Expression of seven carbonic anhydrases in red alga Gracilariopsis chorda and their subcellular localization in a heterologous system, Arabidopsis thaliana.

KEY MESSAGE: Red alga, Gracilariopsis chorda, contains seven carbonic anhydrases that can be grouped into α-, ß- and γ-classes. Carbonic anhydrases (CAHs) are metalloenzymes that catalyze the reversible hydration of CO2. These enzymes are present in all living organisms and play roles in various cellular processes, including photosynthesis. In this study, we identified seven CAH genes (GcCAHs) from the genome sequence of the red alga Gracilariopsis chorda and characterized them at the molecular, cellular and biochemical levels. Based on sequence analysis, these seven isoforms were categorized into four α-class, one ß-class, and two γ-class isoforms. RNA sequencing revealed that of the seven CAHs isoforms, six genes were expressed in G. chorda in light at room temperature. In silico analysis revealed that these seven isoforms localized to multiple subcellular locations such as the ER, mitochondria and cytosol. When expressed as green fluorescent protein fusions in protoplasts of Arabidopsis thaliana leaf cells, these seven isoforms showed multiple localization patterns. The four α-class GcCAHs with an N-terminal hydrophobic leader sequence localized to the ER and two of them were further targeted to the vacuole. GcCAHß1 with no noticeable signal sequence localized to the cytosol. The two γ-class GcCAHs also localized to the cytosol, despite the presence of a predicted presequence. Based on these results, we propose that the red alga G. chorda also employs multiple CAH isoforms for various cellular processes such as photosynthesis.

A proteomic comparison of excretion/secretion products in Fasciola hepatica newly excysted juveniles (NEJ) derived from Lymnaea viatrix or Pseudosuccinea columella.

The characteristics of parasitic infections are often tied to host behavior. Although most studies have investigated definitive hosts, intermediate hosts can also play a role in shaping the distribution and accumulation of parasites. This is particularly relevant in larval stages, where intermediate host's behavior could potentially interfere in the molecules secreted by the parasite into the next host during infection. To investigate this hypothesis, we used a proteomic approach to analyze excretion/secretion products (ESP) from Fasciola hepatica newly excysted juveniles (NEJ) derived from two intermediate host species, Lymnaea viatrix and Pseudosuccinea columella. The two analyzed proteomes showed differences in identity, abundance, and functional classification of the proteins. This observation could be due to differences in the biological cycle of the parasite in the host, environmental aspects, and/or host-dependent factors. Categories such as protein modification machinery, protease inhibitors, signal transduction, and cysteine-rich proteins showed different abundance between samples. More specifically, differences in abundance of individual proteins such as peptidyl-prolyl cis-trans isomerase, thioredoxin, cathepsin B, cathepsin L, and Kunitz-type inhibitors were identified. Based on the differences identified between NEJ ESP samples, we can conclude that the intermediate host is a factor influencing the proteomic profile of ESP in F. hepatica.

Steroids interfere with human carbonic anhydrase activity by using alternative binding mechanisms.

Bile acids have been shown to inhibit human (h) carbonic anhydrases (CA, EC 4.2.1.1) along the gastrointestinal tract, including hCA II. The elucidation of the hormonal inhibition mechanism of the bile acid cholate to hCA II was provided in 2014 by X-ray crystallography. Herein, we extend the inhibition study to a wealth of steroids against four relevant hCA isoforms. Steroids displaying pendants and functional groups of the carboxylate, phenolic or sulfonate types appended at the tetracyclic ring were shown to inhibit the cytosolic CA II and the tumor-associated, transmembrane CA IX in a medium micromolar range (38.9-89.9 µM). Docking studies displayed the different chemotypes CA inhibition mechanisms. Molecular dynamics (MD) gave insights on the stability over time of hyocholic acid binding to CA II.

Dual targeting of cancer-related human matrix metalloproteinases and carbonic anhydrases by chiral N-(biarylsulfonyl)-phosphonic acids.

