College of Biological Sciences and Technology, Weifang Medical University, Weifang 261053, People's Republic of China; Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, People's Republic of China.
Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, People's Republic of China.
Enzyme Microb Technol. 2018 May;112:52-58. doi: 10.1016/j.enzmictec.2017.10.010. Epub 2017 Oct 26.
Phenylalanine ammonia-lyase (PAL) gene plays an important role in the synthesis of flavones, lignin, and other bioactive compounds in living organisms. Inonotus baumii, the only known flavone-producing filamentous fungus, is of great importance in the investigation of flavone metabolic pathways. To study the function of PAL enzyme in I. baumii flavone synthesis, a full-length cDNA of pal gene was cloned from I. baumii using DOP-PCR and RACE-PCR. The 2502-bp PAL coding region encodes an 833 amino acid protein with an approximate MW of 88.2kDa. Three introns and four exons are present in the DNA sequence of IbPAL. Amino acid sequence alignment showed that IbPAL shares 76% similarity with PALs of Inonotus fungi. The three-dimensional structure of IbPAL showed that it is composed of an MIO domain, a core domain and an inserted shielding domain. On this basis, the IbPAL was expressed and purified using the prokaryotic expression vector pSMART-V with a 6xHis-tag in Escherichia coli, and its enzymatic activity was subsequently detected. Our results will aid in understanding the enzymatic properties of PAL and further confirm the mechanism of flavone synthesis in I. baumii.
苯丙氨酸解氨酶（PAL）基因在生物合成类黄酮、木质素和其他生物活性化合物中起着重要作用。桦褐孔菌是唯一已知的产生类黄酮的丝状真菌，对于研究类黄酮代谢途径具有重要意义。为了研究 PAL 酶在桦褐孔菌类黄酮合成中的功能，我们使用 DOP-PCR 和 RACE-PCR 从桦褐孔菌中克隆了 pal 基因的全长 cDNA。2502bp 的 PAL 编码区编码一个 833 个氨基酸的蛋白质，分子量约为 88.2kDa。IbPAL 的 DNA 序列中存在三个内含子和四个外显子。氨基酸序列比对表明，IbPAL 与桦褐孔菌的 PAL 具有 76%的相似性。IbPAL 的三维结构表明它由一个 MIO 结构域、一个核心结构域和一个插入的屏蔽结构域组成。在此基础上，我们使用带有 6xHis 标签的原核表达载体 pSMART-V 在大肠杆菌中表达和纯化了 IbPAL，并检测了其酶活性。我们的研究结果将有助于理解 PAL 的酶学特性，并进一步证实桦褐孔菌类黄酮合成的机制。
