Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
Shandong Energy Institute, Qingdao, China.
Microbiol Spectr. 2022 Apr 27;10(2):e0266021. doi: 10.1128/spectrum.02660-21. Epub 2022 Apr 4.
Phenylpyruvate decarboxylase (PPDC) is a crucial enzyme that plays important roles in 2-phenylethanol (2-PE) biosynthesis. In our previous study, we screened a highly efficient PPDC KDC4427 from the novel 2-PE-producing strain Enterobacter sp. CGMCC 5087. Meanwhile, its decarboxylation activity of indolylpyruvate (IPyA) was also higher than other indolylpyruvate decarboxylases (IPDCs) reported so far. In this study, KDC4427 protein was purified and characterized, and its catalytic mechanisms were analyzed by biological methods. The optimum pH and temperature of KDC4427 was pH 6.5 and 35°C, respectively. The enzyme activity was relatively stable between pH 6 and 8 and over the range of temperatures from 25°C to 45°C. KDC4427 showed the highest catalytic efficiency on phenylpyruvic acid (PPA); meanwhile, it also showed high activity for IPyA and 2-ketobutanoic acid, and it was found that KDC4427 belongs to IPDCs by phylogenetic tree analysis. The coverage of the three-dimensional structure of KDC4427 and IPDC from Enterobacter cloacae was 96%. Leucine 542, one of the residues in the substrate-binding pocket, is replaced by isoleucine in KDC4427 compared with IPDC. Site-directed mutagenesis showed that the transition from leucine to isoleucine was unlikely to make KDC4427 have high catalytic activity for PPA and IPyA; the mutants at glutamate 468 almost completely lost catalytic activities for both PPA and IPyA, indicating that this glutamate was essential for the catalytic activity. Additionally, alanine 387 plays an important role in the substrate selectivity of KDC4427. Compared with the chemical synthesis of 2-phenylethanol (2-PE) by condensation of ethylene oxide and benzene, the biological synthesis of 2-PE is a potential method to replace the traditional process. This makes biotransformation gradually become the main way to produce high-quality 2-PE. Phenylpyruvate decarboxylase (PPDC) is the critical enzyme in 2-PE biosynthesis, and it is a momentous point of penetration to increase the production of 2-PE. In this regard, KDC4427 can catalyze phenylpyruvic acid (PPA) to phenylacetaldehyde more efficiently than any other PPDC previously reported. Moreover, it has high activity of indolepyruvate decarboxylases (IPDCs), which will be a great breakthrough in the synthesis of indole-3-acetic acid (IAA). With this study, we offer insights into the KDC4427 catalytic mechanism and significantly expand the toolbox of available α-ketoacid decarboxylases for application in biosynthesis.
苯丙酮酸脱羧酶（PPDC）是一种重要的酶，在 2-苯乙醇（2-PE）生物合成中发挥重要作用。在我们之前的研究中，我们从新型 2-PE 产生菌株肠杆菌属 CGMCC 5087 中筛选出一种高效的 PPDC KDC4427。同时，其对吲哚丙酮酸（IPyA）的脱羧活性也高于迄今为止报道的其他吲哚丙酮酸脱羧酶（IPDCs）。在本研究中，对 KDC4427 蛋白进行了纯化和表征，并通过生物方法分析了其催化机制。KDC4427 的最适 pH 值和温度分别为 pH6.5 和 35°C。酶活性在 pH6 到 8 之间以及 25°C 到 45°C 的温度范围内相对稳定。KDC4427 对苯丙酮酸（PPA）表现出最高的催化效率；同时，它对 IPyA 和 2-酮丁酸也表现出较高的活性，通过系统发育树分析发现它属于 IPDCs。KDC4427 和肠杆菌 cloacae 的 IPDC 的三维结构覆盖率为 96%。与 IPDC 相比，KDC4427 中底物结合口袋中的残基亮氨酸 542被异亮氨酸取代。定点突变表明，从亮氨酸到异亮氨酸的转变不太可能使 KDC4427 对 PPA 和 IPyA 具有高催化活性；谷氨酸 468 的突变体几乎完全失去了对 PPA 和 IPyA 的催化活性，表明该谷氨酸对催化活性至关重要。此外，丙氨酸 387 在 KDC4427 的底物选择性中起重要作用。与环氧乙烷和苯缩合合成 2-苯乙醇（2-PE）的化学合成相比，2-PE 的生物合成是替代传统工艺的潜在方法。这使得生物转化逐渐成为生产高质量 2-PE 的主要途径。苯丙酮酸脱羧酶（PPDC）是 2-PE 生物合成的关键酶，提高 2-PE 的产量是一个重要的突破点。在这方面，KDC4427 可以比以前报道的任何其他 PPDC 更有效地催化苯丙酮酸（PPA）生成苯乙醛。此外，它具有较高的吲哚丙酮酸脱羧酶（IPDC）活性，这将是吲哚-3-乙酸（IAA）合成的重大突破。通过这项研究，我们深入了解了 KDC4427 的催化机制，并极大地扩展了可用的α-酮酸脱羧酶工具箱，以应用于生物合成。
