title: Alteration of substrate specificity of alanine dehydrogenase.
abstract: 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 mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing 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.
authors.affiliation: Chemistry Department, Hofstra University, Hempstead, NY 11549, USA.
authors.forename: Puja
authors.initials: P
authors.lastname: Fernandes
authors.affiliation: Chemistry Department, Vassar College, Poughkeepsie, NY 12604, USA.
authors.forename: Hannah
authors.initials: H
authors.lastname: Aldeborgh
authors.affiliation: Chemistry Department, Hofstra University, Hempstead, NY 11549, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Carlucci
authors.affiliation: Chemistry Department, Hofstra University, Hempstead, NY 11549, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Walsh
authors.affiliation: Chemistry Department, Hofstra University, Hempstead, NY 11549, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Wasserman
authors.affiliation: Chemistry Department, Hofstra University, Hempstead, NY 11549, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Zhou
authors.affiliation: Chemistry Department, Hofstra University, Hempstead, NY 11549, USA.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Lefurgy
authors.affiliation: Chemistry Department, Hofstra University, Hempstead, NY 11549, USA emily.c.mundorff@hofstra.edu.
authors.forename: Emily C
authors.initials: EC
authors.lastname: Mundorff
mesh_terms: D050788:Alanine Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D050788:Alanine Dehydrogenase
keywords: alanine dehydrogenase; amino acid dehydrogenase; evolvability; saturation mutagenesis; superfamily