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@ARTICLE{Bodourian:642749,
      author       = {Bodourian, Charoutioun S. and Imran, Mohsin and Georgakis,
                      Nikolaos D. and Papageorgiou, Anastassios C. and Labrou,
                      Nikolaos E.},
      title        = {{S}tructural and functional characterization of a
                      metagenomically derived $γ$‐type carbonic anhydrase and
                      its engineering into a hyperthermostable esterase},
      journal      = {Protein science},
      volume       = {34},
      number       = {12},
      issn         = {0961-8368},
      address      = {Hoboken, NJ},
      publisher    = {Wiley},
      reportid     = {PUBDB-2025-05598},
      pages        = {e70396},
      year         = {2025},
      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-CaCA). The sequence
                      of γ-CaCA was amplified by PCR, cloned, and expressed in E.
                      coli. Activity assays showed that γ-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 (Tm)
                      approximately 65–75°C in the pH range between 5.5 and
                      9.0. The structure of γ-CaCA 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-CaCAmut) which possessed a
                      heptapeptide insertion at the active-site loop and two-point
                      mutations. Kinetic analysis demonstrated that γ-CaCAmut was
                      successfully converted into a catalytically active esterase
                      indicating successful activity gain through structure-guided
                      engineering. The thermostability of γ-CaCAmut was
                      significantly increased, aligning with the thermostability
                      typically observed in hyperthermostable enzymes. X-ray
                      crystallographic analysis of the γ-CaCAmut 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.},
      cin          = {EMBL-User},
      ddc          = {610},
      cid          = {I:(DE-H253)EMBL-User-20120814},
      pnm          = {6G3 - PETRA III (DESY) (POF4-6G3)},
      pid          = {G:(DE-HGF)POF4-6G3},
      experiment   = {EXP:(DE-H253)P-P13-20150101},
      typ          = {PUB:(DE-HGF)16},
      doi          = {10.1002/pro.70396},
      url          = {https://bib-pubdb1.desy.de/record/642749},
}