Uno T, Mitchell E, Aida K, Lambert M H, Darden T A, Pedersen L G, Negishi M
Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Biochemistry. 1997 Mar 18;36(11):3193-8. doi: 10.1021/bi962654j.
Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7alpha, 7beta, and 2alpha). An individual P450, however, exhibits high specificity to one of these products. Using site-directed mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7beta-hydroxylase activity, whereas Val at this position defines the 2alpha-hydroxylase activity. Leu at position 209 is essential for high DHEA 7alpha-hydroxylase activity. The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 also depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.
总体而言，细胞色素P450 2a4/2a5系统可在至少三个位置（7α、7β和2α）将脱氢表雄酮（DHEA）羟化。然而，单个细胞色素P450对这些产物之一表现出高度特异性。通过对来自野生小鼠小家鼠的mP450 2a5进行定点诱变和细菌表达，我们已将117、209和481位残基的功能与在每种细胞色素P450中观察到的各自特异性联系起来。117位的丙氨酸决定7β-羟化酶活性，而该位置的缬氨酸则决定2α-羟化酶活性。209位的亮氨酸对高DHEA 7α-羟化酶活性至关重要。用各种疏水氨基酸取代481位残基引发了特定羟化速率的深刻改变，但不影响DHEA三个位置中任何一个位置的区域和立体特异性。481位残基引起的改变也取决于117或209位的残基特性。结果表明，几个关键残基的大小遵循协同的相互关系，由此细胞色素P450的底物口袋进行调整以容纳DHEA。一项有限的分子建模研究成功地将DHEA结合与一系列关键位置突变体的实验性DHEA羟化酶活性相关联。
