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.

Highlights

  • The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme’s unique 1,3-dipolar cycloaddition mechanism and its atypic prenylated flavin mononucleotide (prFMN) cofactor, provided several applications of the ferulic acid decarboxylases (FDCs) 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

  • That compound 1p of the substrate panel was not transformed by wild-type ­FDC7, while for bulky biaryl compounds (1l–1o) no proper active site orientations were obtained by the initial docking predictions

  • We developed a fluorescent phenylalanine ammonia-lyase (PAL) activity assay, that employs FDC as secondary, reporter enzyme, the produced styrene being fluorescently detected upon its reaction with a tetrazole-based fluorogenic p­ robe[51]

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Summary

Introduction

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. The developed cell-plate assay has been employed for the initial, qualitative assessment of the enzyme activities of the FDC mutant library towards the substrate panel, allowing the selection/identification of the best performing variants, based on their relative fluorescence signal intensities (Tables S3–S5, Fig. S2–S17).

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