Highly active extracellular α-class carbonic anhydrase of Cyanothece sp. ATCC 51142

Biochimie. 2019 May:160:200-209. doi: 10.1016/j.biochi.2019.03.009. Epub 2019 Mar 18.

Abstract

Here, for the first time, we report the presence of highly active extracellular carbonic anhydrase (CA) of α-class in cyanobacterial cells. The enzyme activity was confirmed both in vivo in intact cells and in vitro, using the recombinant protein. CA activity in intact cells of Cyanothece sp. ATCC 51142 reached ∼0.6 Wilbur-Anderson units (WAU) per 1 mg of total cell protein, and it was inhibited by a specific CAs inhibitor, ethoxyzolamide. The genes cce_4328 (ecaA) and cce_0871 (ecaB), encoding two potential extracellular CAs of Cyanothece have been cloned, and the corresponding proteins EcaA and EcaB, representing CAs of α- and β-class, respectively, have been heterologously expressed in Escherichia coli. High specific activity (∼1.1 × 104 WAU per 1 mg of target protein) was detected for the recombinant EcaA only. The presence of EcaA in the outer cellular layers of Cyanothece was confirmed by immunological analysis with antibodies raised against the recombinant protein. The absence of redox regulation of EcaA activity indicates that this protein does not possess a disulfide bond essential for some α-class CAs. The content and activity of EcaA in a fraction of periplasmic proteins was higher in Cyanothece cells grown at ambient concentration of CO2 (0.04%) compared to those grown at an elevated CO2 concentration (1.7%). At the same time, the level of ecaA gene mRNA varied insignificantly in response to changes in CO2 supply. Our results indicate that EcaA is responsible for CA activity of intact Cyanothece cells and point to its possible physiological role under low-CO2 conditions.

Keywords: CO(2)-concentrating mechanism; Carbonic anhydrase; Cyanobacteria; Cyanothece sp. ATCC 51142; EcaA; EcaB.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Carbon Dioxide / metabolism*
  • Carbonic Anhydrases / genetics
  • Carbonic Anhydrases / isolation & purification
  • Carbonic Anhydrases / metabolism*
  • Cloning, Molecular
  • Cyanothece / enzymology*
  • Extracellular Space / enzymology*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism*

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Carbon Dioxide
  • Carbonic Anhydrases