REDESIGNING SECONDARY STRUCTURE TO INVERT COENZYME SPECIFICITY IN ISOPROPYLMALATE DEHYDROGENASE

Rational engineering of enzymes involves introducing key amino acids g uided by a knowledge of protein structure to effect a desirable change in function, To date, all successful attempts to change specificity h ave been limited to substituting individual amino acids within a prote in fold. However, the infant field of protein engineering will only re ach maturity when changes in function can be generated by rationally e ngineering secondary structures, Guided by x-ray crystal structures an d molecular modeling, site-directed mutagenesis has been used to syste matically invert the coenzyme specificity of Thermus thermophilus isop ropylmalate dehydrogenase from a 100-fold preference for NAD to a 1000 -fold preference for NADP, The engineered mutant, which is twice as ac tive as wild type, contains four amino acid substitutions and an cy-he lix and loop that replaces the original beta-turn, These results demon strate that rational engineering of secondary structures to produce en zymes with novel properties is feasible.