Engineering TM1459 for Stabilisation against Inactivation by Amino Acid Oxidation

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.

Automated summary

Oxidative alkene cleavage is a promising method for synthesizing aldehydes and ketones. The Mn-dependent protein TM1459 from Thermotoga maritima can undergo alkene cleavage of styrene derivatives in the presence of tert-butyl hydroperoxide. However, the enzyme is prone to inactivation due to auto-oxidation in the oxidative environment required for the cleavage reaction. By replacing oxidizable residues, the stability of TM1459 was improved, leading to increased enzyme activity. However, the residue exchanges also affected metal binding.