Introduction and Motivation

1. *CPD Confidential Crystallization • Crystal structure of F-AmDH solved at 2.5 Å • Truncated versions to eliminate N-and C-terminal loops were not successful so far to get expressed protein • Achieved crystal structure at 3.2 Å with the cFL1-AmDH GOAL: To develop a biocatalyst with novel functionality by creating an NAD+-dependent amine dehydrogenase (AmDH) from an amino acid dehydrogenase (AADH) F-AmDH variants Aqueous Two-Phase System (ATPS) Amine Dehydrogenases Introduction and Motivation • In 2010, 80% of the 200 most prescribed brand name drugs in the U.S. contain nitrogen, with a chiral amine in 40% of these compounds [1] • Chiral amines are notoriously difficult to synthesize often requiring heavy metal catalysts or crystallization Development of a Novel Amine DehydrogenaseSamantha K. Au, Bettina Bommarius, Andreas S. BommariusParker H. Petit Institute for Bioengineering and BioscienceSchool of Chemical and Biomolecular EngineeringGeorgia Institute of Technology, Atlanta, GA GOAL: To purify the protein from cell debris Successfully removed all centrifugation steps Measure specific activity of top and bottom layer to quickly identify how F-AmDH separated • cFL1-AmDH also screened for crystallization, but seems to aggregate at higher protein concentrations • Ongoing collaboration with Russell Judge, Abbvie Figure 1. Subunit of LeuDH Bacillus sphaericus (PDB: 1LEH) Top Layer 1:10 Bottom Layer F-AmDH variants • One of the top aspirational reactions challenging the pharmaceutical industry according to the ACS Pharmaceutical Roundtable Vector aa 1-21 His aa 361-380 • Currently building a pilot-scale ATPS • Apply to other AmDHs and DHS (glucose, formate), charge of enzyme is suspected to play a role in partitioning [1] Mack DJ, Weinrich ML, Vitaku E, Njarðarson JT. Top 200 Brand Name Drugs by US Retail Sales in 2010. [2]Constable, D.J.C., et al.Green Chemistry, 2007, (9), 411-420. *F-AmDH Targeted Reaction: NADH + H+ + NH3 + + NAD+ + H2O Prochiral ketone Chiral primary amine • Developed amine dehydrogenases: • 1. Leucine amine dehydrogenase (L-AmDH)[3] • 2. Phenylalanine amine dehydrogenase (F-AmDH)[4] • 3. Chimeric amine dehydrogenase (cFL1-AmDH)[5] • 4. Valine amine dehydrogenase (V-AmDH) • Currently expanding amine dehydrogenase family • 2 sequence hot spots for protein engineering, loop area around D124 and substrate pocket around V308 (F-AmDH numbering) • Loop1 – Loop6 deletions around D124 to accommodate bigger substrates • V143A, L305A and V308A change substrate specificity • D33A and N145S show activity towards pinacolone in L-AmDH, a novel substrate! Partition coefficients P = [target protein]top/[target protein]bottom Deracemization to (S)-amine • Motivation: Production of the (S)-amine • Successful kinetic resolution of racemic MBA at 97% conversion and pure (S)-MBA achieved • Currently working on protein engineering of NADH oxidase (K147R) to improve temperature stability and improve reaction conditions Pinacolone *Homology model of L. plantarumNox [3] Abrahamson, M.J. et al. AngewanteChemie Int. Ed., 2012. 51: p.3969-3972 [4] Abrahamson, M.J., Wong, J., Bommarius, A. Adv. Synth. Catal., 2013. 355 (9): p. 1780-1786 [5] Bommarius, B. et al. ChemComm 2014.