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Redesign of choline acetyltransferase specificity by protein engineering
1Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA. ciaran@itsa.ucsf.edu
The Journal of Biological Chemistry
|September 12, 1998
Summary
Protein redesign achieved significant success without 3D structure maps. A mutant choline acetyltransferase showed a 1,620-fold increase in catalytic efficiency toward L-carnitine.
Area of Science:
- Biochemistry
- Protein Engineering
- Enzyme Catalysis
Background:
- Site-directed mutagenesis enables enzyme redesign for altered catalytic properties.
- Successful protein redesign has historically relied on 3D protein structure maps.
- 3D structure maps are unavailable for choline acetyltransferase and carnitine acetyltransferase.
Purpose of the Study:
- To redesign choline acetyltransferase (ChAT) to accept L-carnitine as a substrate.
- To demonstrate successful protein redesign in the absence of 3D structural data.
- To investigate empirical approaches for enzyme engineering.
Main Methods:
- Utilized an empirical approach combining substrate structure comparisons and protein alignment data.
- Employed site-directed mutagenesis to introduce four amino acid substitutions into wild-type ChAT.
- Assessed changes in catalytic efficiency (kcat/Km) and substrate discrimination.
Main Results:
- Developed a mutant ChAT with significantly enhanced catalytic efficiency toward L-carnitine (1,620-fold increase).
- Shifted catalytic preference from choline to L-carnitine by over 390,000-fold.
- Demonstrated substantial alterations in enzyme catalytic function.
Conclusions:
- Significant protein redesign is achievable without 3D structural information.
- Empirical strategies can effectively guide enzyme engineering efforts.
- This study provides a novel approach for engineering enzymes with altered substrate specificities.