RESUMO
The implementation of innovative approaches is crucial in an ongoing endeavor to mitigate the impact of COVID-19 pandemic. The present study examines the strategic application of the SARS-CoV-2 Main Protease (Mpro) as a prospective instrument in the repertoire to combat the virus. The cloning, expression, and purification of Mpro, which plays a critical role in the viral life cycle, through heterologous expression in Escherichia coli in a completely soluble form produced an active enzyme. The hydrolysis of a specific substrate peptide comprising a six-amino-acid sequence (TSAVLQ) linked to a p-nitroaniline (pNA) fragment together with the use of a fluorogenic substrate allowed us to determine effective inhibitors incorporating selenium moieties, such as benzoselenoates and carbamoselenoates. The new inhibitors revealed their potential to proficiently inhibit Mpro with IC50-s in the low micromolar range. Our study contributes to the development of a new class of protease inhibitors targeting Mpro, ultimately strengthening the antiviral arsenal against COVID-19 and possibly, related coronaviruses.
Assuntos
COVID-19 , Proteases 3C de Coronavírus , Selênio , Humanos , Selênio/farmacologia , Pandemias , Estudos Prospectivos , SARS-CoV-2 , Escherichia coliRESUMO
Carbonic anhydrases, catalysing the reversible CO2 hydration reaction, contribute in all living organisms to the maintenance of stable metabolic functions depending on intracellular concentrations of carbon dioxide, bicarbonate, and protons. Recent studies have examined how CAs affect bacterial lifecycle, considering these enzymes druggable targets due to interference with their activities by using inhibitors or activators. Here, we propose Escherichia coli cells as a model for testing the effect of acetazolamide (AZA), a potent CA inhibitor, on bacterial survival by evaluating E. coli growth through its glucose consumption. AZA, at concentrations higher than 31.2 µg/mL, was able to impair E. coli growth and glucose uptake. AZA is a good inhibitor of the two recombinant E. coli CAs, the ß-CA CynT2, and the γ-CA EcoCAγ, with KIs of 227 and 248 nM, respectively. This study provides a proof-of-concept, low-cost method for identifying effective CA inhibitors capable of impairing bacterial metabolism.
Assuntos
Anidrases Carbônicas , Acetazolamida/farmacologia , Dióxido de Carbono/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Escherichia coli/metabolismo , Bactérias Gram-Negativas , PrótonsRESUMO
Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO2/HCO3-/pH-sensing in fungi is triggered by HCO3- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the ß-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Malassezia/enzimologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/genética , Relação Dose-Resposta a Droga , Estrutura Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
We here report a study on the activation of the ι-class bacterial CA from Burkholderia territorii (BteCAι). This protein was recently characterised as a zinc-dependent enzyme that shows a significant catalytic activity (kcat 3.0 × 105 s-1) for the physiological reaction of CO2 hydration to bicarbonate and protons. Some amino acids and amines, among which some proteinogenic derivatives as well as histamine, dopamine and serotonin, showed efficient activating properties towards BteCAι, with activation constants in the range 3.9-13.3 µM. L-Phe, L-Asn, L-Glu, and some pyridyl-alkylamines, showed a weaker activating effect towards BteCAι, with KA values ranging between 18.4 µM and 45.6 µM. Nowadays, no information is available on active site architecture, metal ion coordination and catalytic mechanism of members of the ι-group of CAs, and this study represents another contribution towards a better understanding of this still uncharacterised class of enzymes.
