Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst.
Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst.
复制标题
DOI：
10.1002/(sici)1521-3765(20000502)6:9
复制
发表时间：
2000-05
期刊：
影响因子：
--
通讯作者：
Qing-Shan Li;Ulrich Schwaneberg;Peter Fischer;Rolf D. Schmid
中科院分区：
文献类型：
--
作者：
Qing-Shan Li;Ulrich Schwaneberg;Peter Fischer;Rolf D. Schmid
巨大芽孢杆菌的自给细胞色素P450 BM-3酶催化饱和长链脂肪酸和结构相关化合物的亚末端羟基化。由于P450 BM-3的一级结构与哺乳动物P450 II型的一级结构同源，因此它是这个酶家族的一个很好的模型。在P450BM-3定向进化为中链脂肪酸羟基酶的研究中，观察到几个突变体，特别是三重突变体Phe87Val，Leu188Gln，Ala74Gly，在1365M(-1)S(-1)(kcat=2.73 S(-1)，Km=2.0 mM)的催化下，对吲哚进行了羟化反应，生成了靛蓝和靛玉红。两种产物均经核磁共振和质谱分析确证。野生型P450 BM-3不能羟化吲哚。这些结果表明，可以设计一种酶来催化底物的转化，其结构与其天然底物的结构大相径庭。
The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.