Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties

Abstract

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).

Keywords: biocatalysis; phenylalanine ammonia-lyases; protein engineering; saturation mutagenesis.

Figure 1

(a) Saturation mutagenesis using the Megaprimers procedure, representing the gene encoding PcPAL (green), the pET19b-vector backbone (black), and the megaprimers of the different, small, medium, large lengths (blue). In the first PCR stage, the forward mutagenic primer and the reverse, antiprimer anneal to the template resulting the amplified sequences of the corresponding, large, medium, and small-sized megaprimers. In the second PCR stage through annealing of the forward primer and the obtained megaprimers, the mutated plasmid products are obtained. (b) Agarose gel analysis of the PCR products obtained through the different mutagenesis procedures: lanes 1, 2 using the two sets of partially overlapping primers, lane 3 small-megaprimer protocol, lane 4 large-megaprimer protocol, lane 5 medium-megaprimer protocol, lane 6 optimized small-megaprimer protocol, L 10 kb DNA ladder.

Figure 2

Nucleotide-base distribution within randomized position 460 of PcPAL. (a) Theoretical, expected distribution for NNK-degeneracy, with the four bases (A, T, G, C) equally represented in the first two nucleotides, while G, T having the same ratio within the third position); (b) experimental base-distribution from the small megaprimer-transformant library; (c) experimental base-distribution from the transformant library obtained by the partially overlapping primers mutational strategy. For both (a) and (c) cases, the base-distribution was assessed by sequencing the DNA plasmid isolated from the cellular mixture of all colonies pooled from the LB-agar plate of the corresponding transformant library. The G, T, C, A encodes for the corresponding nucleotide bases thymine (T), adenosine (A), cytosine (C), and guanine (G).

Figure 3

Assay optimizations monitoring the relative activity of the different cell lysates of I460V-PcPAL, in the ammonia elimination reaction of p-MeO-Phe. The following lysis buffers have been employed: (a) 1. Tween 20 buffer (50 mM NaH₂PO₄·2H₂O, 300 mM NaCl, 10 mM imidazole, 0.07 mM lysozyme, 400 U/mL DNAse I. 1% Tween20 pH 8), 2. GTE buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris.HCl, pH 8), 3. GTE/Lys. buffer (50 mM glucose, 10 mM EDTA, 0.07 mM lysozyme, 25 mM Tris.HCl, pH 8), 4. 5% glycerol buffer (5 % glycerol, 100 mM NaCl, 1 mM DTT, 50 mM Tris.HCl, pH 7,5), 5. 5% glycerol/Lys. buffer (5 % glycerol, 100 mM NaCl, 1 mM DTT, 0.07 mM lysozyme, 50 mM Tris.HCl, pH 7,5), 6. PMBS/Lys. buffer (0.36 mM PMBS, 0.07 mM lysozyme, 50 mM NaCl, 20 mM Tris.HCl, pH 8.8), 7. Triton X-100 buffer (2% Triton X-100, 50 mM NaCl, 20 mM Tris.HCl, pH 8.8), 8. Triton X-100/PMBS/Lys./Benz. buffer (2% Triton X-100, 0.36 mM PMBS, 0.07 mM lysozyme, 25 U/mL benzonase, 50 mM NaCl, 20 mM Tris.HCl, pH 8.8). (b) Optimizations using the two component PMBS/Lys. buffer and the four-component Triton X-100/PMBS/Lys./Benz. buffer systems with different pHs and/or lysis conditions. Abbreviations: Lys., PMBS, T. X-100 and Benz. denote for lysozyme, polymyxin B sulphate, Triton X-100 and benzonase, correspondingly.

Figure 4

Ammonia elimination from racemic p-MeO-Phe using the induced whole-cells of the corresponding PcPAL variants showing increased activity within the high-throughput activity screens.

Figure 5

(a) The thermal unfolding temperature (T_m) of I460S PcPAL and I460T PcPAL, determined by nanoDSF (Prometheus NT.48). Fluorescence intensity ratios F350/F330 and their first derivative are represented as a function of the applied linear thermal ramp (b) Michaelis–Menten curves obtained by initial reaction velocity measurements using p-MeO-Phe as substrate at different concentration and purified PcPAL variants as catalysts.

Figure 6

View of the catalytic site, including the electrophilic 4-methylideneimidazol-5-one (MIO) prosthetic group; overlay of the (a) p-MeO cinnamic acid and (b) p-MeO-Phe orientations within the catalytic site of I460V and I460T.