LACDR/Division of Molecular Toxicology, Department of Pharmacochemistry, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Biochemistry. 2012 Jan 24;51(3):750-60. doi: 10.1021/bi201433h. Epub 2012 Jan 12.
Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16β. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16β-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16β-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2β.
巨大芽孢杆菌细胞色素 P450 BM3 是一种单加氧酶，具有很大的生物技术应用潜力。在本文中，我们展示了经过工程改造的药物代谢 P450 BM3 突变体，作为一种新型工具，可实现甾体化合物在 16β 位的区域选择性羟化。特别是，我们发现通过在 P450 BM3 突变体 M01 和 M11 中的 82 位丙氨酸替换为色氨酸，对睾酮和诺孕酯的 16β-羟化的选择性得到了显著增强。A82W 突变导致这些甾体化合物 16β-羟化的 Vmax 提高了 ≤42 倍。此外，该突变提高了酶的偶联效率，这可能是由于活性位点中水分子的更有效排除所致。在 M11 中的 82 位色氨酸突变体 M11 中，睾酮的底物亲和力至少提高了 9 倍。与 M11 相比，在 M11 A82W 突变体中观察到睾酮的取向发生了变化。在 M11 中，睾酮的 A 环和 D 环质子与血红素铁最接近。在 82 位用色氨酸取代丙氨酸导致 A 环质子-铁距离增加，与 A 环在 2β 位的羟化水平相对降低一致。