A series of nanomolar phosphonate matrix metalloproteinase (MPP) inhibitors was tested for inhibitory activity against a panel of selected human carbonic anhydrase (CA, EC 4.2.1.1) isozymes, covering the cancer-associated CA IX and XII. None of the reported sulfonyl and sulfonylamino-derivatives sensitively affected the catalytic activity of the cytosolic isoforms CA I and II, which are considered off-target isoforms in view of their physiological role. The most active inhibitors were in the series of chiral N-(sulfonyl)phosphovaline derivatives, which showed good to excellent inhibitory activity over target CAs, with compound 15 presenting the best isoform-selectivity toward CA IX. We suggest here that the phosphonates have the potential as dual inhibitors of MMPs and CAs, both involved in tumor formation, invasion and metastasis.

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

The Antarctic bacterium Colwellia psychrerythraea encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCAγ) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0×10(5) s(-1) and a k(cat)/K(m) of 4.7×10(6) M(-1) s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. The best inhibitor was metanilamide (K(I) of 83.5 nM) followed by indisulam, valdecoxib, celecoxib, sulthiame and hydrochlorothiazide (K(I)s ranging between 343 and 491 nM). Acetazolamide, methazolamide as well as other aromatic/heterocyclic derivatives showed inhibition constants between 502 and 7660 nM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here, by allowing the identification of inhibitors which may be useful as pharmacologic tools.

New light on bacterial carbonic anhydrases phylogeny based on the analysis of signal peptide sequences.

Among protein families, carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes characterized by a common reaction mechanism in all life domains: the carbon dioxide hydration to bicarbonate and protons (CO2+H2O ⇔ HCO3(-)+H(+)). Six genetically distinct CA families are known to date, the α-, ß-, γ-, δ-, ζ- and η-CAs. The last CA class was recently discovered analyzing the amino acid sequences of CAs from Plasmodia. Bacteria encode for enzymes belonging to the α-, ß-, and γ-CA classes and recently, phylogenetic analysis revealed an interesting relationship regarding the evolution of bacterial CA classes. This result evidenced that the three bacterial CA classes, in spite of the high level of the structural similarity, are evolutionarily distinct, but we noted that the primary structure of some ß-CAs identified in the genome of Gram-negative bacteria present a pre-sequence of 18 or more amino acid residues at the N-terminal part. These observations and subsequent phylogenetic data presented here prompted us to propose that the ß-CAs found in Gram-negative bacteria with a periplasmic space and characterized by the presence of a signal peptide might have a periplasmic localization and a role similar to that described previously for the α-CAs.

Synthesis of Schiff base derivatives of 4-(2-aminoethyl)-benzenesulfonamide with inhibitory activity against carbonic anhydrase isoforms I, II, IX and XII.

Schiff base derivatives were obtained by reaction of 4-(2-aminoethyl)benzenesulfonamide with aromatic aldehydes. The corresponding secondary amine derivatives were also prepared by reduction of the imine compounds with NaBH4. These derivatives were investigated as inhibitors of four human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic isozymes hCA I and II, as well as, the transmembrane, tumor-associated hCA IX and XII. Some of the newly synthesised compounds showed effective inhibitory activities against these CA isozymes. Many low nanomolar inhibitors were detected against all isoforms among the secondary amines whereas the Schiff bases were by far less active compared to the corresponding reduced derivatives among all investigated isoforms.

Cloning, characterization and anion inhibition studies of a new γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis.

A new γ-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from the Antarctic bacterium Pseudoalteromonas haloplanktis, PhaCAγ. The enzyme has a medium-low catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a kcat of 1.4×10(5)s(-1) and a kcat/Km of 1.9×10(6)M(-1)s(-1). An anion inhibition study of PhaCAγ with inorganic anions and small molecule inhibitors is also reported. Many anions present in sea water, such as chloride, fluoride, sulfate, iodide, but also others such as azide, perchlorate and tetrafluoroborate did not inhibit this enzyme. Pseudohalides such as cyanate, thiocyanate, cyanide, selenocyanide, and also bicarbonate, nitrate, nitrite and many complex inorganic anions showed inhibition in the millimolar range (KI in the range of 1.7-9.3mM). The best PhaCAγ inhibitors detected in this study were diethyldithiocarbamate (KI of 0.96 mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KI in the range of 82-91 µM). Since γ-CAs are poorly understood at this moment, being present in carboxysomes and thus involved in photosynthesis, this study may be relevant for a better understanding of these processes in Antarctic bacteria/cyanobacteria.

