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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Stabilization of creatinase from Pseudomonas putida by random mutagenesis
J Schumann1, G Böhm, G Schumacher
1Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Protein Science : a Publication of the Protein Society
|October 1, 1993
Summary
Mutations in Pseudomonas putida creatinase (creatine amidinohydrolase) enhanced enzyme stability through subtle structural adjustments. These changes improved local packing without altering quaternary structure or catalytic activity, with stability effects being additive.
Area of Science:
- Enzymology
- Protein Engineering
- Structural Biology
Background:
- Creatinase from Pseudomonas putida is a well-characterized enzyme with a known 3D structure.
- Understanding protein stability through mutagenesis is crucial for enzyme engineering.
Purpose of the Study:
- To investigate the impact of specific point mutations on the physical and enzymological properties of creatinase.
- To compare wild-type creatinase with single, double, and triple mutants.
Main Methods:
- Site-directed mutagenesis to create mutants A109V, V355M, and V182I.
- High-resolution X-ray crystallography to assess structural integrity.
- Physicochemical measurements to evaluate quaternary structure, conformation, and denaturation transitions.
Main Results:
- Mutations did not significantly alter quaternary structure, overall conformation, or catalytic properties compared to wild-type creatinase.
- Point mutations A109V and V355M stabilized the initial phase of biphasic denaturation.
- The stabilizing effects of mutations were additive in combined and triple mutants, suggesting minor local packing improvements.
Conclusions:
- Specific mutations in creatinase can enhance stability through subtle local structural refinements.
- These modifications do not compromise the enzyme's overall structure or catalytic function.
- The additive nature of stabilization increments provides insights into protein stability mechanisms.
