Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János Street 11, 400028, Cluj-Napoca, Romania.
Sci Rep. 2022 Mar 1;12(1):3347. doi: 10.1038/s41598-022-07110-w.
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.
人们对阿魏酸脱羧酶（FDC）产生了浓厚的兴趣，这主要是因为该酶具有独特的 1,3-偶极环加成机制和非典型的 prFMN 辅因子。FDC 介导的脱羧反应具有多种应用，例如合成苯乙烯或其各种衍生物，包括 1,3-丁二烯，以及通过反向卡罗利加反应酶促激活 C-H 键。虽然基于合理设计的蛋白质工程已成功用于针对各种感兴趣的底物对 FDC 进行剪裁，但缺乏高通量的 FDC 活性测定方法阻碍了其基于定向进化的蛋白质工程。在此，我们报告了一个工具箱，可用于基于定向进化和/或结构导向的 FDC 蛋白质工程，该工具箱已在描述良好的源自酿酒酵母（ScFDC）的 FDC 上进行了代表性验证。相应地，开发的荧光板测定法可首次以高通量方式对突变文库进行 FDC 活性筛选。此外，使用该板测定法对包含 16 种不同取代肉桂酸的底物谱对经过合理设计的 23 个成员 ScFDC 变体文库的活性筛选，揭示了几种活性提高的变体。该板测定法揭示的活性提高的变体的优异催化性能，也得到了其分析规模生物转化转化率的支持。计算结果进一步证实了实验结果，表明野生型 ScFDC 活性位点中几个未转化的底物类似物的结合构象是无活性的，而在活性提高的变体的催化位点中则是有利的。结果突出了几个涉及 FDC 底物特异性调节的“热点”残基，例如 I189、I330、F397、I398 或 Q192，将这些残基突变为空间要求较低的残基会增加活性位点的体积，从而有利于各种非天然底物的正确结合和转化率的提高。在揭示哪些突变可以提高 FDC 对特定底物类似物的活性的同时，我们还为 FDC 的合理底物剪裁提供了关键信息。
