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New enzyme lineages by subdomain shuffling

K P Hopfner1, E Kopetzki, G B Kresse

  • 1Abteilung Strukturforschung, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.

Proceedings of the National Academy of Sciences of the United States of America
|August 26, 1998
PubMed
Summary

Protein domain recombination can create new enzymes. Swapping subdomains from coagulation factor X and trypsin generated a potent enzyme (fXYa) with broad substrate specificity and novel properties.

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Area of Science:

  • Protein engineering
  • Enzymology
  • Structural biology

Background:

  • Protein functions evolve through domain recombination, potentially creating new catalytic sites at domain interfaces.
  • The S1 serine protease family likely evolved from recombined domains, with conserved catalytic residues and variable surfaces.

Purpose of the Study:

  • To explore domain recombination as a strategy for engineering novel protein functions.
  • To create and characterize a hybrid enzyme by swapping subdomains from known proteins.

Main Methods:

  • Recombination of N-terminal subdomain from coagulation factor X with C-terminal subdomain from trypsin.
  • Biochemical characterization of the resulting enzyme (fXYa).
  • X-ray crystallography to determine the structure of fXYa.

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Main Results:

  • The hybrid enzyme fXYa exhibits potent activity and broad substrate specificity.
  • The crystal structure reveals plasticity at the subdomain interface, maintaining activity.
  • Surface loops are displaced compared to parent proteins, indicating novel structural features.

Conclusions:

  • Domain swapping is a viable strategy for engineering new protein properties.
  • fXYa represents a new serine proteinase lineage with combined and novel characteristics.
  • The plasticity of the subdomain interface is key to maintaining enzyme function in the hybrid protein.