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

Abstract

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). With some exceptions, the results support that the positions of substrate specificity modulating residues are conserved among PALs, thus the mutation with beneficial effect for PAL-activity enhancement can be predicted using the established rational design model. Accordingly, the study supports that tailoring PALs of different origins and different substrate scope, can be performed through a general method. Moreover, the fact that AtPAL variants I461V, L133A and L257V, all outperformed in terms of catalytic efficiency the corresponding, previously reported, highly efficient PcPAL variants, of identical catalytic site, suggests that not only catalytic site differences influence the PAL-activity, thus for the selection of the optimal PAL-biocatalysts for a targeted process, screening of PALs from different origins, should be included.

Engineered, Scalable Production of Optically Pure l-Phenylalanines Using Phenylalanine Ammonia-Lyase from Arabidopsis thaliana

“…Note that the assessment of the intracellular PAL concentration of whole-cell biocatalysts is challenging; therefore, direct comparison of conversion-based catalytic efficiencies of whole-cell biocatalysts and purified PALs should be treated cautiously. In general, the conversion values provided by the purified Pc PALs and the corresponding At PALs are of similar range (Table ), in accordance with their similar k cat values (equivalent to turnover frequencies), determined by enzyme kinetic measurements . However, whole-cell At PALs outperformed their Pc PAL homologues in terms of conversion values (Table ), despite both recombinant PALs being expressed under identical conditions from similar recombinant plasmid constructs, supporting a higher operational stability of the At PAL whole-cell biocatalysts.…”

“…While the catalytic site residues of At PAL and Pc PAL are identical and share high 81% sequence identity, our previous and current study revealed that At PAL variants I461V, L133A, and L257V outperformed in terms of catalytic efficiency (including substrate/product inhibition issues) the corresponding PcPAL variants. Since no structural data for At PAL exists, and our protein crystallization efforts have been hindered by the high aggregation tendency at elevated concentration of the purified enzyme, we can only suppose that structural differences not related to the catalytic site influence the different behavior of the two PALs.…”

“…At the initial stage of our study, we tested the At PAL variants of improved catalytic efficiency in comparison to the corresponding Pc PAL variants within the biotransformation of several monosubstituted cinnamic acids ( 2a – l ) (Figure ), employing the optimal reaction conditions reported for the Pc PAL-catalyzed processes. Besides monitoring the final, stationary conversions (the maximal conversions remaining quasi-stationary in time when using a single batch of biocatalysts) and product enantiomeric excess (ee) values, the effect of substrate concentration on the conversion values of the At PAL-catalyzed reactions was also assessed, since in several cases the corresponding Pc PAL variants were subjected to substrate inhibition .…”

“…Preparation of Whole-Cell Biocatalysts for Enzymatic Reactions: General Procedure. The overnight preculture was prepared in Erlenmeyer flasks containing LB (Luria−Bertani) medium supplemented with carbenicillin (50 μg/mL) and chloramphenicol (30 μg/mL) and inoculated with glycerol stocks of E. coli Rosetta (DE3) pLysS cells harboring the pET-19b vector carrying the wt-or mutant atpal gene, 24 followed by overnight incubation at 37 °C and shaking at 200 rpm. The obtained preculture (5 mL) was further used to inoculate shake flasks (2 L) containing 500 mL of LB.…”

“…23 However, within this gram-scale production of L-phenylalanine analogues, in several cases substrate or product inhibition occurred, hindering its scalability and the development of a cost-effective PAL-based synthesis. 23 Our recent work, by expanding the mutational strategy to PALs from other origins such as Arabidopsis thaliana (AtPAL) and Rhodosporidium toruloides (RtPAL), 24 revealed several AtPAL variants of high catalytic efficiency, which outperformed in terms of conversions their corresponding, previously reported, PcPAL variants. 23 Based on these results, our attention turned toward the applicability of AtPAL variants for the improvement of PAL-based technologies, 23,25 providing rigorously engineered biocatalytic procedures of high efficiency and low environmental footprint, ready to scale up by industrial partners.…”