Evolution of Novel Amine Dehydrogenase Properties

The reductive amination of ketones to produce chiral amines is an important transformation in the production of pharmaceutical intermediates. Recently, we obtained the first amine dehydrogenases (L-AmDH) from a leucine dehydrogenase [1] and from phenylalanine dehydrogenase (F-AmDH).[2]  Here, we report on successful domain shuffling efforts to create further variants of amine DH with altered substrate specificity.  As an example, we now can accommodate sterically very demanding side chains, such as adamantly groups, in the ketone substrate, which did not react at all beforehand.  Next, we will elucidate the role of restricted libraries towards higher activity and broader specificity.

We also report on progress in processing AmDHs and in fusing them to cofactor regeneration enzymes.  On large-scale, IMAC purification with his-tagged proteins is problematic.  We have developed a fractionated precipitation, two-phase extraction purification protocol for the untagged AmDHs.  In parallel, we have developed a fusion protein of AmDH with formate dehydrogenase (FDH) to regenerate NADH and report on the status.          

References:

[1]   M.J. Abrahamson, E. Vazquez-Figueroa, N.B. Woodall, J.C. Moore, A.S. Bommarius, Development of a Novel Amine Dehydrogenase for Synthesis of Chiral Amines“, Angew. Chem. Intl. Ed. 2012, 51, 3969-3972

[2]   M.J. Abrahamson, J.W. Wong, and A.S. Bommarius, “The rapid evolution of an amine dehydrogenase biocatalyst for the asymmetric production of chiral amines”, Adv. Synth. Catal. 2013, 000 (in press)