Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase.
Rational engineering of enzymes involves introducing key amino acids guided by a knowledge of protein structure to effect a desirable change in function. To date, all successful attempts to change specificity have been limited to substituting individual amino acids within a protein fold. However, the infant field of protein engineering will only reach maturity when changes in function can be generated by rationally engineering secondary structures. Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to systematically invert the coenzyme specificity of Thermus thermophilus isopropylmalate dehydrogenase from a 100-fold preference for NAD to a 1000-fold preference for NADP. The engineered mutant, which is twice as active as wild type, contains four amino acid substitutions and an alpha-helix and loop that replaces the original beta-turn. These results demonstrate that rational engineering of secondary structures to produce enzymes with novel properties is feasible.[1]References
- Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase. Chen, R., Greer, A., Dean, A.M. Proc. Natl. Acad. Sci. U.S.A. (1996) [Pubmed]
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