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Structural insights into CYP107G1 from rapamycin-producing Streptomyces rapamycinicus.
Archives of Biochemistry and Biophysics ( IF 3.8 ) Pub Date : 2020-08-18 , DOI: 10.1016/j.abb.2020.108544
Vitchan Kim 1 , Young-Ran Lim 1 , Inho Lee 1 , Jong-Ha Lee 1 , Sangjun Han 1 , Tan-Viet Pham 1 , Harim Kim 1 , Rowoon Lee 1 , Lin-Woo Kang 1 , Donghak Kim 1
Affiliation  

Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 μM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.



中文翻译:

产生雷帕霉素的链霉菌雷帕霉素对CYP107G1的结构见解。

雷帕霉素是一种临床上重要的大环内酯类药物,由放线杆菌链霉菌链霉菌产生,具有免疫抑制和抗增殖特性。雷帕霉素的生物合成涉及两种细胞色素P450酶。CYP107G1和CYP122A2分别催化雷帕霉素的C27和C9的氧化反应。为了了解雷帕霉素生物合成中P450酶的结构和生化特性,克隆了CYP107G1CYP122A2基因,并在大肠杆菌中表达了它们的重组蛋白。,并鉴定了纯化的酶。两种酶在铁形式的绝对光谱中均显示出低自旋状态,并且雷帕霉素滴定引起的I型光谱随K d变化CYP107G1和CYP122A2的分别为4.4±0.4和3.0±0.3μM。CYP107G1的X射线晶体结构及其与临床雷帕霉素衍生物依维莫司的共晶复合物的分辨度分别为2.9和3.0。CYP107G1的整体结构采用细胞色素P450的典型支架,并具有较大的底物袋。血红素基团的远端面暴露于溶剂中以适应大环内酯类药物的进入。当将依维莫司结合的CYP107G1复合物(CYP107G1-Eve)的结构与无配体的CYP107G1形式的结构进行比较时,未观察到明显的构象变化。因此,CYP107G1具有相对刚性的结构,带有通用的环以容纳庞大的基板。依维莫司分子以挤压的甜甜圈形状结合到基质结合袋上,

更新日期:2020-08-25
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