Enzymes as traps in the elucidation of complex biochemical pathways

酶作为阐明复杂生化途径的陷阱

基本信息

  • 批准号:
    BB/I013334/1
  • 负责人:
  • 金额:
    $ 51.55万
  • 依托单位:
  • 依托单位国家:
    英国
  • 项目类别:
    Research Grant
  • 财政年份:
    2012
  • 资助国家:
    英国
  • 起止时间:
    2012 至 无数据
  • 项目状态:
    已结题

项目摘要

In this application we outline a method that will allow a step-change in our ability to study complex biochemical pathways, provide molecular detail on fascinating enzyme mechanisms and to rewrite the metabolic control of pathways involving labile intermediates. The elucidation of biochemical pathways is a challenging area that is often complicated by low levels of inherently unstable metabolic intermediates. We have developed a method that allows for the isolation of enzyme-bound metabolites, permitting their characterisation and thereby providing an opportunity to gain atomic resolution of a number of fascinating enzyme-mediated transformations. The application is based on the finding that in some biochemical pathways the product of one reaction is passed directly onto the next in a process known as substrate channelling. Key to this is a tight association between an enzyme and its product, which allows for the isolation of highly stable enzyme-product complexes. We will exploit these properties to unravel the mysteries surrounding the biosynthesis of vitamin B12 (cobalamin). By using His-tagged enzymes of the pathway it is now possible to isolate many of the hitherto ephemeral intermediates, trapped and stabilised on the tagged enzymes as tightly bound enzyme-product complexes. Characterisation of these intermediates will allow the complete elucidation of the corrin pathway. Moreover, a combination of enzymology and X-ray crystallography will permit a detailed understanding of the mechanism of the enzymes that mediate the synthesis of the corrin framework, including the ring contraction process that involves the extrusion of an integral carbon atom in a reaction that has no parallel in nature. Our preliminary data is consistent with the B12 pathway operating by direct metabolite channelling. We outline experiments to investigate this further and to determine whether enzyme rather than substrate concentration controls this metabolic process.
在这个应用程序中,我们概述了一种方法,将允许我们的能力,以研究复杂的生化途径的一步变化,提供分子细节迷人的酶机制和重写代谢控制的途径,涉及不稳定的中间体。生物化学途径的阐明是一个具有挑战性的领域,通常由于低水平的固有不稳定的代谢中间体而变得复杂。我们已经开发出一种方法,允许酶结合的代谢产物的分离,允许他们的表征,从而提供了一个机会,获得原子分辨率的一些迷人的酶介导的转换。这一应用是基于这样的发现,即在某些生化途径中,一个反应的产物在一个称为底物通道的过程中直接传递到下一个反应中。其关键是酶与其产物之间的紧密结合,这允许分离高度稳定的酶-产物复合物。我们将利用这些特性来揭开维生素B12(钴胺素)生物合成的奥秘。通过使用该途径的His标记的酶,现在可以分离许多迄今为止短暂的中间体,这些中间体被捕获并稳定在标记的酶上作为紧密结合的酶-产物复合物。这些中间体的特性将允许咕啉途径的完整阐明。此外,酶学和X射线晶体学的结合将允许详细了解介导咕啉框架合成的酶的机制,包括环收缩过程,该过程涉及在自然界中没有平行反应的完整碳原子的挤出。我们的初步数据与B12途径通过直接代谢物通道运作一致。我们概述了实验,以进一步研究这一点,并确定是否酶,而不是底物浓度控制这一代谢过程。

项目成果

期刊论文数量(7)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Anatomy of secretin binding to the Dickeya dadantii type II secretion system pilotin.
An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.
  • DOI:
    10.1038/nchembio.1086
  • 发表时间:
    2012-11
  • 期刊:
  • 影响因子:
    14.8
  • 作者:
  • 通讯作者:
FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR).
FAD结合,Corin还原酶(COBR)中的核酰胺结合和主动位点通信。
  • DOI:
    10.1042/bsr20140060
  • 发表时间:
    2014-07-04
  • 期刊:
  • 影响因子:
    4
  • 作者:
    Lawrence AD;Taylor SL;Scott A;Rowe ML;Johnson CM;Rigby SE;Geeves MA;Pickersgill RW;Howard MJ;Warren MJ
  • 通讯作者:
    Warren MJ
The structure, function and properties of sirohaem decarboxylase--an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway.
  • DOI:
    10.1111/mmi.12656
  • 发表时间:
    2014-07
  • 期刊:
  • 影响因子:
    3.6
  • 作者:
    Palmer DJ;Schroeder S;Lawrence AD;Deery E;Lobo SA;Saraiva LM;McLean KJ;Munro AW;Ferguson SJ;Pickersgill RW;Brown DG;Warren MJ
  • 通讯作者:
    Warren MJ
Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap.
  • DOI:
    10.1038/srep16943
  • 发表时间:
    2015-11-30
  • 期刊:
  • 影响因子:
    4.6
  • 作者:
    Gu S;Sushko O;Deery E;Warren MJ;Pickersgill RW
  • 通讯作者:
    Pickersgill RW
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Richard Pickersgill其他文献

Richard Pickersgill的其他文献

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{{ truncateString('Richard Pickersgill', 18)}}的其他基金

Structure of the assembly platform of the bacterial type II secretion system
II型细菌分泌系统组装平台的结构
  • 批准号:
    BB/W006693/1
  • 财政年份:
    2022
  • 资助金额:
    $ 51.55万
  • 项目类别:
    Research Grant
Cryo-electron microscope for structural and cell biology
用于结构和细胞生物学的冷冻电子显微镜
  • 批准号:
    BB/R000514/1
  • 财政年份:
    2017
  • 资助金额:
    $ 51.55万
  • 项目类别:
    Research Grant
Elucidation and evolution of substrate recognition and reaction mechanism in the methyltransferases of cobalamin biosynthesis
钴胺素生物合成甲基转移酶底物识别和反应机制的阐明和进化
  • 批准号:
    BB/E002137/1
  • 财政年份:
    2007
  • 资助金额:
    $ 51.55万
  • 项目类别:
    Research Grant

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