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A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity
1Department of Biological Chemistry, Chicago Medical School, North Chicago, IL 60064, USA.
Summary
Researchers engineered Escherichia coli isocitrate dehydrogenase to prefer NAD over NADP. This enzyme engineering achieved a significant shift in coenzyme preference, creating a valuable tool for biochemical studies.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Structural Biology
Background:
- Escherichia coli isocitrate dehydrogenase lacks the typical Rossmann fold.
- The native enzyme exhibits a strong preference for NADP+ over NAD+.
Purpose of the Study:
- To engineer the Escherichia coli isocitrate dehydrogenase to shift its coenzyme preference from NADP+ to NAD+.
- To investigate the role of specific amino acid residues in coenzyme binding and specificity.
Main Methods:
- Utilized X-ray crystallography and molecular modeling to guide enzyme engineering.
- Employed site-directed mutagenesis to introduce six specific amino acid substitutions.
- Characterized the kinetic properties and coenzyme preference of the engineered enzyme.
Main Results:
- Engineered enzyme showed an 850-fold preference for NAD+ over NADP+, a significant shift from the native enzyme.
- The engineered enzyme's NAD+ preference surpassed that of a homologous NAD+-dependent enzyme.
- Identified that homology-guided substitutions alone were insufficient; remote mutations were also crucial for optimal function.
Conclusions:
- Enzyme engineering can effectively alter the coenzyme specificity of isocitrate dehydrogenase.
- A combination of targeted and remote mutations is key to achieving desired kinetic characteristics and coenzyme preference.
- The study provides insights into enzyme structure-function relationships and the design of novel biocatalysts.