Mutational analysis of phenylalanine ammonia lyase to improve reactions rates for various substrates

Abstract

Phenylalanine ammonia lyases (PAL) catalyze the reversible, non-reductive amination of trans-cinnamic acid to l-phenylalanine in the presence of high ammonia concentrations. Since neither cofactor recycling nor other additives are needed and by this asymmetric synthesis theoretical yields of 100% can be reached, it is an interesting reaction for industrial processes. In this study we demonstrate the superior properties of p-nitro-cinnamic acid (p-n-CA) in the amination reaction using the PAL from Petroselinum crispum (pcPAL). By focused-directed evolution, three mutants were identified showing increased reaction rates and decreased substrate inhibition. Together, the F137V mutant with p-n-CA showed a 15-fold increased reaction rate compared with the pcPAL WT with the natural cinnamic acid. The high reaction rates were also proven in preparative scale experiments. Activities towards other p-substituted cinnamic acids showing different electronic effects of the substituent were analyzed. Focused-directed evolution around the carboxylic acid- and amine-binding site always decreased PAL activity, due to a sensitive H-bond network.