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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.
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.
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.
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.