The Characterization of an Efficient Phenylpyruvate Decarboxylase KDC4427, Involved in 2-Phenylethanol and IAA Production from Bacterial Enterobacter sp. CGMCC 5087

• Published in: Microbiology spectrum Vol. 10; no. 2; p. e0266021
• Main Authors: Bao, Wenzhi, Li, Xing, Liu, Jinfeng, Zheng, Rong, Liu, Lijuan, Zhang, Haibo
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 27.04.2022
• Subjects: 2-phenylethanol; Applied and Industrial Microbiology; Carboxy-Lyases - chemistry; Carboxy-Lyases - genetics; Carboxy-Lyases - metabolism; Enterobacter - genetics; Enterobacter - metabolism; Glutamates; Indoleacetic Acids; indolepyruvate decarboxylase; Isoleucine; KDC4427; Leucine; Phenylethyl Alcohol; phenylpyruvate decarboxylase; Phylogeny; Research Article; 2-phenylethanol; KDC4427; phenylpyruvate decarboxylase; indolepyruvate decarboxylase
• DOI: 10.1128/spectrum.02660-21
• ISSN: 2165-0497

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.