Amine Transaminase fromExophiala Xenobiotica—Crystal Structure and Engineering of a Fold IV Transaminase that Naturally Converts Biaryl Ketones

Abstract

Amine transaminases are frequently used for the production of chiral amines starting from prochiral ketones. These amines can be applied as active pharmaceutical ingredients or drug precursors. However, there are still limitations to the use of amine transaminases when it comes to bulky ketone substrates, such as biaryl ketones. Using data mining, an (R)-selective amine transaminase from Exophiala xenobiotica was identified which naturally converts biaryl ketone substrates to the corresponding amines with up to 85% conversion and excellent enantioselectivity (>99% ee). Its protein crystal structure was obtained with a resolution of 1.52 Å, which enabled us to explain this interesting substrate acceptance. Structure-guided protein engineering resulted in a quintuple variant with increased stability. Moreover, the amino acid exchange T273S increased the activity and broadened the substrate scope enabling conversions of various biaryl ketones with up to >99%. A preparative biotransformation of 1-(4-(pyridin-3-yl)phenyl)ethenone at 75 mM (15 g/L) resulted in 96% of isolated yield of the respective amine.

An (R)-Selective Transaminase From Thermomyces stellatus: Stabilizing the Tetrameric Form

“…SEC unexpectedly revealed that 90% of Ts RTA in solution exists as a tetramer, with only 10% adopting the dimeric form ( Figure S5 ). Other highly similar RTAs, including At RTA (4ce5, 81% identity) (Łyskowski et al, 2014 ), and RTAs from Nectria haematococca (4cmd, 78% identity) (Sayer et al, 2014 ), Exophiala xenobiotica (6fte, 70% identity) (Telzerow et al, 2019 ), and Aspergillus fumigatus (4uug, 73% identity) (Thomsen et al, 2014 ) are reported as dimers based on their crystal packing. Two thermostable BCATs, belonging to the same fold type IV as RTAs, with hexameric structures have recently been reported by Isupov et al ( 2019 ).…”

Enantioselective One-Pot Synthesis of Biaryl-Substituted Amines by Combining Palladium and Enzyme Catalysis in Deep Eutectic Solvents

“…For example, the rationally engineered (S)-selective ATA from V. fluvialis catalyzed an ortho-biaryl ketone to the corresponding (S)-biaryl amine. 47 Bornscheuer and coworkers, 16 in parallel to us, 48…”

Non‐Conventional Media as Strategy to Overcome the Solvent Dilemma in Chemoenzymatic Tandem Catalysis

“…In 2016, a rationally engineered ( S )‐selective ATA from V. fluvialis catalyzed an ortho‐biaryl ketone to the corresponding biaryl amine . Similarly, two biocatalysts recently found by data mining, namely the ( R )‐selective ATA from Exophiala xenobiotica (EX‐ωTA) and Asperguillus fumigatus (4CHI‐TA), catalyzed efficiently the amination of meta ‐ and para ‐biaryl ketones. Further structure‐guided protein engineering resulted in broader substrate scope and improved stability which paved the way for the straightforward approach A in Scheme .…”