Creation of a (R)-β-Transaminase by Directed Evolution of d-Amino Acid Aminotransferase

Abstract

β-Transaminases (β-TAs) have shown considerable potential as biocatalysts for the synthesis of chiral βand γ-amino acid. Herein, a (R)-β-TA was successfully created by the directed evolution of D-amino acid aminotransferase. The specific activities of created (R)-β-TA for β-phenylalanine (1.74 U/mg) and γ-phenylbutanoic acid (1.67 U/mg) were comparable with those of naturally occurring (S)-β-TAs. Moreover, the designed (R)-β-TA also showed potential for the biocatalytic synthesis of a δ-amino acid with specific activity (0.23 U/mg), which has not yet been explored. Enantiopure (S)-and (R)-forms of various β-, γand δ-amino acids (>99% ee) were synthesized using engineered (R)-β-TA-mediated kinetic resolution and asymmetric synthesis, respectively.

Exemplifying Natural (R)-β-Transamination Potential of Fold Type-IV Transaminase for Kinetic Resolution of rac-β-Amino Acids Synthesized from Aldehydes

“…At the same reaction conditions, the activity of TATT toward 1a (42 mU/mg) was ∼28-fold higher than ( R )-PEA (1.5 mU/mg). In line with our previous reports, mutation to F39 had detrimental effects on both activities, indicating it is highly conserved for the TA activity of fold type-IV TAs (Figure A). , On the other hand, mutation of M103 moderately decreased ( R )-β-TA activity with a 4-fold improvement in ( R )-ATA activity. Mutation to F114 showed slightly improved activities toward both substrates, while S115 remained unaffected by the mutation.…”

“…In our previous study, T242 from DATA played a crucial role in the coordination of the γcarboxylate group of α-ketoglutarate and α-carboxylate group of (R)-β-amino acid. 13 Active site models for DATAs and BCATs differ entirely in regard to substrate acceptance (Figure S15). The γ-carboxylate group is accepted in the O-pocket of the BCATs, while it is accepted in the P-pocket of DATA.…”

“…To demonstrate the broader substrate scope of TATT than reported ( R )-β-TA, we performed kinetic resolution of 10 mM bulkier β-amino acid 1d . TATT completely resolved the 1d into its ( S )-enantiomer, whereas in the case of reported ( R )-β-TA (Mab) the substrate remained unchanged. These results demonstrate that TATT possesses a broader substrate scope than reported ( R )-β-TA.…”

“…To this end, our research group has recently created (R)-β-TA by rational design and the directed evolution of D-amino acid aminotransferase (DATA), which displayed an activity comparable with natural (S)-β-TAs. 13 Unlike its (S)-selective counterparts, the substrate scope of created (R)-β-TA was comparatively narrow and could only resolve the monosubstituted β-amino acids at the p-position. Thus, discovering an (R)-β-TA with a broader substrate scope is highly desired to access a wide range of enantiopure (S)-β amino acids by kinetic resolution.…”

“…Synthesis of ( S )-β-amino acids becomes impossible using this method since ( R )-β-TAs are not naturally occurring. To this end, our research group has recently created ( R )-β-TA by rational design and the directed evolution of d -amino acid aminotransferase (DATA), which displayed an activity comparable with natural ( S )-β-TAs . Unlike its ( S )-selective counterparts, the substrate scope of created ( R )-β-TA was comparatively narrow and could only resolve the monosubstituted β-amino acids at the p -position.…”