Abdulmughni Ammar, Jóźwik Ilona K, Brill Elisa, Hannemann Frank, Thunnissen Andy-Mark W H, Bernhardt Rita
Department of Biochemistry, Saarland University, Saarbrücken, Germany.
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
FEBS J. 2017 Nov;284(22):3881-3894. doi: 10.1111/febs.14276. Epub 2017 Oct 16.
Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D (VD ) metabolites are of pharmaceutical importance and are applied for the treatment of VD deficiency and other disorders. However, the chemical synthesis of VD derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24 h and allowed to identify the main product by NMR analysis as 25-hydroxylated VD . Product yield amounted to 54.9 mg·L ·day , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for VD binding as the related VD hydroxylases CYP109E1 from B. megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of VD hydroxylation.
The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate-free CYP109A2).
Cytochrome P450 monooxygenase CYP109A2, EC 1.14.14.1, UniProt ID: D5DF88, Ferredoxin, UniProt ID: D5DFQ0, cytochrome P450 reductase, EC 1.8.1.2, UniProt ID: D5DGX1.
细胞色素P450酶因其作为生物催化剂在温和条件下具有高区域选择性和/或立体选择性的潜在应用而受到越来越多的研究。维生素D（VD）代谢产物具有药物重要性，可用于治疗VD缺乏症和其他疾病。然而，VD衍生物的化学合成显示出低特异性和低产率。在本研究中，来自巨大芽孢杆菌DSM319的细胞色素P450 CYP109A2被表达、纯化，并显示出对VD具有高区域选择性氧化作用。使用来自同一菌株的细胞色素P450还原酶（BmCPR）和铁氧还蛋白（Fdx2）进行的体外转化显示出典型的米氏反应动力学。在过表达CYP109A2的巨大芽孢杆菌中的全细胞系统在24小时后转化率达到76±5%，并通过核磁共振分析确定主要产物为25-羟基化VD。产物产量为54.9 mg·L·天，使所建立的全细胞系统成为生产这种有价值代谢产物的极具前景的生物催化途径。无底物CYP109A2的晶体结构在2.7 Å分辨率下测定，显示出开放构象。结构分析预测，CYP109A2与来自巨大芽孢杆菌的相关VD羟化酶CYP109E1和来自自养诺卡氏菌的CYP107BR1（Vdh）一样，使用一组高度相似的残基进行VD结合。然而，这三种P450中BC环的折叠和序列高度不同，导致它们活性位点口袋的形状和非极性/极性表面分布存在差异，这可能解释了观察到的底物特异性差异和VD羟基化的区域选择性。
原子坐标和结构因子已存入蛋白质数据库，登录号为5OFQ（无底物CYP109A2）。
细胞色素P450单加氧酶CYP109A2，EC 1.14.14.1，UniProt ID：D5DF88，铁氧还蛋白，UniProt ID：D5DFQ0，细胞色素P450还原酶，EC 1.8.1.2，UniProt ID：D5DGX1。