Overview of the carbonic anhydrase family.

The purpose of this collection of chapters is to provide a glimpse of where the carbonic anhydrase (CA) field is. This book is by no means fully inclusive, as only a few of the lead researchers around the world contributed; it serves only to show that the CA field is still pushing the boundaries of research as it has done since its discovery, and will do for a long time to come.

Physiological functions of the alpha class of carbonic anhydrases.

Carbonic anhydrases are ubiquitous enzymes that catalyze the reversible hydration of carbon dioxide. These enzymes are of ancient origin as they are found in the deepest of branches of the evolutionary tree. Of the five different classes of carbonic anhydrases, the alpha class has perhaps received the most attention because of its role in human pathology. This review focuses on the physiological function of this class of carbonic anhydrases organized by their cellular location.

Thermal-stable carbonic anhydrases: a structural overview.

The potential of carbonic anhydrase (CA) family as target for the drug design of inhibitors with various medicinal chemistry applications has been recognized from long time, whereas the industrial interest in using these enzymes as biocatalysts for carbon dioxide sequestration and biofuel production is only recently emerging. However, an efficient utilization in these processes often requires stable enzymes, able to work in the harsh conditions typical of the CO2 capture process. In this context CAs active at very high temperatures are of extreme interest. In this chapter we have summarized in a comparative manner all existing data on thermostable CAs both isolated by extremophiles and obtained by protein engineering studies. Among the five CA-classes, the biochemical and structural features of thermostable α-, ß- and γ-CAs have been discussed. Data show that so far α-CAs isolated from thermophilic organisms are the best candidates to be used in biotechnological processes, even if plenty of work can be still done in this field also with help of protein engineering.

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

The carbonic anhydrases (CAs; EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide (CO2) and dehydration of bicarbonate (HCO3 (-)) in a two-step ping-pong mechanism: [Formula: see text] CAs are ubiquitous enzymes and are categorized into five distinct classes (α, ß, γ, δ and ζ). The α-class is found primarily in vertebrates (and the only class of CA in mammals), ß is observed in higher plants and some prokaryotes, γ is present only in archaebacteria whereas the δ and ζ classes have only been observed in diatoms.The focus of this chapter is on α-CAs as the structure-function relationship is best understood for this class, in particular for humans. The reader is referred to other reviews for an overview of the structure and catalytic mechanism of the other CA classes. The overall catalytic site structure and geometry of α-CAs are described in the first section of this chapter followed by the kinetic studies, binding of CO2, and the proton shuttle network.

Renal plasticity in response to feeding in the Burmese python, Python molurus bivittatus.

Burmese pythons are sit-and-wait predators that are well adapted to go long periods without food, yet subsequently consume and digest single meals that can exceed their body weight. These large feeding events result in a dramatic alkaline tide that is compensated by a hypoventilatory response that normalizes plasma pH; however, little is known regarding how plasma HCO3(-) is lowered in the days post-feeding. The current study demonstrated that Burmese pythons contain the cellular machinery for renal acid-base compensation and actively remodel the kidney to limit HCO3(-) reabsorption in the post-feeding period. After being fed a 25% body weight meal plasma total CO2 was elevated by 1.5-fold after 1 day, but returned to control concentrations by 4 days post-feeding (d pf). Gene expression analysis was used to verify the presence of carbonic anhydrase (CA) II, IV and XIII, Na(+) H(+) exchanger 3 (NHE3), the Na(+) HCO3(-) co-transporter (NBC) and V-type ATPase. CA IV expression was significantly down-regulated at 3 dpf versus fasted controls. This was supported by activity analysis that showed a significant decrease in the amount of GPI-linked CA activity in isolated kidney membranes at 3 dpf versus fasted controls. In addition, V-type ATPase activity was significantly up-regulated at 3 dpf; no change in gene expression was observed. Both CA II and NHE3 expression was up-regulated at 3 dpf, which may be related to post-prandial ion balance. These results suggest that Burmese pythons actively remodel their kidney after feeding, which would in part benefit renal HCO3(-) clearance.

Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas.

Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.

Biochemistry and physiology of the ß class carbonic anhydrase (Cpb) from Clostridium perfringens strain 13.

The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the ß class, the first ß class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO2 and 4.8 × 106 s⁻¹ M⁻¹, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO2. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO2 in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO2 fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.

A highly catalytically active γ-carbonic anhydrase from the pathogenic anaerobe Porphyromonas gingivalis and its inhibition profile with anions and small molecules.

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the γ-class are present in archaea, bacteria and plants but, except the Methanosarcina thermophila enzymes CAM and CAMH, they were poorly characterized so far. Here we report a new such enzyme (PgiCA), the γ-CA from the oral cavity pathogenic bacterium Porphyromonas gingivalis, the main causative agent of periodontitis. PgiCA showed a good catalytic activity for the CO2 hydration reaction, comparable to that of the human (h) isoform hCA I. Inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate were effective PgiCA inhibitors with inhibition constants in the range of 41-97 µM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, and phenylboronic acid, with KIs of 4.0-9.8 µM. The role of this enzyme as a possible virulence factor of P. gingivalis is poorly understood at the moment but its good catalytic activity and the possibility to be inhibited by a large number of compounds may lead to interesting developments in the field.

Kinetic and anion inhibition studies of a ß-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis.

Several ß-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C4 plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO2 hydrase catalytic activity (kcat of 1.2×10(5) and kcat/Km of 7.5×10(6)M(-1)×s(-1)) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (KIs in the range of 4-60µM). Such inhibitors may be used as tools to better understand the role of various ß-CA isoforms in photosynthesis.

Carbonic anhydrase inhibitors: inhibition of the ß-class enzyme from the pathogenic yeast Candida glabrata with sulfonamides, sulfamates and sulfamides.

The fungal pathogen Candida glabrata encodes for a ß-carbonic anhydrase (CA, EC 4.2.1.1), CgNce103, recently discovered. Only anions have been investigated as CgNce103 inhibitors up until now. Here we report the first sulfonamides inhibition study of this enzyme. Simple sulfonamides showed weak or moderate CgNce103 inhibitory properties, whereas acetazolamide, and a series of 4-substituted ureido-benzene-sulfonamides, sulfamates and sulfamides showed effective CgNce103 inhibitory properties, with KIs in the range of 4.1-115 nM, being also ineffective as human CA II inhibitors. As there is significant resistance of C. glabrata clinical isolates to many classical antifungal agents, inhibition of the ß-CA from this organism may allow an interesting means of controlling the pathogen growth, eventually leading to antifungals with a novel mechanism of action.

An α-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction.

We report the biochemical characterization of a new carbonic anhydrase (CA, EC 4.2.1.1), named SazCA, identified by translated genome inspection in Sulfurihydrogenibium azorense, a thermophilic bacterium from terrestrial hot springs of the Azores. SazCA is an α-CA showing kinetic parameters that make it the fastest enzyme of the CA family described so far. The biochemical properties, thermostability and inhibition of SazCA were compared with those of the thermophilic and mesophilic counterparts, demonstrating the special features of this unique enzyme.

Analysis of evolution of carbonic anhydrases IV and XV reveals a rich history of gene duplications and a new group of isozymes.

Carbonic anhydrase (CA) isozymes CA IV and CA XV are anchored on the extracellular cell surface via glycosylphosphatidylinositol (GPI) linkage. Analysis of evolution of these isozymes in vertebrates reveals an additional group of GPI-linked CAs, CA XVII, which has been lost in mammals. Our work resolves nomenclature issues in GPI-linked fish CAs. Review of expression data brings forth previously unreported tissue and cancer types in which human CA IV is expressed. Analysis of collective glycosylation patterns of GPI-linked CAs suggests functionally important regions on the protein surface.