Towards a general approach for tailoring the hydrophobic binding site of phenylalanine ammonia-lyases.

Unnatural substituted amino acids play an important role as chiral building blocks, especially for pharmaceutical industry, where the synthesis of chiral biologically active molecules still represents an open challenge. Recently, modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) resulted in specifically tailored PcPAL variants, contributing to a rational design template for PAL-activity enhancements towards the differently substituted substrate analogues. Within this study we tested the general applicability of this rational design model in case of PALs, of different sources, such as from Arabidopsis thaliana (AtPAL) and Rhodosporidium toruloides (RtPAL).

The production of L- and D-phenylalanines using engineered phenylalanine ammonia lyases from Petroselinum crispum.

The biocatalytic synthesis of L- and D-phenylalanine analogues of high synthetic value have been developed using as biocatalysts mutant variants of phenylalanine ammonia lyase from Petroselinum crispum (PcPAL), specifically tailored towards mono-substituted phenylalanine and cinnamic acid substrates. The catalytic performance of the engineered PcPAL variants was optimized within the ammonia elimination and ammonia addition reactions, focusing on the effect of substrate concentration, biocatalyst:substrate ratio, reaction buffer and reaction time, on the conversion and enantiomeric excess values. The optimal conditions provided an efficient preparative scale biocatalytic procedure of valuable phenylalanines, such as (S)-m-methoxyphenylalanine (Y = 40%, ee > 99%), (S)-p-bromophenylalanine (Y = 82%, ee > 99%), (S)-m-(trifluoromethyl)phenylalanine (Y = 26%, ee > 99%), (R)-p-methylphenylalanine, (Y = 49%, ee = 95%) and (R)-m-(trifluoromethyl)phenylalanine (Y = 34%, ee = 93%).