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The metalloenzyme, carbonic anhydrase (CA), catalyzes the reversible hydration of carbon dioxide into bicarbonate, and is responsible for biomineralization processes in animals. In the Annelida, the marine worms in the family Serpulidae are typified by the construction of calcium carbonate tubes. Hydroides elegans, a common member of warmwater biofouling communities around the world, provides an outstanding model for studies of calcification. To better understand the molecular process of biomineralization in H. elegans, we searched transcriptomes for CA genes at several life-history stages. Twelve CA genes were recovered in the transcriptomes. Whole mount in situ hybridization was performed for two of those genes on larvae and calcifying juveniles. A cytosolic CA isoform, HeCA1, and a secreted CA isoform, HeCA2, were expressed within the collar segment corresponding to the location of glands involved in formation of the calcified tube. Expression of these genes within collar segment tissues supports the role of CAs in generating bicarbonate for biomineralization processes. A phylogenetic tree of the α-CA gene family was constructed to increase understanding of CAgene evolution within the family and its relationship to CA genes among the Metazoa. [ABSTRACT FROM AUTHOR]

Melioidosis is a severe disease caused by the highly pathogenic gram-negative bacterium Burkholderia pseudomallei . Several studies have highlighted the broad resistance of this pathogen to many antibiotics and pointed out the pivotal importance of improving the pharmacological arsenal against it. Since γ-carbonic anhydrases (γ-CAs) have been recently introduced as potential and novel antibacterial drug targets, in this paper, we report a detailed characterization of BpsγCA, a γ-CA from B . pseudomallei by a multidisciplinary approach. In particular, the enzyme was recombinantly produced and biochemically characterized. Its catalytic activity at different pH values was measured, the crystal structure was determined and theoretical pKa calculations were carried out. Results provided a snapshot of the enzyme active site and dissected the role of residues involved in the catalytic mechanism and ligand recognition. These findings are an important starting point for developing new anti-melioidosis drugs targeting BpsγCA., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Acidic extracellular pH (pHe) is a typical attribute of a tumor microenviroment, which has an impact on cancer development and treatment outcome. It was believed to result from an accumulation of lactic acid excessively produced by glycolysis. However, metabolic profiles of glycolysis-impaired tumors have revealed that CO2 is a significant source of acidity, thereby indicating a contribution of carbonic anhydrase (CA). The tumor-associated CA IX isoform is the best candidate, because its extracellular enzyme domain is highly active, expression is induced by hypoxia and correlates with poor prognosis. This study provides the first evidence for the role of CA IX in the control of pHe. We show that CA IX can acidify the pH of the culture medium in hypoxia but not in normoxia. This acidification can be perturbed by deletion of the enzyme active site and inhibited by CA IX-selective sulfonamides, which bind only to hypoxic cells containing CA IX. Our findings suggest that hypoxia regulates both expression and activity of CA IX in order to enhance the extracellular acidification, which may have important implications for tumor progression. [Copyright &y& Elsevier]

The thylakoid membrane containing photosystem II (PSII membranes) from pea and wheat leaves catalyzed the reaction of CO2 hydration with low rate, which increased after their incubation either with Triton X-100, up to Triton/chlorophyll ratio 1:1, or 1 M CaCl2. The presence of the inhibitor of CAs, p-aminomethylbenzensulfonamide (mafenide), at the start line in the course of electrophoresis of PSII membranes solubilized by n-dodecyl-β-maltoside (DM) decreased the amount of PSII core complex in the gel. The elution of PSII core complex from the column with immobilized mafenide occurred only either by mafenide or another inhibitor of CAs, ethoxyzolamide. The above results led to a conclusion that membrane-bound CA activity associated with PSII is situated in the core complex. [Copyright &y& Elsevier]

Bombyx mori (Shunrei × Shogetsu) is sensitive to Cry1Aa and resistant to Cry1Ac, both insecticidal proteins of Bacillus thuringiensis. Cry1Aa passed through the peritrophic membrane (PM) much faster (0.37 μg/mm2 PM/h) than Cry1Ac (0.05 μg/mm2 PM/h) during the initial observation period. Both Cry1Aa and Cry1Ac bound to the PM but only the binding of Cry1Ac was specifically inhibited by N-acetylgalactosamine (GalNAc). When Cry1Ac was pretreated with GalNAc, Cry1Ac permeated the PM much faster. These results suggested that Cry1Ac bound a PM protein via GalNAc on a sugar side chain. The role of the PM on Cry1Ac resistance of B. mori was briefly discussed. [Copyright &y& Elsevier]

