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

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.

Site-Specific Incorporation of Photocrosslinking Non-Canonical Amino Acid at Dimeric Interface of the Transaminase from Vibrio fluvialis.

“…2(B)). The expression levels of the mutants containing ncAAs are generally com-promised compared to their native counterparts [35]. However, pBpA mutants of the TAVF showed a comparable expression level with wild-type TAVF as determined by SDS-PAGE (Fig.…”

“…The incorporation of bulky pBpA at the 55 th and 91 st positions might disrupt the proper folding of the enzyme leading to insoluble variants. In many instances, it has been observed that the expression levels and amount of soluble protein may also vary from residue to residue [35]. Therefore, we further generated two more stop codon variants at the 73 rd and 77 th positions, and pBpA was successfully incorporated.…”

Engineering of enzymes using non-natural amino acids

“…Transaminases (TAms) are one of the most promising biocatalysts in organic synthesis for the preparation of chiral amino compounds. Replacement of the Phe88 residue in the active site of ( R )-amine TAm with p -benzoylphenylalanine ( p BzF, 4 ) showed a pronounced improvement in the activity towards 1-phenylpropan-1-amine and benzaldehyde [ 29 ]. The introduction of a single p BzF into the active residue reshaped the size of the active pocket while maintaining hydrophobicity, thus further enhancing the range of substrates.…”

Protein engineering of amine transaminases

“…Incorporation of non-canonical amino acids has been widely used in protein engineering to improve and introduce a new catalytic activity (Drienovská and Roelfes, 2020;Pagar et al, 2021). This rational strategy was applied to expand the substrate scope of ATAs and an (R)-selective ATA that was previously engineered from a D-amino acid transaminase was selected as a scaffold (Voss et al, 2020;Pagar et al, 2022). The active site of this template enzyme is highly hydrophobic, mainly because of three phenylalanine residues (Phe31, Phe86, and Phe88).…”