Crystallization and preliminary X-ray diffraction analysis of fatty-acid hydroxylase cytochrome P450BSβ from Bacillus subtilis
Crystallization and preliminary X-ray diffraction analysis of fatty-acid hydroxylase cytochrome P450BSβ from Bacillus subtilis
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DOI：
10.1107/s0907444902001762
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发表时间：
2002-04-01
影响因子：
2.2
通讯作者：
Shiro, Y
中科院分区：
文献类型：
--
作者：
Lee, DS;Yamada, A;Shiro, Y
从枯草芽孢杆菌中分离到的细胞色素P450(P450BSbeta；MW 48 kDa)以过氧化氢为氧化剂，催化α和β位长链脂肪酸的羟基化。用10%(w/v)PEG4000和50 mM MES pH 6.8的沉淀液，用坐滴气相扩散法合成了无底物形式的P450BSbeta晶体，属于P3(2)21或P3(1)21空间群。在2 mM肉豆蔻酸存在下，由10%PEG4000，0.15 mM醋酸镁和50 mM MES pH 6.5组成的沉淀液中得到了另一种晶型，属于菱面体空间群R3或R32。使用同步辐射，两个P450BSbeta晶体的衍射分辨率都达到了2.5埃。Bijvoet图和色散异常差Patterson图显示了与血红素铁相对应的一个明显的峰。
Cytochrome P450 isolated from Bacillus subtilis (P450BSbeta; MW 48 kDa) catalyzes the hydroxylation of long-chain fatty acids at the alpha and beta positions using H2O2 as an oxidant. Crystals of the substrate-free form of P450BSbeta belonging to the trigonal space group P3(2)21 or P3(1)21 were obtained by the sitting-drop vapour-diffusion method using a precipitate solution consisting of 10%(w/v) PEG 4000 and 50 mM MES pH 6.8. Another crystal form, belonging to the rhombohedral space group R3 or R32, was obtained from precipitate solution consisting of 10% PEG 4000, 0.15 mM magnesium acetate and 50 mM MES pH 6.5 in the presence of 2 mM myristic acid (substrate). Using synchrotron radiation, both P450BSbeta crystals diffracted to 2.5 Angstrom resolution. Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem iron.