Engineering TM1459 for Stabilisation against Inactivation by Amino Acid Oxidation

Abstract

Oxidative alkene cleavage is a highly interesting reaction to obtain aldehydes and ketones. The Mn‐dependent protein TM1459 from Thermotoga maritima can catalyse alkene cleavage of styrene derivatives in the presence of tert‐butyl hydroperoxide. Despite the high thermal stability of the enzyme, it gets inactivated during the reaction. The data reported here indicate that auto‐oxidation is responsible for the low stability of TM1459 in the oxidative environment required for the alkene cleavage reaction. By targeting the exchange of residues prone to oxidation, this phenomenon was successfully prevented. Importantly, the stability to oxidation conveyed by the amino acid exchanges led to increased enzyme activity. However, the exchanges resulted in slightly modified positions of two of the four metal‐binding amino acids, thereby strongly impacting metal binding.

Can a Hydroxynitrile Lyase Catalyze an Oxidative Cleavage?

“…A total of 21 ± 0.3% of rac- 2a was formed after 2 h compared to 14 ± 0.66% for the blank reaction. The potential Tm 1459 catalyzed synthesis of 2a was not further investigated given the low yields and lack of selectivity displayed by either Tm 1459 or Tm 1459-C106L and the large number of selective HNLs available. , The lack of activity and enantioselectivity cannot be ascribed to the absence of a base such as His106 in Gt HNL, as work with copper variants of Tm 1459 has demonstrated successful deprotonation of nitroalkanes and methyl acetoacetate. − …”