Esteves Francisco, Campelo Diana, Gomes Bruno Costa, Urban Philippe, Bozonnet Sophie, Lautier Thomas, Rueff José, Truan Gilles, Kranendonk Michel
Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Huma Toxicology, NOVA Medical School, Faculty of Medical Sciences, Universidade NOVA de Lisboa, Lisbon, Portugal.
Centre National de la Recherche, Scientifique, Institut National de la Recherche Agronomique, Institut National des Sciences Appliqu es de Toulouse, Toulouse Biotechnology Institute, Universit de Toulouse, Toulouse, France.
Front Pharmacol. 2020 Mar 18;11:299. doi: 10.3389/fphar.2020.00299. eCollection 2020.
NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches - formerly indicated to be involved in CPR:CYP interactions - or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.
烟酰胺腺嘌呤二核苷酸磷酸细胞色素P450氧化还原酶（CPR）是维持微粒体细胞色素P450（CYP）酶活性的必需电子供体。微粒体CYP同工型特异性近端界面的变异性质表明，CPR能够通过不同的结合机制与CYP进行多种退化相互作用以进行电子转移，而这些机制仍未得到充分理解。最近，我们发现CPR的动力学允许形成开放构象，其结构多样的氧化还原伙伴可以以CYP同工型依赖的方式对其进行采样。为了进一步研究CPR黄素单核苷酸（FMN）结构域在CPR与多种受体有效结合中的作用，并阐明潜在的分子机制，我们创建了五个不同的CPR-FMN结构域随机突变文库。筛选这些文库以寻找与特定CYP同工型结合时活性增加的突变体。鉴定出七个CPR-FMN结构域突变体，支持CYP1A2（P117H、G144C、A229T）、2A6（P117L/L125V、G175D、H183Y）或3A4（N151D）活性的增加。使用扩展酶动力学分析、细胞色素竞争、离子强度对CYP活性的影响以及结构分析来评估效果。突变残基位于几个酸性氨基酸序列中或其附近——以前表明这些序列参与CPR:CYP相互作用——或靠近两个推测参与FMN结合的酪氨酸残基。几个已鉴定的位置与天然存在的CPR变体中发现的突变共定位，这些变体先前已显示会导致CYP同工型依赖性效应。这些突变似乎不会显著改变FMN结构域的几何形状，但可能会导致非常细微的改变，从而改善与特定CYP的相互作用。总体而言，这些数据表明CYP使用FMN结构域的几个结合基序的同工型特异性组合与CPR相互作用。
