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 β-CA, and CynS gene, encoding for the cyanase. CynT (β-CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second β-CA (CynT2 or Can or yadF), as well as a γ and ι-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: kcat = 5.3 × 105 s−1 and kcat/KM = of 4.1 × 107 M−1 s−1. Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (KI = 2.5 to 84 µM). 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.
Highlights
Escherichia coli is a bacterium discovered in 1885 by the German bacteriologist Theodor Escherich who isolated it from the feces of a newborn [1]
The common genus Escherichia coli contains a broad variety of different forms: (i) pathogenic microorganisms, which can lead to death, or triggering severe disease outbreaks worldwide as well as serious infections, such as watery diarrhea, bloody diarrhea, urinary tract infection, meningitis, and sepsis [6–8]; (ii) opportunistic pathogens, which can cause disease if the host defenses are weakened [9]; and (iii) commensal microorganisms that innocuously colonize the healthy intestine of warm-blooded animals, including humans, with mutual benefits [10–12]
Here, using a stopped-flow technique, we investigated the kinetic constants of the recombinant CynT2, a β-CA identified in the genome of Escherichia coli, for which the kinetic characterization has not yet been reported
Summary
Escherichia coli is a bacterium discovered in 1885 by the German bacteriologist Theodor Escherich who isolated it from the feces of a newborn [1]. Escherichia coli is a harmless microbe, which typically colonizes the infant gastrointestinal tract within the first hours of life, establishing a mutual benefit with its host [2–4]. The common genus Escherichia coli contains a broad variety of different forms: (i) pathogenic microorganisms, which can lead to death, or triggering severe disease outbreaks worldwide as well as serious infections, such as watery diarrhea, bloody diarrhea, urinary tract infection, meningitis, and sepsis [6–8]; (ii) opportunistic pathogens, which can cause disease if the host defenses are weakened [9]; and (iii) commensal microorganisms that innocuously colonize the healthy intestine of warm-blooded animals, including humans, with mutual benefits [10–12]
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