An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.
An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.
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DOI：
10.1038/nchembio.1086
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发表时间：
2012-11
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
由于途径中间体的低丰度和动力学不稳定性，许多维生素和辅酶的生物合成常常被证明是难以阐明的。通过对大肠杆菌中钴胺素（维生素B12）途径的一系列重建，并通过his标记反应序列中的末端酶，我们观察到许多不稳定的中间体可以作为紧密结合的酶产物复合物被分离出来。总之，这些方法已被用于提取游离酸形式的预科林斯-4和氢氢酸之间的中间体，并允许描述CobL催化的整个反应，包括正式阐明预科林斯-7作为代谢物。此外，已经鉴定出一种底物载体蛋白CobE，它也可以用来稳定一些瞬时代谢中间体并增强它们的向前转化。途径中间体与酶的紧密关联为代谢物通道的一种形式提供了证据。
The biosynthesis of many vitamins and coenzymes has often proved difficult to elucidate due to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B12) pathway in E. coli, and by His-tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly-bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalysed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, has been identified, which can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.
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DOI：
10.1107/s0907444904016427
发表时间：
2004-12-01
影响因子：
2.2
作者：
Blanc, E;Roversi, P;Bricogne, G
通讯作者：
Bricogne, G
影响因子：
3.2
作者：
BLANCHE, F;THIBAUT, D;CROUZET, J
通讯作者：
CROUZET, J
影响因子：
15
作者：
Lee, Chang Hoon;Dogutan, Dilek K.;Nocera, Daniel G.
通讯作者：
Nocera, Daniel G.
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
影响因子：
2
作者：
PETTERSSON, G
通讯作者：
PETTERSSON, G