Discovery and evolution of an (R)-selective transaminase for production of sitagliptin

Abstract

We report the discovery and engineering of a new (R)-selective transaminase (RTA-223, UniProt W9Z089), identified from Capronia coronata. The wild-type enzyme was found to display broad activity on a range of bulky aryl and alkyl ketone substrates, with high enantioselectivity using both d-alanine and isopropyl amine as amine donors. We then investigated activity towards pro-sitagliptin ketone, a well-known challenging pharmaceutical target of industrial significance. Although no forward amination of pro-sitagliptin was initially detected by wild-type RTA-223, low-level activity in the reverse deamination reaction enabled engineering without the need for truncated sitagliptin analogues. Adopting this reverse screening strategy, two active-site mutations unlocked forward amination activity, and subsequent rounds of directed evolution delivered a quadruple mutant (H53L/V60G/F113A/V148A) capable of converting pro-sitagliptin to (R)-sitagliptin with high levels of stereoselectivity (e.r. 93 : 7). We further performed a kinetic analysis of the candidates from across the evolution process, using a previously reported coupled assay system linked to d-alanine oxidation. This work validates reverse screening as an effective approach for evolving transaminases toward sterically hindered substrates and highlights RTA-223 as a promising candidate for further biocatalyst development.