Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana
Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana
复制标题
结构引导操作 Sebekia benihana 环孢菌素 A 羟化酶 CYP-sb21 的区域选择性
DOI：
10.1016/j.synbio.2020.07.004
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发表时间：
2020-09-01
影响因子：
4.8
通讯作者：
Li, Shengying
中科院分区：
文献类型：
--
作者：
Li, Fengwei;Ma, Li;Li, Shengying
来自稀有放线菌Sebekia Benihana的细胞色素P450酶CYP-sb21能够使免疫抑制药物环孢素A(CsA)分子在第4位N-甲基亮氨酸(MeLeu(4))上羟基化，生成伽马-羟基-N-甲基-L-Leu(4)-CsA(CsA-4-OH)。CsA的这种氧化修饰导致免疫抑制活性显著降低，同时保留了促进头发生长的副作用，因此在制药和化妆品行业都显示出巨大的应用潜力。然而，这种P450酶也使CsA在第9位N-甲基亮氨酸(MeLeu(9))的不需要的位置羟化，这表明CsA-4-OH的区域选择性需要提高，才能将CsA-4-OH开发成商品毛发生长刺激剂。在这里，我们报道了CYP-sb21在1.85埃的无底物形式的晶体结构。结合序列和三维结构比较、基于Autodock的底物对接、分子动力学(MD)模拟和定点突变，我们确定了一些关键残基，包括R294、E264和M179，它们可以提高CYP-sb21对CsA的催化效率或改变其区域选择性，为更好地酶法制备CsA-4-OH奠定了基础。这项研究还为适应大型底物的P450酶的底物识别和结合机制提供了新的见解。
The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A (CsA) primarily at the 4th N-methyl leucine (MeLeu(4)), giving rise to gamma-hydroxy-N-methyl-L-Leu(4)-CsA (CsA-4-OH). This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect, thus demonstrating great application potential in both pharmaceutical and cosmetic industries. However, this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine (MeLeu(9)), indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator. Herein, we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85 angstrom. Together with sequence and 3D structure comparisons, Autodock-based substrate docking, molecular dynamics (MD) simulation, and site-directed mutagenesis, we identified a number of key residues including R294, E264, and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA, setting the stage for better enzymatic preparation of CsA-4-OH. This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.