Assuntos
Aminas/farmacologia , Aminoácidos/farmacologia , Burkholderia/enzimologia , Anidrases Carbônicas/metabolismo , Aminas/química , Aminoácidos/química , Anidrases Carbônicas/análise , Relação Dose-Resposta a Droga , Estrutura Molecular , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Fungi are exposed to various environmental variables during their life cycle, including changes in CO2 concentration. CO2 has the potential to act as an activator of several cell signaling pathways. In fungi, the sensing of CO2 triggers cell differentiation and the biosynthesis of proteins involved in the metabolism and pathogenicity of these microorganisms. The molecular machineries involved in CO2 sensing constitute a promising target for the development of antifungals. Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial enzymes in the CO2 sensing systems of fungi, because they catalyze the reversible hydration of CO2 to proton and HCO3-. Bicarbonate in turn boots a cascade of reactions triggering fungal pathogenicity and metabolism. Accordingly, CAs affect microorganism proliferation and may represent a potential therapeutic target against fungal infection. Here, the inhibition of the unique ß-CA (MpaCA) encoded in the genome of Malassezia pachydermatis, a fungus with substantial relevance in veterinary and medical sciences, was investigated using a series of conventional CA inhibitors (CAIs), namely aromatic and heterocyclic sulfonamides. This study aimed to describe novel candidates that can kill this harmful fungus by inhibiting their CA, and thus lead to effective anti-dandruff and anti-seborrheic dermatitis agents. In this context, current antifungal compounds, such as the azoles and their derivatives, have been demonstrated to induce the selection of resistant fungal strains and lose therapeutic efficacy, which might be restored by the concomitant use of alternative compounds, such as the fungal CA inhibitors.
Assuntos
Anidrase Carbônica I/antagonistas & inibidores , Malassezia/efeitos dos fármacos , Micoses/tratamento farmacológico , Sulfonamidas/farmacologia , Animais , Animais Domésticos/microbiologia , Antifúngicos/farmacologia , Anidrase Carbônica I/química , Inibidores da Anidrase Carbônica/farmacologia , Humanos , Malassezia/enzimologia , Malassezia/patogenicidade , Estrutura Molecular , Micoses/enzimologia , Micoses/microbiologia , Micoses/veterinária , Relação Estrutura-AtividadeRESUMO
There is significant interest in increasing the microalgal efficiency for producing high-quality products that are commonly used as food additives in nutraceuticals. Some natural substances that can be extracted from algae include lipids, carbohydrates, proteins, carotenoids, long-chain polyunsaturated fatty acids, and vitamins. Generally, microalgal photoautotrophic growth can be maximised by optimising CO2 biofixation, and by adding sodium bicarbonate and specific bacteria to the microalgal culture. Recently, to enhance CO2 biofixation, a thermostable carbonic anhydrase (SspCA) encoded by the genome of the bacterium Sulfurihydrogenibium yellowstonense has been heterologously expressed and immobilised on the surfaces of bacteria. Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyse the physiologically reversible reaction of carbon dioxide hydration to bicarbonate and protons: CO2 + H2O â HCO3- + H+. Herein, we demonstrate for the first time that the fragments of bacterial membranes containing immobilised SspCA (M-SspCA) on their surfaces can be doped into the microalgal culture of the green unicellular alga, Chlorella sorokiniana, to significantly enhance the biomass, photosynthetic activity, carotenoids production, and CA activity by this alga. These results are of biotechnological interest because C. sorokiniana is widely used in many different areas, including photosynthesis research, human pharmaceutical production, aquaculture-based food production, and wastewater treatment.
Assuntos
Bactérias/enzimologia , Anidrases Carbônicas/metabolismo , Chlorella/metabolismo , Bactérias/citologia , Bactérias/crescimento & desenvolvimento , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , HumanosRESUMO
Proteins are relevant antimicrobial drug targets, and among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central metabolism, or are necessary for the pathogen vitality. Genomic exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant ß-CA (CynT2) identified in the genome of the Gram-negative bacterium Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO2. Surprisingly, this enzyme has not been investigated for its inhibition with any class of CA inhibitors. Here, we show that CynT2 was strongly inhibited by some substituted benzene-sulfonamides and the clinically used inhibitor sulpiride (KIs in the range of 82-97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure-activity relationship for this class of enzyme inhibitors.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Proteínas de Escherichia coli/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Proteínas de Transporte de Ânions/antagonistas & inibidores , Proteínas de Transporte de Ânions/genética , Antibacterianos/química , Benzeno/química , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Humanos , Relação Estrutura-AtividadeRESUMO
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO2 hydration/dehydration reaction significantly. Here, we propose the marine unicellular diatom Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors (CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Diatomáceas/efeitos dos fármacos , Sulfonamidas/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/isolamento & purificação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Diatomáceas/enzimologia , Diatomáceas/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Bactérias Gram-Negativas Quimiolitotróficas/enzimologia , Coloração e Rotulagem/métodos , Temperatura , Estabilidade Enzimática , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the ß-glycoside hydrolase (SsßGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Enzimas Imobilizadas/metabolismo , Escherichia coli/enzimologia , Transferases/metabolismo , Enzimas Imobilizadas/química , Escherichia coli/metabolismo , Humanos , Coloração e Rotulagem , Propriedades de Superfície , Transferases/químicaRESUMO
The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and ß-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Anidrases Carbônicas/análise , Anidrases Carbônicas/metabolismo , Helicobacter pylori/enzimologia , Helicobacter pylori/metabolismoRESUMO
We report the cloning, purification and characterization of BpsßCA, a ß-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 γ-CA from the same organism, BpsγCA, recently investigated by our groups. BpsßCA showed a significant catalytic activity for the physiologic, CO2 hydration reaction, with the following kinetic parameters, kcat of 1.6×105s-1 and kcat/Km of 3.4×107M-1×s-1. The inhibition of BpsßCA with a group of anions and small molecules was also investigated. The best inhibitors were sulfamide, sulfamic acid and phenylarsonic acid, which showed KIs in the range of 83-92µM, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate and hexafluorophosphate showed KIs>100mM. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32-8.6mM. 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.