Current understanding of chloride cells (CCs) is briefly reviewed with emphasis on molecular aspects of their channels, transporters and regulators. Seawater-type and freshwater-type CCs have been identified based on their shape, location and response to different ionic conditions. Among the freshwater-type CCs, subpopulations are emerging that are implicated in the uptake of Na+, Cl− and Ca2+, respectively, and can be distinguished by their shape of apical crypt and affinity for lectins. The major function of the seawater CC is transcellular secretion of Cl−, which is accomplished by four major channels and transporters: (1) CFTR Cl− channel, (2) Na+,K+-ATPase, (3) Na+/K+/2Cl− cotransporter and (4) a K+ channel. The first three components have been cloned and characterized, but concerning the K+ channel that is essential for the continued generation of the driving force by Na+,K+-ATPase, only one candidate is identified. Although controversial, freshwater CCs seem to perform the uptake of Na+, Cl− and Ca2+ in a manner analogous to but slightly different from that seen in the absorptive epithelia of mammalian kidney and intestine since freshwater CCs face larger concentration gradients than ordinary epithelial cells. The components involved in these processes are beginning to be cloned, but their CC localization remains to be established definitively. The most important yet controversial issue is the mechanism of Na+ uptake. Two models have been postulated: (i) the original one involves amiloride-sensitive electroneutral Na+/H+ exchanger (NHE) with the driving force generated by Na+,K+-ATPase and carbonic anhydrase (CA) and (ii) the current model suggests that Na+ uptake occurs through an amiloride-sensitive epithelial sodium channel (ENaC) electrogenically coupled to H+-ATPase. While fish ENaC remains to be identified by molecular cloning and database mining, fish NHE has been cloned and shown to be highly expressed on the apical membrane of CCs, reviving the original model. The CC is also involved in acid–base regulation. Analysis using Osorezan dace (Tribolodon hakonensis) living in a pH 3.5 lake demonstrated marked inductions of Na+,K+-ATPase, CA-II, NHE3, Na+/HCO3− cotransporter-1 and aquaporin-3 in the CCs on acidification, leading to a working hypothesis for the mechanism of Na+ retention and acid–base regulation. [Copyright &y& Elsevier]

The concept of structure-based drug design is based upon an in-depth understanding of the principles of molecular recognition. Despite our lack of a thorough comprehension of these principles, the wealth of protein structures available opens up unprecedented possibilities for new insights from the analysis of these data. Unravelling universal rules of molecular recognition is certainly one of the most appealing goals. But our knowledge is enhanced also when studying the specific determinants that characterise single targets or target families only, and the factors governing and discriminating their recognition properties.Here, we illustrate how the structure-based design process can benefit from the consequent incorporation of database query tools. We discuss representative examples to address issues such as protein flexibility, water molecules in binding pockets, and ligand specificity as some of the most critical aspects of drug design. All studies are carried out using our database system Relibase. We also show the application of Relibase in searching for preferred geometrical patterns between interacting molecular fragments. [Copyright &y& Elsevier]

To help understand the biological functions of carbonic anhydrase-related proteins (CA-RPs VIII, X, and XI), we obtained cDNA clones of murine CA-RPs X and XI and studied the tissue distribution of all three CA-RPs and their developmental expression in the murine embryonic brain. The amino acid sequences of murine CA-RPs are highly conserved with their human homologues, indicating a fundamental biological role of CA-RPs. Among a panel of vital organs, the strongest mRNA expression of all three CA-RPs was consistently observed in the brain. Immunohistochemical analysis revealed the expression of all three CA-RPs in the neural cell body and neurites. CA-RP X was also observed in the myelin sheath, as demonstrated using the shiverer demyelinated mouse. In murine embryos, CA-RP VIII and X messages first appeared in the middle of the gestation period, while the CA-RP XI message was seen at an early gestational stage and expressed to a lesser extent as gestation progressed. All CA-RPs were expressed in the neuroprogenitor cells in the subventricular area and subsequently detected in the neural cells migrating to the cortex. Although the exact function of CA-RPs is still undefined, these findings suggest an important role of CA-RPs in the CNS. [Copyright &y& Elsevier]

