桃儿七苯丙氨酸解氨酶的基因克隆及其酶活分析

Gene cloning and enzymatic activity analysis of phenylalanine ammonia-lyase from Sinopodophyllum hexandrum (Royle) Ying

摘要:

苯丙氨酸解氨酶（phenylalanine ammonia-lyase，PAL）作为植物苯丙烷类途径的入口关键酶，对抗肿瘤木脂素——鬼臼毒素的生物合成具有重要影响。桃儿七[Sinopodophyllum hexandrum（Royle） Ying]是鬼臼毒素的主要天然来源，以四川阿坝地区桃儿七植株的根为材料，基于桃儿七公共SRA转录组数据包，通过反转录聚合酶链式反应（reverse transcription-polymerase chain reaction，RT-PCR）克隆其苯丙氨酸解氨酶基因ShPAL。生物信息学分析表明，ShPAL编码蛋白由711个氨基酸组成，包含PAL保守结构域，有芳香族氨基酸解氨酶活性中心的特征性基序，二级结构主要由α-螺旋和无规则卷曲组成，单体三级结构呈现典型的“海马状”，Swiss建模空间结构为同源四聚体，在系统发育谱系上与同属小蘖科（Berberidaceae）的三枝九叶草（Epimedium sagittatum） PAL序列相似度最高、进化距离最短。亚细胞定位实验结果显示ShPAL蛋白主要定位于细胞质中、少量定位于内质网膜上。ShPAL蛋白通过大肠杆菌（Escherichia coli）重组表达和组氨酸标签亲和纯化，其酶活高达20.91 U/mg，最适温度为41℃，最适pH为9.0；其F130H突变体酶活降低约23.6%，但随温度、pH变化的趋势不变，证实该位置的苯丙氨酸确为影响PAL底物特异性的残基；两者的热稳定性均较差，但pH稳定性较好。这些结果将有助于进一步分析ShPAL在鬼臼毒素生物合成过程中的调控作用，以及促进鬼臼毒素异源合成，从而保护桃儿七种质资源，同时也表明ShPAL可在生物化工和生物医学方面具有潜在应用价值。

Abstract:

Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical ‘seahorse’ shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.