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.

Rational Engineering ofBacillus cereusLeucine Dehydrogenase Towards α-keto Acid Reduction for Improving Unnatural Amino Acid Production

“…AADH superfamily show relative strict substrate specificity and have a long and rich history of being targeted in protein engineering to alter substrate specificity due to its members’ usefulness in diagnostic kits and in the biocatalysis of chiral amines and unnatural amino acids . However, as the most used industrial AADH, there is still little progress in LeuDHs’ engineering for industrial use, especially engineering LeuDHs with residues in or affected substrate entrance tunnel which could be of vital importance for enzymatic activities and stability.…”

Characterization of Alanine Dehydrogenase and Its Effect on <b><i>Streptomyces coelicolor</i></b>A3(2) Development in Liquid Culture

“…The natural substrates of ALD enzymes for oxidative deamination and reductive amination reactions are believed to be L-alanine and pyruvate, respectively. However, there have been reports of promiscuity of such substrate specificity [Fernandes et al, 2015;Giffin et al, 2012;Phogosee et al, 2018]. In order to characterize the substrate specificity for ScALD, several different substrates have been explored.…”

Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity

“…The closest relative, with 51 % amino acid sequence identity, was AlaDH from M. tuberculosis (MtAlaDH) [38,39]. The crystal structures of both the apo-form and the NADH complex of this enzyme (PDB IDs: 2VHY and 2VHW) were determined to 2.3 and 2.0Å resolution, respectively [27].…”