The crystal structures of sulfanilamide (4-aminobenzene-sulfonamide) complexed to the enzyme variant human carbonic anhydrase I Michigan 1 (CA I M1) and to its (Zn)2 adduct refined at 2.2 and 2.6 A˚ resolution respectively are described. Comparisons among these structures and the corresponding sulfonamide adduct of human CA I show significant differences in the orientation of the inhibitor molecule and in its interactions with active site residues such as His200, Thr199, Leu198, Gln92, and Arg/His67 which are known to play important roles in substrate or inhibitor recognition and binding. In CA I M1 molecule B a lengthening of the Zn&z.sbnd;N1 sulfanilamide bond distance and a corresponding shortening of the distance between the sulfonamido group and Thr199 are observed compared with the values for native CA I. When the second Zn(II) ion is present in the active site, the p-amino group and the aromatic ring of the inhibitor molecule appear to tilt towards Gln92 and Arg67, moving away from residues His200 and Leu198. The structural differences in inhibitor binding between the CA I isozyme and the CA I M1 variant are discussed in terms of the different inhibition constants measured for a variety of aromatic and heterocyclic sulfonamides. [Copyright &y& Elsevier]

Three cDNA homologues of carbonic anhydrase with unknown biological functions have been reported: carbonic anhydrase-related proteins (CA-RP) VIII, X, and XI. In the present study, we produced monoclonal antibodies to these CA-RPs and studied their regional and cellular distributions in the human adult and fetal brains by immunohistochemical analysis. In the adult brain, CA-RP VIII was expressed in the neural cell body spreading to most parts of the brain. CA-RP X was expressed in the myelin sheath and its expression was shown in the cytoplasm of cultured tumor cells by immunocytochemical analysis. CA-RP XI was expressed in the neural cell body, neurites, and astrocytes in relatively limited regions of the brain. In the fetal brain, CA-RP VIII and XI were expressed in the neuroprogenitor cells in the subventricular zone as early as the 84th day of gestation and subsequently detected in the neural cells migrating to the cortex. CA-RP X first appeared in the neural cells in the cortex at the 141st day. In the choroid plexus, the epithelial cells gave CA-RP VIII and XI expressions in both adult and fetal brains.From the findings in the present study on the distribution and the developmental expression of CA-RP VIII, X, and XI in the human brain we suggest that these CA-RPs play roles in various biological process of the CNS. [Copyright &y& Elsevier]

Carbonic anhydrases fall into three distinct evolutionary and structural classes: α, β, and γ. The β-class carbonic anhydrases (β-CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a β-CA from Escherichia coli, to a resolution of 2.0 Å. In agreement with the structure of the β-CA from the chloroplast of the red alga Porphyridium purpureum, the active-site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some β-CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH-dependent in the physiological range, and its expression in yeast complements an oxygen-sensitive phenotype displayed by a β-CA-deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other β-CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase (CA) catalyzes reversible hydration of CO 2 to HCO 3 - to mediate pH and ion homeostasis. Some chemical pollutants have been reported to have inhibitory effects on fish CA. In this study, we investigated effects of a CA inhibitor ethoxyzolamide (EZA) on neuromasts development during zebrafish embryogenesis, since embryogenesis in aquatic organisms can be particularly sensitive to water pollution. EZA caused alteration of pH and calcium concentration and production of reactive oxygen species (ROS) in larvae, and induced apoptosis in hair cells especially in the otic neuromast, in which CA2 was distributed on the body surface. mRNA levels of apoptotic genes and caspase activities were increased by EZA, whereas anti-oxidants and apoptotic inhibitors, Bax, NF-κB, and p53 inhibitors significantly relieved the induction of hair cell death. Also, mRNA levels of Bip and CHOP, which are induced in response to ER stress, were upregulated by EZA, suggesting that EZA induces otic hair cell apoptosis via the intrinsic mitochondrial pathway and ER stress. Our results demonstrated an essential role of CA in neuromast development via maintenance of ion transport and pH, and that the CA, which is directly exposed to the ambient water, shows marked sensitivity to EZA., Competing Interests: The authors report no declarations of interest., (© 2021 The Author(s).)

