Expression, purification, and crystallization of a plant polyketide cyclase from Cannabis sativa
Expression, purification, and crystallization of a plant polyketide cyclase from Cannabis sativa
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大麻植物聚酮化合物环化酶的表达、纯化和结晶
DOI：
10.1107/s2053230x15020385
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
H. Morita
中科院分区：
文献类型：
--
作者：
X. Yang;T. Matsui;T. Mori;F. Taura;H. Noguchi;I. Abe;H. Morita
植物聚酮化合物是一类结构多样的天然产物。在植物聚酮化合物的生物合成中，碳环骨架的构建是聚酮化合物结构多样化的关键步骤。来自大麻的橄榄油酸环化酶（OAC）。是唯一已知的植物聚酮环化酶，其催化线性戊基四-β-酮-CoA的C2-C7分子内羟醛环化以在大麻素的生物合成中产生橄榄酚酸。该酶也被认为属于二聚体α+β桶（DABB）蛋白家族。然而，由于缺乏对其他植物DABB蛋白的功能分析以及与功能不同的细菌DABB蛋白的低序列同一性，OAC的催化机制仍然不清楚。为了阐明OAC的密切催化机制，该酶在大肠杆菌中过表达，并使用气相扩散法结晶。晶体衍射X射线分辨率为1.40 μ m，属于空间群P3121或P3221，晶胞参数a = B = 47.3，c = 176.0 μ m。 进一步的晶体学分析将为OAC的结构-功能关系和催化机理提供有价值的见解。
Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2–C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure–function relationship and catalytic mechanism of OAC.