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

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

doi:10.3389/fchem.2022.839636

Pritam Giri, Taresh P Khobragade + Show 7 more

Open Access

https://doi.org/10.3389/fchem.2022.839636

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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.

Highlights

Ω-TAs can accept aliphatic ketones and amines as substrates. ω-TAs can be further divided into two subgroups, β-TAs, and amine transaminases (ATAs), the latter being commonly used as a synonym for all ω-TAs (Rocha et al, 2019)
We report ncAAbased engineering of (R)-ATA previously created from D-amino acid aminotransferases (DATAs) scaffold
The rational design approach based on canonical amino acids (cAAs) limits to create a more hydrophobic environment into the active site as Phe is highly hydrophobic among all cAAs

Summary

Introduction

Chiral amines are valuable and versatile building blocks for the pharmaceutical, agricultural, and fine chemical industries (Mathew and Yun, 2012; Kelly et al, 2018; Patil et al, 2018). Broad substrate scope, ncAA-based (R)-ATA Engineering and no need for external cofactor are the beneficial properties of TAs in an industrial context (Steffen-Munsberg et al, 2015). (R)-ATAs catalyze the transfer of an amino group from (R)-aromatic or (R)-primary aliphatic amines to pyruvate producing ketones or aldehydes and D-Ala (Schätzle et al, 2011; Iwasaki et al, 2012; Sayer et al, 2014; Iglesias et al, 2017; Lakó et al, 2020). Several research groups contributed to this field by identification and characterization of new members and employing protein engineering tools to expand the substrate scope and enhance the thermostability (Iwasaki et al, 2006, 2012; Łyskowski et al, 2014; Guan et al, 2015; Hou et al, 2016; Pavkov-Keller et al, 2016; Bezsudnova et al, 2019; Zeifman et al, 2019; Cheng et al, 2020a; Telzerow et al, 2021). It is of interest to identify, characterize and engineer more (R)-ATAs to provide broad diversity of applications in the chiral amine synthesis

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