Emily C Mundorff
Hofstra University, USA
Title: Alteration of the substrate specificity of alanine dehydrogenase
Biography
Biography: Emily C Mundorff
Abstract
What determines the evolvability of an enzyme?Is an enzyme’s apparent natural history a good guide to determine the ease of evolving new substrate specificities? The L-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: It is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for L-alanine over all other substrates. By contrast, engineering of the Amino Acid Dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH. A single mutationintroduced intothe AlaDH from Mycobacterium tuberculosis(MtAlaDH) completely alters its substrate specificity pattern enabling activity towards a range of larger amino acids.Saturation mutagenesis libraries in this mutant background additionally identified a double mutant with improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the Amino Acid Dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates.