Search

We report the kinetic properties and sulfonamide inhibition profile of an a-carbonic anhydrase (CA, EC 4.2.1.1), named CruCA4, identified in the red coral Corallium rubrum. This isoform is involved in the biomineralization process leading to the formation of a calcium carbonate skeleton. Experiments performed on the recombinant protein show that the enzyme has a "moderate activity" level. Our results are discussed compared to values obtained for other CA isoforms involved in biomineralization. This is the first study describing the biochemical characterization of an octocoral CA. (C) 2017 Elsevier Ltd. All rights reserved.

A panel of 22 phenols was investigated as inhibitors of the 8-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa (MgCA), a validated anti-dandruff drug target. The displayed inhibitory activities were compared to the ones previously reported against the off-target widely distributed human (h) isoforms hCA I and II. All tested phenols possessed a better efficacy in inhibiting MgCA than the clinically used sulfonamide acetazolamide, with Kis in the range of 2.5 and 65.0 mu M. A homology-built model of MgCA was also used for understanding the binding mode of phenols to the fungal enzyme. Indeed, a wide network of hydrogen bonds and hydrophobic interactions between the phenol and active site residues were evidenced. The OH moiety of the inhibitor was observed anchored to the zinc-coordinated water, also making hydrogen bonds with Ser48 and Asp49. The diverse substituents at the phenolic scaffold were observed to interact with different portions of the hydrophobic pocket according to their nature and position. Considering the effective MgCA inhibitory properties of phenols, beside to the rather low inhibition against the off-target hCA I and II, this class of compounds might be of considerable interest in the cosmetics field as potential anti-dandruff drugs. (C) 2017 Elsevier Ltd. All rights reserved.

We report the cloning, purification and characterization of Bps beta CA, a beta-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, the etiological agent of melioidosis, and compare its activity and inhibition with those of the gamma-CA from the same organism, Bps gamma CA, recently investigated by our groups. Bps beta CA showed a significant catalytic activity for the physiologic, CO2 hydration reaction, with the following kinetic parameters, k(cat) of 1.6 x 10(5) s(-1) and k(cat)/K-m of 3.4 x 10(7) M-1 x s(-1). The inhibition of Bps beta CA with a group of anions and small molecules was also investigated. The best inhibitors were sulfamide, sulfamic acid and phenylarsonic acid, which showed K(I)s in the range of 83-92 mu M, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate and hexafluorophosphate showed K(I)s > 100 mM. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32-8.6 mM. As little is known of the life cycle and virulence of this bacterium, this type of study may bring information of interest for the development of novel strategies to fight bacterial infection and drug resistance to commonly used antibiotics. (C) 2017 Elsevier Ltd. All rights reserved.

We have cloned, purified and characterized the gamma-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with the following kinetic parameters: k(cat) of 6.0 x 10(5) s(-1) and a k(cat)/K-m of 4.7 x 10(6) M-1 x s(-1). This activitywas inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a K-I of 502 nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed K(I)s in the range of 1.4-4.4 mM. Diethyldithiocarbamate was a better inhibitor (K-I of 0.58 mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with K(I)s ranging between 8 and 38 mu M. The present study may shed some more light regarding the role that gamma-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here. (c) 2016 Elsevier Ltd. All rights reserved.

With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to alpha-, beta-, and gamma-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 degrees C, suggesting a possible role of the two coupled enzymes in biotechnological processes. (c) 2015 Elsevier Ltd. All rights reserved.

A new ?-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from the Antarctic bacterium Pseudoalteromonas haloplanktis, PhaCA?. 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 × 105 s-1 and a kcat/Km of 1.9 × 106 M-1 s-1. An anion inhibition study of PhaCA? 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 1.7-9.3 mM). The best PhaCA? 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 ?M). Since ?-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.