Toolbox for the structure-guided evolution of ferulic acid decarboxylase (FDC)

Abstract

The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme's unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC). Accordingly, the developed fluorescent plate-assay allows in premiere the FDC-activity screens of a mutant library in a high-throughput manner. Moreover, using the plate-assay for the activity screens of a rationally designed 23-membered ScFDC variant library against a substrate panel comprising of 16, diversely substituted cinnamic acids, revealed several variants of improved activity. The superior catalytic properties of the hits revealed by the plate-assay, were also supported by the conversion values from their analytical scale biotransformations. The computational results further endorsed the experimental findings, showing inactive binding poses of several non-transformed substrate analogues within the active site of the wild-type ScFDC, but favorable ones within the catalytic site of the variants of improved activity. The results highlight several 'hot-spot' residues involved in substrate specificity modulation of FDC, such as I189, I330, F397, I398 or Q192, of which mutations to sterically less demanding residues increased the volume of the active site, thus facilitated proper binding and increased conversions of diverse non-natural substrates. Upon revealing which mutations improve the FDC activity towards specific substrate analogues, we also provide key for the rational substrate-tailoring of FDC.

Figure 1

(A) substrate panel, including cinnamic acid derivatives 1a–p; (B) active site model of ScFDC (PDB: 4ZAC) accommodating trans-cinnamic acid as substrate, highlighting in blue the residues selected for individual replacement with alanine/valine (C) top view of active site model with respect to the substrate plane: proper substrate binding requires the location of the α − β double bond of the substrate (highlighted in brown) in the proximity of carbon C1’ and C4a atoms of the prFMN cofactor, that facilitates the 1,3-cycloaddition step of the reaction mechanism^,. Hydrogen bonds between residues E280 and R175, as well as Q192 and the cofactor are shown as yellow dashed lines. Softwares used for the preparation of images are listed in Supporting information, Section 1.

Figure 2

Initial FDC activity screens using the fluorescent cell-plate assay allowing rapid identification of enzyme variants with improved activity, that upon selection have been characterized by the conversion values from their analytical scale decarboxylations. Representative results are presented in case of substrate analogue 3,4,5-trimethoxy-cinnamic acid 1i (for details of image preparations see Supplementary Information, Chapter 6) The cell-plate activity screens has been performed on the whole substrate panel 1a–1p (see Fig. S2–S17 and Tables S3–S5 for results of cell plate assays, while their detailed discussions in Sect. 2.3), while the conversions of the biotransformations of 1a–1p were determined by HPLC (Tables S7–S21) (Softwares used for image preparation are listed in Supporting information, Section 1).

Figure 3

HPLC conversion values obtained from the analytical scale biotransformation of the substrate panel 1a–1p, using ScFDC whole-cell biocatalyst. Results of the best performing three variants and of the wild-type ScFDC are shown. Softwares used for the preparation of images are listed in Supporting information, Section 1.

Figure 4

In each figure the side chains of the preserved active site residues of ScFDC1 (green) and the mutant residues (blue) overlaid with their original counterparts from the wild-type enzyme (magenta) are represented as stick models. The substrates 1e, 1i, 1m, 1o are colored in brown within Fig. 4a–d, respectively. Hydrogen bonds between the carboxyl group of the substrate and the backbone nitrogen atom of residue M286 and side chain of residue R175 are indicated as orange dashed lines, whereas the blue dash corresponds to the pi–pi interactions of the substrate’s aromatic ring and residue F440 and the prFMN cofactor, respectively. All these interactions were considered within the selection process of the proper binding state, required for the 1,3-cycloaddition mechanism. Softwares used for the preparation of images are listed in Supporting information, Section 1.