C_igrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-C_i cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-C_i algae. After that time their activities were similar to the corresponding CA values in low-C_i-grown cells. In contrast, plastids from high-C_i cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-C_i and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-C_i air-adapted and low-C_i cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.This work is dedicated to Professor A. Wild on the occasion of his 65th birthday     

The activation state of Rubisco directly limits photosynthesis at low CO2 and low O2 partial pressures

Using gas exchange, enzyme assays, and theoretical modeling of photosynthetic responses to light and CO₂, we investigated whether decarbamylation of the active site of Rubisco at low CO₂ and low light leads to a condition where the activation state of Rubisco directly limits the rate of net CO₂ assimilation. Photosynthetic limitation by a reduction in the activation state of Rubisco would be indicated as a decline in the initial slope of the photosynthetic CO₂ response relative to what is predicted using theoretical models. In bean (Phaseolus vulgaris) and oat (Avena sativa), we saw no discrepancy between predicted and observed initial slope values at 200 and 400 mbar O₂, indicating no limitation by the carbamylation state of Rubisco. At 30 mbar O₂ and light saturation, we also saw no discrepancy between predicted and observed initial slope values; however, at subsaturating light intensity, our observed initial slope values were less than the modeled initial slope values that corresponded to an RuBP regeneration limitation. Moreover, significant reduction of the Rubisco activation state occurred in both species at 30 mbar O₂ and 30 μbar CO₂. When the model was reprogrammed to account for observed levels of Rubisco deactivation, the predicted and measured initial slope values at low O₂ and low PPFD were similar, indicating the reduction in carbamylation state accounted for the discrepancy. We interpret this as evidence for a direct limitation of the carbamylation state of Rubisco, probably because of a CO₂ limitation for carbamate formation. This limitation was only observed at intercellular CO₂ levels below what is encountered in vivo. At physiologically relevant CO₂ levels in situ, the leaves maintained sufficient Rubisco activity to avoid cabamylation state limitations in the steady state. This revised version was published online in June 2006 with corrections to the Cover Date.     

A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

C-glycosides incorporating the 6-methoxy-2-naphthyl moiety are selective inhibitors of fungal and bacterial carbonic anhydrases

Abstract

A small series of C-glycosides containing the methoxyaryl moieties was tested for the inhibition of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from Cryptococcus neoformans and Brucella suis. Many compounds showed activities in the micromolar or submicromolar range and excellent selectivity for pathogen CAs over human isozymes. The deprotected glycosides incorporating the 6-methoxy-2-naphthyl moiety showed the best inhibition profile and therefore represent leads for the development of novel anti-infectives with a new mechanism of action.     

Evidence for recycling of inorganic phosphate by wheat chloroplasts during photosynthesis at air levels of CO2 and O2

Phosphate recycling under photorespiratory conditions was investigated using intact wheat chloroplasts from Triticum aestivum (cv. Maris dove). A decline in the optimal Pi level needed to support steady-state photosynthesis was observed (a) as the bicarbonate supply became limiting, or (b) as oxygen concentrations were increased. Further, at subsaturating CO2 and elevated O2 (52%), photosynthetic induction periods were shortest in the absence of exogenous Pi, and severely extended by its addition. Thus, photosynthesis under low CO2 levels which favor ribulose 1,5 bisphosphate (RuBP) oxygenase activity and glycolate synthesis by chloroplasts decreases their dependency on exogenous Pi from the initial illumination of chloroplasts through to the attainment of steady state rates of O2 evolution. Uptake of phosphate (Pi) was directly measured at ambient O2 concentrations and showed the stoichiometry of O2 evolved to Pi consumed at 10 mmol/L bicarbonate (saturating) had a mean value of 3.0, and was increased to 5.4 at 2.5 mmol/L bicarbonate and to > 8.0 at 1.0 mmol/L bicarbonate. The observation is consistent with enhanced stromal recycling of Pi released during hydrolysis of phosphoglycolate produced in greater quantities as the ratio of RuBP carboxylase relative to oxygenase activities (vc/vo) declines. The theoretical relationship between vc/vo and O2/Pi stoichiometries was derived and compared favorably to experimental data obtained.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO(2) atmosphere

This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the BleR (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO2 atmosphere for optimal growth. The DNA flanking the BleR insert of one of the high-CO2-requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO2 atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO2-grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO2-concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii.     

Glucose and insulin are needed for optimal defensin expression in human cell lines

Many infections are associated with diabetes, as the ability of the body to fight pathogens is impaired. Recently, low levels of defensins have been found in diabetic rodents. However, whether hyperglycemia and/or insulin deficiency/insensitivity is the reason for the reduced defensin levels is still unknown. To study the functionality of the innate immune system during hyperglycemia, the expression levels of human beta-defensin-1 (hBD-1) was measured in human embryonic kidney (HEK-293) and colon adenocarcinoma (HCT-116) cells treated with different concentrations of glucose and insulin. Increasing concentrations of glucose enhanced hBD-1 expression and these levels were further elevated after insulin treatment. Insulin treatment also led to the up-regulation of human sodium/glucose transporter 1 (hSGLT1), which further increases intracellular glucose levels. Thus, our findings suggest for the first time that insulin signaling is important for hBD-1 optimal expression by elevating intracellular glucose levels and by mediating gene expression.