Assuntos
Antibacterianos/farmacologia , Burkholderia pseudomallei/enzimologia , Inibidores da Anidrase Carbônica/farmacologia , Sequência de Aminoácidos , Anidrases Carbônicas/química , Catálise , Cinética , Filogenia , Homologia de Sequência de AminoácidosRESUMO
We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s-1 and kcat/Km of 3.5 × 107 M-1 ×s-1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.
Assuntos
Anidrases Carbônicas/genética , Anidrases Carbônicas/isolamento & purificação , Mytilus/enzimologia , Animais , Clonagem Molecular , Mytilus/genéticaRESUMO
We have cloned, purified, and characterized a ß-carbonic anhydrase (CA, EC 4.2.1.1), BpsCAß, from the pathogenic bacterium Burkholderia pseudomallei, responsible for the tropical disease melioidosis. The enzyme showed high catalytic activity for the physiologic CO2 hydration reaction to bicarbonate and protons, with the following kinetic parameters: kcat of 1.6 × 105 s-1 and kcat/KM of 3.4 × 107 M-1 s-1. An inhibition study with a panel of 38 sulfonamides and one sulfamate-including 15 compounds that are used clinically-revealed an interesting structure-activity relationship for the interaction of this enzyme with these inhibitors. Many simple sulfonamides and clinically used agents such as topiramate, sulpiride, celecoxib, valdecoxib, and sulthiame were ineffective BpsCAß inhibitors (KI > 50 µM). Other drugs, such as ethoxzolamide, dorzolamide, brinzolamide, zonisamide, indisulam, and hydrochlorothiazide were moderately potent micromolar inhibitors. The best inhibition was observed with benzene-1,3-disulfonamides-benzolamide and its analogs acetazolamide and methazolamide-which showed KI in the range of 185-745 nM. The inhibition profile of BpsCAß is very different from that of the γ-class enzyme from the same pathogen, BpsCAγ. Thus, identifying compounds that would effectively interact with both enzymes is relatively challenging. However, benzolamide was one of the best inhibitors of both of these CAs with KI of 653 and 185 nM, respectively, making it an interesting lead compound for the design of more effective agents, which may be useful tools for understanding the pathogenicity of this bacterium.
