(12h) Expanding the Utility of Amine Dehydrogenases Toward the Biocatalytic Production of Chiral Amines

Franklin, R. D., Georgia Institute of Technology
Mount, C., Georgia Institute of Technology
Alvi, U., Georgia Institute of Technology
Bommarius, B., Georgia Institute of Technology
Bommarius, A. S., Georgia Institute of Technology

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Expanding the utility
of amine dehydrogenases toward the biocatalytic production of chiral amines

normal">Robert D. Franklin‡, Conner M.
Mount#, Umair S. Alvi‡, Bettina R.
Bommarius‡, 11.5pt">Andreas S. Bommarius‡#

6.5pt">‡School of Chemical and
Biomolecular Engineering, Georgia Institute of Technology,

10.0pt">Parker H. Petit Institute for Bioengineering and Bioscience,

10.0pt">Engineered Biosystems Building, 950 Atlantic Drive N.W., GA 30322, USA

6.5pt">#School of Chemistry and
Biochemistry, Georgia Institute of Technology,

10.0pt">950 Atlantic Drive, Atlanta, GA 30332-2000, USA

Chiral amine functional groups are commonly found in active
pharmaceutical ingredients, and their efficient synthesis poses important
challenges to the industry. Of particular interest in recent years has been the
biocatalytic manufacture of enantiopure amine compounds. Enzymes tend to have
higher enantioselectivities when compared to traditional heterogeneous catalysts
and can be operated in milder conditions. Six years ago, a small family of
enzymes called amine dehydrogenases (AmDHs) were
engineered from wild amino acid dehydrogenases. yes">1-3AmDHs catalyze the reductive
amination of prochiral ketones to form enantiomerically pure (R)-amines. This is accomplished through
the addition of aqueous ammonia, concomitant with the oxidation of NADH. In the
present work, the authors report a broad effort to expand the utility of AmDHs by increasing both the specific activity and
substrate scope of the enzymes. Site directed mutagenesis, guided by previously
reported results, as well as structural insights resulted in activity towards
much larger substrate than could previously be converted. Mutations away from
the active site significantly increased specific activity without affecting
substrate specificity. Finally, Site saturation mutagenesis (SSM), substrate
walking, and screening of error-prone PCR libraries yielded AmDH
variants which could convert an interesting variety of substrates, including
ketones containing polar functional groups (except acids). This activity has
not been previously reported.

1.            Abrahamson, M. J.; Vazquez-Figueroa,
E.; Woodall, N. B.; Moore, J. C.; Bommarius, A. S., Development of an amine
dehydrogenase for synthesis of chiral amines. normal">Angew Chem Int Ed Engl 2012,
51 (16), 3969-72.

2.            Bommarius, B. R.; Schurmann, M.;
Bommarius, A. S., A novel chimeric amine dehydrogenase shows altered substrate
specificity compared to its parent enzymes. Chem
Commun (Camb)
2014, 50 (95), 14953-5.

3.            Bommarius,
B. R.; Au, S. K.; Abrahamson, M. J.; Wong, J. W.; Bommarius, A. S., Rapid
evolution of an amine dehydrogenase biocatalyst to chiral amines. Abstracts of Papers of the American Chemical
2013, 245, 1.