We herein describe AncPhore, a versatile tool for drug discovery, which is characterized by pharmacophore feature analysis and anchor pharmacophore ( i.e. , most important pharmacophore features) steered molecular fitting and virtual screening. Comparative analyses of numerous protein-ligand complexes using AncPhore revealed that anchor pharmacophore features are biologically important, commonly associated with protein conservative characteristics, and have significant contributions to the binding affinity. Performance evaluation of AncPhore showed that it had substantially improved prediction ability on different types of target proteins including metalloenzymes by considering the specific contributions and diversity of anchor pharmacophore features. To demonstrate the practicability of AncPhore, we screened commercially available chemical compounds and discovered a set of structurally diverse inhibitors for clinically relevant metallo- β -lactamases (MBLs); of them, 4 and 6 manifested potent inhibitory activity to VIM-2, NDM-1 and IMP-1 MBLs. Crystallographic analyses of VIM-2: 4 complex revealed the precise inhibition mode of 4 with VIM-2, highly consistent with the defined anchor pharmacophore features. Besides, we also identified new hit compounds by using AncPhore for indoleamine/tryptophan 2,3-dioxygenases (IDO/TDO), another class of clinically relevant metalloenzymes. This work reveals anchor pharmacophore as a valuable concept for target-centered drug discovery and illustrates the potential of AncPhore to efficiently identify new inhibitors for different types of protein targets., Competing Interests: The authors declare no competing financial interest., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

CDCA1 is a very peculiar member of the Carbonic Anhydrase (CA) family. It has been the first enzyme to show an efficient utilization of Cd(II) ions in Nature and a unique adaptation capability to live on the surface ocean. Indeed, in this environment, which is extremely depleted in essential metal ions, CDCA1 can utilize Zn(II) or Cd(II) as catalytic metal to support the metabolic needs of fast growing diatoms. In this paper we demonstrate a further catalytic versatility of this enzyme by using a combination of X-ray crystallography, molecular dynamics simulations and enzymatic experiments. First we identified the CO 2 binding site and the way in which this substrate travels from the environment to the enzyme active site. Then, starting from the observation of a structural similarity with the substrate entry route of CS 2 hydrolase from Acidanius A1-3 , we hypothesized and demonstrated that also CS 2 is a substrate for CDCA1. This finding is new and unexpected since until now only few CS 2 hydrolases have been characterized, and none of them is reported to have any CO 2 hydratase action. The physiological implications of this supplementary catalytic activity still remain to be unveiled. We suggest here that it could represent another ability of diatoms expressing CDCA1 to adapt to the external environment. Indeed, the ability of this enzyme to convert CS 2 could represent an alternative source of carbon acquisition for diatoms, in addition to CO 2 ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Background & Aims: Human enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl - and HCO 3 - secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation., Methods: Human duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl -  and HCO 3 - /CO 2 , to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s)., Results: Differentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na + /K + /2Cl - co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na + /HCO 3 - co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl - and HCO 3 - secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl - or HCO 3 - /CO 2 . The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl - secretion with no significant change in HCO 3 - secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl - or HCO 3 - /CO 2 . Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K + channel(s), and Na + /K + -adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids., Conclusions: Human enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl - secretion but no significant change in HCO 3 - secretion.

Carbon isotope fractionation (ε p ) between the inorganic carbon source and organic matter has been proposed to be a function of p CO 2 . To understand the CO 2 -dependency of ε p and species-specific differences therein, inorganic carbon fluxes in the four dinoflagellate species Alexandrium fundyense, Scrippsiella trochoidea, Gonyaulax spinifera and Protoceratium reticulatum have been measured by means of membrane-inlet mass spectrometry. In-vivo assays were carried out at different CO 2 concentrations, representing a range of p CO 2 from 180 to 1200 μatm. The relative bicarbonate contribution (i.e. the ratio of bicarbonate uptake to total inorganic carbon uptake) and leakage (i.e. the ratio of CO 2 efflux to total inorganic carbon uptake) varied from 0.2 to 0.5 and 0.4 to 0.7, respectively, and differed significantly between species. These ratios were fed into a single-compartment model, and ε p values were calculated and compared to carbon isotope fractionation measured under the same conditions. For all investigated species, modeled and measured ε p values were comparable ( A. fundyense, S. trochoidea, P. reticulatum ) and/or showed similar trends with p CO 2 ( A. fundyense, G. spinifera, P. reticulatum ). Offsets are attributed to biases in inorganic flux measurements, an overestimated fractionation factor for the CO 2 -fixing enzyme RubisCO, or the fact that intracellular inorganic carbon fluxes were not taken into account in the model. This study demonstrates that CO 2 -dependency in ε p can largely be explained by the inorganic carbon fluxes of the individual dinoflagellates.