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Dangosydd eitem ddigidol (DOI)

  • Isabel Cea-Rama
    Institute of Physical Chemistry Rocasolano, CSIC
  • Cristina Coscolín
    Institute of Catalysis, Madrid, Spain
  • Pangiotis Katsonis
    Baylor College of Medicine, Houston
  • Rafael Bargiela
  • Peter Golyshin
  • Olivier Lichtarge
    Baylor College of Medicine, Houston
  • Manuel Ferrer
    Institute of Catalysis, Madrid, Spain
  • Julia Sanz-Aparicio
    Institute of Physical Chemistry Rocasolano, CSIC
Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH3) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.


Iaith wreiddiolSaesneg
Tudalennau (o-i)2307-2317
Nifer y tudalennau11
CyfnodolynComputational and Structural Biotechnology Journal
Dyddiad ar-lein cynnar18 Ebr 2021
Dynodwyr Gwrthrych Digidol (DOIs)
StatwsCyhoeddwyd - 2021

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