State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong 266237, China.
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China.
J Steroid Biochem Mol Biol. 2023 May;229:106268. doi: 10.1016/j.jsbmb.2023.106268. Epub 2023 Feb 9.
Steroid drugs, the second largest class of pharmaceuticals after antibiotics, have shown significant anti-inflammatory, anti-allergic, and endocrine-regulating effects. A group of cytochrome P450 enzymes, namely, CYP11A1 isoenzymes from different organisms are capable of converting cholesterol into pregnenolone, which is a pivotal reaction in both steroid metabolism and (bio)synthetic network of steroid products. However, the low activity of CYP11A1s greatly restricts the industrial application of these cholesterol side-chain cleavage enzymes. Herein, we investigate ten CYP11A1 enzymes of different origins and in vitro characterize two CYP11A1s with a relatively higher expression level from Capra hircus and Sus scrofa, together with the CYP11A1s from Homo sapiens and Bos taurus as references. Towards five selected sterol substrates with different side chain structures, S. scrofa CYP11A1 displays relatively higher activities. Through redox partners combination screening, we reveal the optimal redox partner pair of S. scrofa adrenodoxin and C. hircus adrenodoxin reductase. Moreover, the semi-rational mutagenesis for the active sites and substrate entrance channels of human and bovine CYP11A1s is performed based on comparative analysis of their crystal structures. The mutant mBtCYP11A1-Q377A derived from mature B. taurus CYP11A1 shows a 1.46 times higher activity than the wild type enzyme. These results not only demonstrate the tunability of the highly conserved CYP11A1 isoenzymes, but also lay a foundation for the following engineering efforts on these industrially relevant P450 enzymes.
甾体药物是继抗生素之后第二大类药物，具有显著的抗炎、抗过敏和内分泌调节作用。一组细胞色素 P450 酶，即不同生物体的 CYP11A1 同工酶，能够将胆固醇转化为孕烯醇酮，这是甾体代谢和甾体产物（生物）合成网络中的关键反应。然而，CYP11A1 的低活性极大地限制了这些胆固醇侧链裂解酶在工业中的应用。在此，我们研究了来自不同生物体的 10 种 CYP11A1 酶，并对表达水平相对较高的来自山羊和猪的两种 CYP11A1 酶进行了体外特征分析，同时以人类和牛的 CYP11A1 酶作为参考。对于具有不同侧链结构的 5 种选定甾醇底物，猪 CYP11A1 显示出相对较高的活性。通过氧化还原伴侣组合筛选，我们揭示了猪肾上腺皮质酮和山羊肾上腺皮质酮还原酶的最佳氧化还原伴侣对。此外，还基于人 CYP11A1 和牛 CYP11A1 晶体结构的比较分析，对其活性位点和底物进入通道进行了半理性突变。源自成熟牛 CYP11A1 的突变体 mBtCYP11A1-Q377A 比野生型酶的活性高 1.46 倍。这些结果不仅证明了高度保守的 CYP11A1 同工酶的可调节性，而且为进一步对这些具有工业相关性的 P450 酶进行工程改造奠定了基础。
