GUIDED EVOLUTION OF ENZYMES WITH NEW SUBSTRATE SPECIFICITIES

A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alph a(E)-beta(D)) Of Bacillus stearothermophilus L-lactate dehydrogenase ( bsLDH). All but one of 38 enzyme variants examined were thermally stab le and had native-like hydrodynamic properties. In this sample, there was no bias detected in either the DNA or amino acid sequences encoded . We argue that the alpha(E)-beta(D) surface loop sequence is unimport ant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities. The selection of NAD-depend ent dehydrogenases with specificity for: malate, phenyllactate, hydrox yisocaproate and 4-phenyl-2-hydroxy butanoate from two bsLDH libraries is described. This required a highly discriminatory screen for 2-hydr oxy acid dehydrogenase activity to select enzymes which, in the absenc e of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition.In general the amino acid residues at positions 1 01 and 102 which determined substrate specificity were as expected fro m hydrophobic and ionic complementarity to the substrate. For example, a bsLDH variant with Asn101Val102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate Using molecul ar modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate. Similarly, nine out of ten malate dehydr ogenases (MDHs) selected had an arginine residue at position 102 to co mplement the negatively charged carboxyl group in oxaloacetate. One, A rg101Arg102 (k(cat)/K-M oxaloacetate = 1.6 x 10(6) M(-1) s(-1)) is 25% more active than the previous best synthetic MDH. There were surprise s: present understanding would not have predicted the oxaloacetate tra nsforming activity of Ser101Leu102 or the phenylpyruvate activity of P ro101Lys102. The former is about one-third as efficient as the best ma late dehydrogenase selected, whilst the latter had about one-seventh o f the best phenylpyruvate dehydrogenase activity. (C) 1996 Academic Pr ess Limited