Expanding the Toolbox of R‐Selective Amine Transaminases by Identification and Characterization of New Members

Abstract

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R‐selective ATAs belong to the fold type IV PLP‐dependent enzymes, and different sequence‐, structure‐ and substrate scope‐based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)‐ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)‐ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)‐ATAs. The ATA from Exophiala sideris contains a motif characteristic for d‐ATAs, which was previously believed to be a disqualifying factor for (R)‐ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram‐negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)‐ATAs with a shortened/missing N‐terminal helix. The active‐site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.

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

“…( 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 ). Thermodynamically, deamination of amines is preferable; nonetheless, amination of ketones is possible if a sufficient strategy to shift the equilibrium to amine synthesis is utilized ( Schätzle et al, 2011 ; Iglesias et al, 2017 ; Telzerow et al, 2019 , 2021 ). 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 ).…”

“…Thermodynamically, deamination of amines is preferable; nonetheless, amination of ketones is possible if a sufficient strategy to shift the equilibrium to amine synthesis is utilized ( Schätzle et al, 2011 ; Iglesias et al, 2017 ; Telzerow et al, 2019 , 2021 ). 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 ). For instance, ( R )-ATA from Exophiala xenobiotica has been identified to synthesize biaryl amines, which are considered privileged scaffolds for pharmaceuticals ( Telzerow et al, 2019 ).…”

“…( R )-ATAs are not the only fold type IV PLP dependent enzymes. ( Telzerow et al, 2021 ). d -amino acid aminotransferases (DATAs), l -branched chain aminotransferases (BCATs), and 4-Amino-4-deoxychorismate lyases (ADCLs) hold an overall structural similarity, therefore, belong to the same family ( Telzerow et al, 2021 ).…”

“…( Telzerow et al, 2021 ). d -amino acid aminotransferases (DATAs), l -branched chain aminotransferases (BCATs), and 4-Amino-4-deoxychorismate lyases (ADCLs) hold an overall structural similarity, therefore, belong to the same family ( Telzerow et al, 2021 ). Still, their amino acid sequence, activity, stereo preference, and substate scope considerably differ ( Bezsudnova et al, 2020 ).…”

Characterization of proteins from the 3N5M family reveals an operationally stable amine transaminase

“…These patterns — also referred to as active site fingerprints — can be compared easily with a given protein of unknown function to guide its annotation. A variety of different ( S )- and ( R )-selective wild-type ATA were identified by this approach from protein databases or metagenomic resources (Baud et al 2017 ; Ferrandi et al 2017 ; Gao et al 2017 ; Iglesias et al 2017 ; Kelly et al 2018 ; Leipold et al 2018 ; Pawar et al 2018 ; Seo et al 2012 ; Wilding et al 2015 ; Wu et al 2017 ; Zhai et al 2019 ; Telzerow et al 2021 ; Wang et al 2021 ).…”