Döhr O, Paine M J, Friedberg T, Roberts G C, Wolf C R
Biomedical Research Center, University of Dundee, and Imperial Cancer Research Fund Molecular Pharmacology Unit, Ninewells Hospital and Medical School, Dundee, DD1 9SY, United Kingdom.
Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):81-6. doi: 10.1073/pnas.98.1.81.
A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system.
通过改变人NADPH细胞色素P450还原酶（CPR，P450的氧化还原伴侣）的辅因子偏好，已开发出一种功能性人NADH依赖性细胞色素P450系统。这是通过保守芳香族氨基酸Trp-676的单个氨基酸变化实现的，该氨基酸覆盖了烟酰胺结合位点中FAD异咯嗪环的Re侧。在所进行的突变中，用丙氨酸替代Trp-676（W676A）产生了一种功能性NADH依赖性酶，该酶催化细胞色素c和铁氰化物的还原，并促进CYP1A2对7-乙氧基试卤灵的代谢。测量细胞色素c活性的动力学分析表明，W676A的NADH依赖性k(cat)与野生型酶的NADPH依赖性k(cat)相当（90%），W676A对NADH的特异性高约1000倍。W676A的表观K(M)(NADPH)和K(M)(NADH)值分别降低了80倍和150倍。根据显示NADPH二分结合模式的结构数据，如野生型CPR和W676A突变体的抑制作用所示，Trp-676的替代不影响2'-AMP结合。此外，NADPH是W676A NADH依赖性细胞色素c还原和CYP1A2活性的有效抑制剂。总体而言，结果表明人CPR的Trp-676在辅因子识别中起主要作用，用丙氨酸替代这个保守的芳香族残基可产生一种有效的NADH依赖性细胞色素P450系统。
