The Search for Novel Substrate Specificity in Mutants of L‐Alanine Dehydrogenase

Abstract

The enzyme L‐Alanine dehydrogenase from M. tuberculosis (AlaDH) catalyzes the conversion of L‐alanine to pyruvate by replacing the amino group with a carbonyl. AlaDH belongs to a superfamily of alcohol dehydrogenases that reversibly convert alcohols to carbonyls rather than the Glu/Phe/Leu amino acid dehydrogenase superfamily. Molecular modeling of the AlaDH binding pocket suggested that mutating the 94‐phenylalanine residue to alanine or serine would open up the binding pocket to accommodate larger, bulkier substrates. We predicted that these mutations would enable the enzyme to catalyze the same reaction on larger amino acids. An assay of the F94A and F94S mutants with different substrates showed novel activity on leucine and norleucine in comparison to the wild type, as well as greatly increased activity on norvaline and methionine. The Km values of both mutants are comparable to that of the wild type value, while the kcat values are <1% of the wild type value indicating that the mutant enzyme binds to the substrate well, but potentially in a less reactive conformation. We thank the HHMI for support for this project.