9qev | pdb_00009qev

A native carbonic anhydrase from Caloramator australiacus

spinqualitylabelsall models 9qev, resolution 1.11Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 9qev is a 3 chain structure with sequence from Caloramator australicus. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.11Å Ligands: FMT, NA, ZN Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function G0V3M6_9CLOT Publication Abstract from PubMed 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 gamma-type carbonic anhydrase (gamma-CA) from Caloramator australicus (gamma-CaCA). The sequence of gamma-CaCA was amplified by PCR, cloned, and expressed in E. coli. Activity assays showed that gamma-CaCA 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 degrees C in the pH range between 5.5 and 9.0. The structure of gamma-CaCA was determined by X-ray crystallography at 1.11 A resolution, the highest resolution reported so far for a gamma-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 (gamma-CaCAmut) which possessed a heptapeptide insertion at the active-site loop and two-point mutations. Kinetic analysis demonstrated that gamma-CaCAmut was successfully converted into a catalytically active esterase indicating successful activity gain through structure-guided engineering. The thermostability of gamma-CaCAmut was significantly increased, aligning with the thermostability typically observed in hyperthermostable enzymes. X-ray crystallographic analysis of the gamma-CaCAmut structure at 2.1 A 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 gamma-CAs and demonstrate a strategy for converting an inactive enzyme into a catalytically active form through rational design. Structural and functional characterization of a metagenomically derived gamma-type carbonic anhydrase and its engineering into a hyperthermostable esterase.,Bodourian CS, Imran M, Georgakis ND, Papageorgiou AC, Labrou NE Protein Sci. 2025 Dec;34(12):e70396. doi: 10.1002/pro.70396. PMID:41294346 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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