No 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.

The enzyme L‐Alanine dehydrogenase (AlaDH) from Mycobacterium tuberculosis catalyzes the reversible conversion of L‐alanine to pyruvate. AlaDH, however, belongs to the formate‐glycerate dehydrogenase superfamily, whose other members generally reversibly interconvert carbonyls to alcohols, not amines as in the AlaDHs. In a case of convergent evolution, enzymes in the Phe/Glu/Val/Leu amino acid dehydrogenase (AADH) superfamily catalyze the same reaction as AlaDH on larger amino acids, but bare no structural resemblance to AlaDH. Can AlaDH be altered in a way that will allow it to act on different amino acid substrates, or has evolution make it too specific to alanine to alter its activity? Our objective is to find novel activity on various substrates in the highly specific AlaDH. Previously, it has been shown that the replacement of the 94‐phenylalanine binding pocket residue with alanine and serine leads to novel activity on leucine and norleucine in comparison to the wild type, as well as greatly increased activity on norvaline and methionine. Here we present the additional mutation of 133‐methionine to alanine. We successfully express and purify this double mutant, and find additional novel activity.

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.