Department of Biophysics, School of Basic Medical Sciences, Peking University, Beijing, 100191, China.
Institute of Physical Science and Information Technology, Anhui University, Hefei, 230601, China.
Biochem Biophys Res Commun. 2019 Apr 16;511(4):813-819. doi: 10.1016/j.bbrc.2019.02.103. Epub 2019 Mar 2.
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 Å. 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.
丹参酮是丹参中重要的二萜类次生代谢产物，广泛用于心脑血管药物。CYP76AH1 是一种膜结合细胞色素 P450 酶，在丹参酮的生物合成途径中起着关键作用。为了阐明 CYP76AH1 的结构与功能关系，我们最近构建了 CYP76AH1 的表达载体并对其进行了纯化。该工程 CYP76AH1 在 E. coli Trans-blue 细胞中表达，表达量和可溶性均得到增强。通过 CO 差光谱测定评估了工程 CYP76AH1 的正确折叠。通过用纯化的 CYP76AH1 在底物、辅因子 NADPH 和纯化的 SmCPR1（来自丹参的细胞色素 P450 还原酶）存在下进行酶反应，并通过随后通过 GC-MS 分析反应提取物，对重组酶进行了功能鉴定。CYP76AH1 与抑制剂 4-苯咪唑（4-PI）的 X 射线晶体复合物结构在 2.6Å 的分辨率下确定。在结合 4-PI 的 CYP76AH1 的配体结合腔中，抑制剂 4-PI 形成氢键与水分子配位，水分子与血红素在第六配位位置配位。有两个开放通道，底物和产物部位可能进入和离开活性部位。在 CYP76AH1/4-PI 复合物结构中，4-PI 的咪唑环与 I 螺旋平行，而不是与大多数结合咪唑的 P450s 中的 I 螺旋垂直。4-PI 可能在 CYP76AH1 晶体结构的稳定性中起作用。这些研究提供了 CYP76AH1 的功能表达和纯化以及与 4-PI 结合的 CYP76AH1 总体结构的信息。
