Liu Kang-Cheng, Hughes John M X, Hay Sam, Scrutton Nigel S
Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM), School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, UK.
FEBS J. 2017 Jul;284(14):2302-2319. doi: 10.1111/febs.14129. Epub 2017 Jun 30.
The haem-containing mono-oxygenase cytochrome P450 3A4 (CYP3A4) and its redox partner NADPH-dependent cytochrome P450 oxidoreductase (CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane-bound in the endoplasmic reticulum (ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady-state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7-benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono-oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4-nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR-CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR-CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity.
含血红素的单加氧酶细胞色素P450 3A4（CYP3A4）及其氧化还原伴侣NADPH依赖性细胞色素P450氧化还原酶（CPR）是人类肝脏中代谢药物和外源性化合物最重要的酶之一。它们在内质网（ER）中与膜结合。内质网的共定位以及复杂的内质网磷脂组成如何影响酶活性，目前尚不清楚。CPR和CYP3A4分别以1:1的比例单独或一起被整合到磷脂双层纳米盘中，以研究膜插入的意义以及不同膜组成对稳态反应动力学的影响。使用以7-苄氧基喹啉为CYP3A4底物的荧光测定法分析反应动力学。只有当CPR和CYP3A4共定位在同一纳米盘中时，单加氧酶系统才能通过CPR从NADPH进行电子转移，从而恢复全部活性。当CPR和CYP3A4分别被整合到纳米盘中然后混合在一起时，或者当CPR的可溶性形式与预先组装好的CYP3A4纳米盘混合时，未观察到活性。因此，膜整合和共定位对于电子转移至关重要。与磷脂酰胆碱相比，肝微粒体脂质对纳米盘中单独的CPR活性具有增强作用，对CPR-CYP3A4纳米盘复合物的活性具有更大的增强作用，而模拟内质网组成的磷脂混合物则没有这种效果。此外，肝脂质增强了系统内的氧化还原偶联。因此，天然内质网脂质具有对CPR-CYP3A4纳米盘复合物增强催化作用重要的特性或包含重要成分。我们的研究结果证明了使用天然脂质制剂对膜蛋白活性进行详细分析的重要性。
