Kumar Santosh, Scott Emily E, Liu Hong, Halpert James R
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031, USA.
J Biol Chem. 2003 May 9;278(19):17178-84. doi: 10.1074/jbc.M212515200. Epub 2003 Feb 27.
The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.
基于细胞色素P450 2C5的X射线晶体结构，对细胞色素P450 2B1催化孕酮羟基化的区域选择性进行了重新设计。2B1是一种高K(m)的孕酮16α-羟化酶，而2C5是一种低K(m)的孕酮21-羟化酶。最初，将2B1活性位点的9个单个残基替换为相应的2C5残基，并从大肠杆菌表达系统中纯化突变体，然后检测其孕酮羟基化活性。在150 μM孕酮浓度下，I114A、F297G和V363L表现出2C5的21-羟化酶活性的5%-15%，而F206V对一种未知产物表现出高活性，且K(m)降低了13倍。因此，构建了一个四重突变体I114A/F206V/F297G/V363L（Q），其表现出2C5的孕酮21-羟化酶活性的60%和57%的区域选择性。基于它们类似2C5的睾酮羟基化谱，将S294D和I477F单独及组合添加到四重突变体中。所有这三个突变体对孕酮21-羟化均表现出增强的区域选择性（70%），而只有Q/I477F具有更高的催化常数（k(cat)）。最后，将其余三个单个突变体V103I、V367L和G478V添加到Q/I477F和Q/S294D/I477F中，得到另外七个多重突变体。其中，Q/V103I/S294D/I477F表现出最高的催化常数（比2C5高3倍）和80%的孕酮21-羟化区域选择性。将孕酮对接至该突变体的三维模型表明，21-羟化是有利的。总之，一种跨亚家族转换P450区域选择性的系统方法表明，活性位点残基主要负责2B1和2C5之间的区域选择性差异，并验证了基于2C5晶体结构的2B1模型的可靠性。
