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).

中文翻译：

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增强催化性能的苯丙氨酸氨分解酶的饱和诱变。

苯丙氨酸氨裂合酶（PAL）是用于非天然苯丙氨酸立体选择性合成的有吸引力的生物催化剂。底物范围扩大的PAL的合理设计突显了Petroselinum crispum PAL残基I460的底物特异性调节剂作用。在本文中，进行了关键残基I460的饱和诱变，以鉴定活性增强的Pc PAL变体或验证合理探索的I460V Pc的优异催化性能PAL与其他可能的突变体相比。经过优化后，利用NNK简并性进行的饱和诱变产生了高质量的转化子文库。对于突变库的高通量酶活性筛选，开发了一种PAL活性测定法，可以鉴定在非天然底物p -MeO-苯丙氨酸的反应中显示活性的命中。在命中中，除了已知的I460V Pc PAL外，还鉴定了一些突变体，并通过使用全细胞或纯化的PAL生物催化剂进行生物转化证实了其提高的催化效率。在对-MeO-Phe的反应中的催化效率方面，变体I460T和I460S优于I460V- Pc PAL 。而且，I460TPc PAL保持了野生型酶对天然底物1- Phe的高特异性常数。分子对接支持的有利基板取向p -MeO-肉桂酸I460T变体的活性位点内，类似地如前所示用于I460V PC PAL（PDB ID：6RGS）。