Assuntos
Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/enzimologia , Inibidores da Anidrase Carbônica/farmacologia , Sulfanilamidas/farmacologia , Burkholderia pseudomallei/genética , Ativação Enzimática/efeitos dos fármacos , Cinese , Estrutura Molecular , Proteínas Recombinantes , SulfanilamidaRESUMO
Carbonic anhydrases (CA, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyze the conversion of carbon dioxide (CO2) to bicarbonate (HCO3(-)) and protons (H(+)). In prokaryotes, the existence of genes encoding for α-, ß- and γ-classes suggests that these enzymes play an important role in the prokaryotic physiology. It has been demonstrated, in fact, that their inhibition in vivo leads to growth impairment or growth defects of the microorganism. Ultimately, we started to investigate the biochemical properties and the inhibitory profiles of the α- and ß-CAs identified in the genome of Vibrio cholerae, which is the causative agent of cholera. The genome of this pathogen encodes for CAs belonging to α, ß and γ classes. Here, we report a sulfonamide inhibition study of the γ-CA (named VchCAγ) comparing it with data obtained for the α- and ß-CA enzymes. VchCAγ activity (kcat=7.39 × 10(5)s(-1)) was significantly higher than the other γ-CAs. The inhibition study with a panel of sulfonamides and one sulfamate led to the detection of a large number of nanomolar VchCAγ inhibitors, including simple aromatic/heterocyclic sulfonamides (compounds 2-9, 11, 13-15, 24) as well as EZA, DZA, BRZ, BZA, TPM, ZNS, SLP, IND (KIs in the range of 66.2-95.3 nM). As it was proven that bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit this virulence in vivo, we propose that VchCA, VchCAß and VchCAγ may be a target for antibiotic development, exploiting a mechanism of action rarely considered up until now, i.e., interference with bicarbonate supply as a virulence factor.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Sulfonamidas/farmacologia , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/genética , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Vibrio cholerae/genéticaRESUMO
The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, ß- and γ-classes. Here we report and anion inhibition study of the ß-CA, VchCAß with anions and other small molecules which inhibit metalloenzymes. The best VchCAß anion inhibitors were sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed KIs in the range of 54-86µM. Diethyldithiocarbonate was also an effective VchCAß inhibitor, with an inhibition constant of 0.73mM. The halides, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrate, nitrite, stannate, selenate, tellurate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanide, trithiocarbonate, and fluorosulfonate showed affinity in the low millimolar range, with KIs of 2.3-9.5mM. Identification of selective inhibitors of VchCAß (over the human CA isoforms) may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzyme.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Vibrio cholerae/enzimologia , Ânions/química , Ânions/farmacologia , Arsenicais/química , Arsenicais/farmacologia , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Modelos Moleculares , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologiaRESUMO
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.
Assuntos
Alteromonadaceae/enzimologia , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Sulfanilamidas/química , Antibacterianos/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/classificação , Anidrases Carbônicas/metabolismo , Humanos , Cinética , Filogenia , Ligação Proteica , Sulfanilamida , Sulfanilamidas/metabolismoRESUMO
We report the cloning, purification and characterization of the full domain of carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, which incorporates 358 amino acid residues (from 181 to 538, in the sequence of this 600 amino acid long protein), called PfCAdom. The enzyme, which belongs to the η-CA class showed the following kinetic parameters: kcat of 3.8×10(5)s(-1) and kcat/Km of 7.2×10(7)M(-1)×s(-1), being 13.3 times more effective as a catalyst compared to the truncated form PfCA. PfCAdom is more effective than the human (h) isoform hCA I, being around 50% less effective compared to hCA II, one of the most catalytically efficient enzymes known so far. Intriguingly, the sulfonamides CA inhibitors generally showed much weaker inhibitory activity against PfCAdom compared to PfCA, prompting us to hypothesize that the 69 amino acid residues insertion present in the active site of this η-CA is crucial for the active site architecture. The best sulfonamide inhibitors for PfCAdom were acetazolamide, methazolamide, metanilamide and sulfanilamide, with KIs in the range of 366-808nM.
Assuntos
Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/metabolismo , Clonagem Molecular , Plasmodium falciparum/enzimologia , Sulfonamidas/química , Sequência de Aminoácidos , Anidrase Carbônica I/antagonistas & inibidores , Anidrase Carbônica I/metabolismo , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Humanos , Cinética , Dados de Sequência Molecular , Plasmodium falciparum/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Relação Estrutura-Atividade , Sulfonamidas/farmacologiaRESUMO
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 α-, ß-, and γ-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 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.
Assuntos
Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Synechococcus/enzimologia , Temperatura , Dióxido de Carbono/química , Anidrases Carbônicas/isolamento & purificação , Cinética , Estrutura Molecular , Ácido Oxaloacético/química , Fosfoenolpiruvato Carboxilase/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
We have cloned, purified and characterized the γ-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: kcat of 6.0×10(5)s(-1) and a kcat/Km of 4.7×10(6)M(-1)×s(-1). This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a KI of 502nM. 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 KIs in the range of 1.4-4.4mM. Diethyldithiocarbamate was a better inhibitor (KI of 0.58mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with KIs ranging between 8 and 38µM. 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.