Expression, purification and structural characterization of resveratrol synthase from Polygonum cuspidatum

文献类型： 外文期刊

第一作者： Dai, Mei

作者： Dai, Mei;Yuan, Daopeng;Li, Jiangtao;Ren, Yangjie;Zhang, Yitong;Gao, Wei;Lei, Yangmei;Tang, Yixiong;Cang, Huaixing

作者机构：

关键词： Resveratrol synthase; Dynamic light scattering; Molecular simulations

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：2.025； 五年影响因子：1.634 ）

ISSN： 1046-5928

年卷期： 2022 年 191 卷

页码：

收录情况： SCI

摘要： Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.

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