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The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
Published on: February 1, 2018
Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox
Julien Hiblot1, Guillaume Gotthard, Mikael Elias
1URMITE UMR CNRS-IRD 6236, Faculté de Médecine et de Pharmacie, Université de la Méditerranée, Marseille, France.
Plos One
|October 3, 2013
Summary
Enzyme active site flexibility influences substrate specificity and promiscuous functions. Mutations in the SsoPox lactonase active site loop enhanced its catalytic activities by increasing flexibility.
Area of Science:
- Enzymology
- Structural Biology
- Protein Engineering
Background:
- Enzymes catalyze reactions through specific conformational states.
- Active site flexibility is crucial for enzyme function and can mediate promiscuous activities.
Purpose of the Study:
- To investigate the role of active site loop flexibility in enzyme substrate specificity and promiscuity.
- To engineer the lactonase SsoPox for enhanced phosphotriesterase and oxo-lactonase activities.
Main Methods:
- Site-directed mutagenesis at position W263 in the SsoPox active site loop.
- Structural and kinetic characterization of enzyme variants.
- Analysis of enzyme flexibility and substrate specificity.
Main Results:
- Mutations at W263 induced distinct conformational states of the active site loop.
- Increased loop flexibility enhanced both promiscuous phosphotriesterase and oxo-lactonase activities.
- Substrate specificity was modulated by altering active site loop dynamics.
Conclusions:
- Conformational diversity of enzyme active sites governs enzymatic promiscuity.
- Active site flexibility is a key determinant of protein evolvability and enzyme engineering potential.
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