Engineered amine dehydrogenase exhibits altered kinetic mechanism compared to parent with implications for industrial application

Leucine amine dehydrogenase (L-AmDH) was engineered from the leucine dehydrogenase (LeuDH) from Bacillus stearothermophilus through the introduction of two key mutations to the substrate binding pocket, thus removing affinity for keto-acids in favor of methyl ketones and enabling the production of chiral amines, important intermediates for pharmaceuticals. The present work examines the effects of the mutations on the kinetic properties of these two enzymes. We conclude that L-AmDH exhibits a random order of association of ketone and cofactor in the reductive amination direction, compared to the ordered sequential mechanism of LeuDH. In the oxidative deamination direction, an ordered sequential mechanism was confirmed for LeuDH, while a more complicated situation was found for L-AmDH. Kinetic solvent viscosity effects revealed an isomerization of enzyme-substrate complexes for both LeuDH reactions, but not for L-AmDH. Inhibition experiments showed stronger product inhibition for L-AmDH than for LeuDH, an important consideration for large-scale synthesis.