The interconversion of CO2 and HCO3- is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO2 and HCO3-. In the genome of Escherichia coli, a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a beta -CA, and CynS gene, encoding for the cyanase. CynT (beta -CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second beta -CA (CynT2 or Can or yadF), as well as a gamma and iota -CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO2 hydratase reaction with the following parameters: k(cat) = 5.3 x 10(5) s(-1) and k(cat)/K-M = of 4.1 x 10(7) M-1 s(-1). Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (K-I = 2.5 to 84 mu M). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.

Anion Inhibition Studies of the Beta-Carbonic Anhydrase from Escherichia coli

Del Prete Sonia;Scaloni Andrea;Capasso Clemente
2020

Abstract

The interconversion of CO2 and HCO3- is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO2 and HCO3-. In the genome of Escherichia coli, a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a beta -CA, and CynS gene, encoding for the cyanase. CynT (beta -CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second beta -CA (CynT2 or Can or yadF), as well as a gamma and iota -CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO2 hydratase reaction with the following parameters: k(cat) = 5.3 x 10(5) s(-1) and k(cat)/K-M = of 4.1 x 10(7) M-1 s(-1). Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (K-I = 2.5 to 84 mu M). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.
2020
Istituto di Bioscienze e Biorisorse
Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo - ISPAAM
carbonic anhydrase
anions
inhibitors
antibacterials
Escherichia coli
stopped-flow assay
protonography
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14243/405279
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