Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties

Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of Petroselinum crispum PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PcPAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PcPAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, p-MeO-phenylalanine. Among the hits, besides the known I460V PcPAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-PcPAL in terms of catalytic efficiency within the reaction of p-MeO-Phe. Moreover, I460T PcPAL maintained the high specificity constant of the wild-type enzyme for the natural substrate, l-Phe. Molecular docking supported the favorable substrate orientation of p-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V PcPAL (PDB ID: 6RGS).


Materials
The commercial chemicals and solvents were products of Sigma Aldrich or Alfa-Aesar. The primers used for the mutagenesis were purchased through services of Genomed (Debrecen, Hungary). IPTG, Phusion Hot Start DNA Polymerase, dNTPs, DpnI, XhoI, Bpu1102I, agarose were all products of Thermo Fischer Scientific (Waltham, MA, USA). Plasmid extraction kit and ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). LB medium was from Liofilchem (Roseto, Italy), protease inhibitor cocktail from Hoffman La-Roche (Basel, Switzerland), while the Ni-NTA Superflow resin used for affinity chromatography was from Qiagen (Hilden, Germany).

Instrumentation
The differential scanning fluorimetry (nanoDSF) measurements were performed using Prometheus NT.48 instrument (NanoTemper Technologies, Munich, Germany). High performance liquid chromatography (HPLC) analyses were conducted with Agilent (Santa Clara, CA, USA) 1200, 1260 and 1100 systems. Kinetic measurements were performed on TECAN Spark 10M equipped with a TE-cool module. Mastercyler proS from Eppendorf (Hamburg, Germany) was used to perform the PCR reactions, while the electroporation was carried out using 1 mm cuvettes and an Eppendorf electroporator (Hamburg, Germany). Gene sequencing services were performed through BIOMI Ltd. (Gödöllő, Hungary).

ISM Primers
The PcPAL mutants were obtained through site-saturation mutagenesis. The primers containing the NNK degenerate codons were synthesized by Invitrogen (Germany) and are listed in Table S1 and S2.

Estimation of Parental Content From the Sequencing Chromatogram of the PCR Products
Peak intensities of the four different chromophores representing adenine (A), cytosine (C), guanidine (G) and thymine (T) from each sequencing result were evaluated with Chromas Lite and represented in pie diagrams ( Figure S1) followed by their comparison to the theoretical NNK randomization of position 460 in pET19b_pcpal. Figure S1. Sequencing chromatogram and base distribution for colony pooling after the transformation of the PCR product using (A) partially overlapping primers (B) small megaprimer.

Determination of Conversion Values by HPLC
The conversions of the PcPAL-catalyzed ammonia elimination reactions were calculated using the relative response factor of the 4-MeO-cinnamic acid compared to 4-MeO-Phe, that was determined by injecting the mixture of known composition of the racemic 4-methoxy-phenylalanine and the 4-MeO-cinnamic acid onto Gemini NX-C18 column (150 × 4.5 mm; 5 μm).

Enzyme Kinetics
The kinetic measurements were based on UV-spectroscopy by monitoring the production of the cinnamic acid or 4-methoxy cinnamic acid at 290 nm, where the corresponding amino acids (phenylalanine and 4-methoxy-phenylalanine) showed no absorption.