Crystal structure of CYP76AH1 in 4-PI-bound state from Salvia miltiorrhiza
Crystal structure of CYP76AH1 in 4-PI-bound state from Salvia miltiorrhiza
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丹参 4-PI 结合态 CYP76AH1 的晶体结构
DOI：
10.1016/j.bbrc.2019.02.103
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发表时间：
2019-04-16
影响因子：
3.1
通讯作者：
Chang, Zhenzhan
中科院分区：
文献类型：
--
作者：
Gu, Mingyue;Wang, Mingzhu;Chang, Zhenzhan
丹参酮是丹参中重要的二萜类次生代谢产物，广泛用作心脑血管药物。CYP76AH1是一种与膜相关的细胞色素P450酶，在丹参酮的生物合成途径中起关键作用。为了明确CYP76AH1的结构与功能之间的关系，我们最近构建了CYP76AH1的表达载体并纯化了该酶。工程CYP76AH1在大肠杆菌Trans-blue细胞中表达，并表现出增强的表达和溶解性。用CO差谱法评价基因工程CYP76AH1的适当折叠。将纯化的CYP76AH1在底物、辅因子NADPH和纯化的SmCPR1（丹参细胞色素P450还原酶）存在的条件下进行酶促反应，对重组酶进行功能鉴定，并对反应提取物进行GC-MS分析。在2.6埃分辨率下测定了CYP76AH1与抑制剂4-苯咪唑（4-PI）的x射线晶体配合物结构。在4-PI结合CYP76AH1的配体结合腔中，抑制剂4-PI与一个在第六个配位上与血红素配位的水分子形成氢键。有两个开放通道，底物和产物位点可以进出活性位点。在CYP76AH1/4-PI络合物结构中，大多数p450结合的咪唑中，4-PI的咪唑环平行于螺旋I，而不是垂直于螺旋I。4-PI可能对CYP76AH1晶体结构的稳定性起作用。这些研究为CYP76AH1的功能表达和纯化，以及CYP76AH1与4-PI络合的整体结构提供了信息。(C) 2019由爱思唯尔公司出版。
Tanshinones are important diterpenoid secondary metabolites from Salvia miltiorrhiza, widely used as cardiovascular and cerebrovascular medicines. CYP76AH1 is a membrane-associated cytochrome P450 enzyme and plays a critical role in the biosynthetic pathway of tanshinones. To clarify the relationship between structure and function of CYP76AH1, we recently constructed the expression vector of CYP76AH1 and purified the enzyme. The engineered CYP76AH1 was expressed in E. coli Trans-blue cells and exhibited enhanced expression and solubility. The proper folding of the engineered CYP76AH1 was assessed by CO difference spectrum assay. Functional identification of the recombinant enzyme was performed by conducting enzymatic reaction with the purified CYP76AH1 in presence of substrate, the co-factor NADPH and the purified SmCPR1 (cytochrome P450 reductase from Salvia miltiorrhiza), and by subsequently analyzing the reaction extract through GC-MS. X-ray crystal complex structure of CYP76AH1 with inhibitor 4-phenylimmidazole (4-PI) was determined at the resolution of 2.6 angstrom. In the ligand-binding cavity of 4-PI bound CYP76AH1, the inhibitor 4-PI forms a hydrogen bound with a water molecule which coordinates with heme at the sixth coordination position. There are two open channels which substrate and product site may access and leave the active site. In the CYP76AH1/4-PI complex structure, the imidazole ring of 4-PI is parallel to helix I instead of perpendicular to helies I in most P450s bound imidazole. 4-PI may be work in the stability of CYP76AH1 crystal structure. These studies provide information on functional expression and purification of CYP76AH1, and overall structure of CYP76AH1 complexed with 4-PI. (C) 2019 Published by Elsevier Inc.