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. 1996 Oct 29;93(22):12171–12176. doi: 10.1073/pnas.93.22.12171

Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase.

R Chen 1, A Greer 1, A M Dean 1
PMCID: PMC37962  PMID: 8901552

Abstract

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.

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Selected References

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