Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

Amol D Pagar; Hyunwoo Jeon; Taresh P Khobragade; Sharad Sarak; Pritam Giri; Seonga Lim; Tae Hyeon Yoo; Byoung Joon Ko; Hyungdon Yun

doi:10.3389/fchem.2022.839636

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

Front Chem. 2022 Feb 28:10:839636. doi: 10.3389/fchem.2022.839636. eCollection 2022.

Authors

Amol D Pagar¹, Hyunwoo Jeon¹, Taresh P Khobragade¹, Sharad Sarak¹, Pritam Giri¹, Seonga Lim¹, Tae Hyeon Yoo², Byoung Joon Ko³, Hyungdon Yun¹

Affiliations

¹ Department of Systems Biotechnology, Konkuk University, Seoul, South Korea.
² Department of Molecular Science and Technology, Ajou University, Suwon, South Korea.
³ School of Biopharmaceutical and Medical Sciences, Sungshin Women's University, Seoul, South Korea.

Abstract

Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of p-benzoyl phenylalanine (pBpA) to engineer (R)-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with pBpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively. Reshaping of the enzyme's active site afforded an another variant F86A/F88pBpA, with 30% higher thermostability at 55°C without affecting parent enzyme activity. Moreover, various racemic amines were successfully resolved by transaminase variants into (S)-amines with excellent conversions (∼50%) and enantiomeric excess (>99%) using pyruvate as an amino acceptor. Additionally, kinetic resolution of the 1-phenylpropan-1-amine was performed using benzaldehyde as an amino acceptor, which is cheaper than pyruvate. Our results highlight the utility of ncAAs for designing enzymes with enhanced functionality beyond the limit of 20 canonical amino acids.

Keywords: (R)-amine transaminase; enzyme engineering; genetic code expansion; non-canonical amino acid; p-benzoyl-l-phenylalanine.