Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093-0703, USA.
Arch Biochem Biophys. 2010 Feb 15;494(2):151-8. doi: 10.1016/j.abb.2009.11.026. Epub 2009 Nov 26.
Rational mutagenesis was used to improve the thermal stability of human cytochrome P450 2B6 and canine P450 2B11. Comparison of the amino acid sequences revealed seven sites that are conserved between the stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. P334S was the only mutant that showed increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure-perturbation spectroscopy. Compressibility of the heme pocket in variants of all four CYP2B enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterpart. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Improved stability of P334S 2B6 and 2B11 may facilitate the studies of these enzymes by X-ray crystallography and biophysical techniques.
理性突变被用于提高人细胞色素 P450 2B6 和犬细胞色素 P450 2B11 的热稳定性。氨基酸序列的比较揭示了在稳定的 2B1 和 2B4 之间保守的七个位点，但与在不太稳定的 2B6 和 2B11 中发现的位点不同。P334S 是唯一一种在 2B6 和 2B11 中均表现出增加的异源表达水平和热稳定性的突变体。利用压力扰动光谱法探讨了这种效应的机制。在所有四种包含脯氨酸 334 的 CYP2B 酶的变体中，血红素口袋的可压缩性的特征是比其更稳定的丝氨酸 334 对应物的可压缩性更低。因此，P334S 的稳定作用与血红素口袋区域的构象灵活性增加有关。P334S 2B6 和 2B11 的稳定性提高可能有助于通过 X 射线晶体学和生物物理技术研究这些酶。
