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.
Department of Biological Chemistry, Chicago Medical School, IL 60064-3095, USA.
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Chen
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Greer
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Dean
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003067:Coenzymes / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D003067:Coenzymes; D009249:NADP; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase