Structural and functional characterization of a metagenomically derived γ‐type carbonic anhydrase and its engineering into a hyperthermostable esterase

Abstract

Abstract The 16S microbial community profiling of a metagenomics library from geothermal spring at Lisvori (Lesvos island, Greece) enabled the identification of a putative sequence exhibiting 95% identity to the γ‐type carbonic anhydrase (γ‐CA) from Caloramator australicus (γ‐ Ca CA). The sequence of γ‐ Ca CA was amplified by PCR, cloned, and expressed in E. coli . Activity assays showed that γ‐ Ca CA possesses very low, but detectable, anhydrase activity, while exhibiting no measurable esterase activity. Differential scanning fluorimetry (DSF) revealed that the enzyme shows high thermal stability with a melting temperature ( T m ) approximately 65–75°C in the pH range between 5.5 and 9.0. The structure of γ ‐Ca CA was determined by X‐ray crystallography at 1.11 Å resolution, the highest resolution reported so far for a γ ‐ CA. The enzyme was crystallized as a trimer in the crystallographic asymmetric unit and contains three zinc‐binding sites, one at each interface of neighboring subunits of the trimer. Structure‐based rational design enabled the design and creation of a mutant enzyme (γ ‐Ca CAmut) which possessed a heptapeptide insertion at the active‐site loop and two‐point mutations. Kinetic analysis demonstrated that γ‐ Ca CAmut was successfully converted into a catalytically active esterase indicating successful activity gain through structure‐guided engineering. The thermostability of γ‐ Ca CAmut was significantly increased, aligning with the thermostability typically observed in hyperthermostable enzymes. X‐ray crystallographic analysis of the γ‐ Ca CAmut structure at 2.1 Å resolution, provided detailed structural insights into how the mutations impact the overall enzyme structure, function, and thermostability. These findings provide valuable structural and functional insights into γ‐CAs and demonstrate a strategy for converting an inactive enzyme into a catalytically active form through rational design.