Engineering nickel supply to cyanobacterial hydrogenase to test the relationship between enzyme metallation and metal-sensing

对蓝藻氢化酶进行工程镍供应，以测试酶金属化和金属传感之间的关系

• 批准号: BB/K00817X/1
• 负责人: Nigel Robinson
• 金额: $ 41.05万
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK00817X%2F1
• 关键词: Engineering; nickel; supply; cyanobacterial; hydrogenase

A large proportion of the proteins that biotechnology aims to exploit, need metals. Such proteins somehow acquire the correct metals inside cells. Efforts in synthetic biology (for example to engineer organisms for the sustainable manufacture of compounds for industry) must consider how to also engineer an adequate supply of metal cofactors, because so much of biological catalysis is driven by metals. Hypotheses about how cells help proteins to acquire the correct metals have been introduced for a general audience in a podcast which can be downloaded from the Nature web-site (search for 'nature podcast pick your partners') linked to review article (Nature (2009) 460, 823-830). This research will test these hypotheses. There is a tendency for proteins to form partnerships with unsuitable metals. It is thought that cells maintain each metal at optimal buffered concentrations to overcome this challenge. It is hypothesised that the most competitive metals are kept at the lowest buffered concentrations. Under this regime proteins compete with other proteins for the competitive metals, rather than metals competing with other metals for a limited pool of proteins. If this model is true, then the mechanisms which maintain the correct buffered concentrations of each metal are vital for the fidelity of metal co-factoring of a large proportion of proteins. It raises the possibility that the buffered metal levels may change from cell to cell with widespread implications for metabolism. Moreover, the model suggests an opportunity to engineer optimal metal-levels to assist co-factoring of useful proteins in, for example, synthetic biology.Metal sensor proteins detect fluctuations in the buffered concentrations of metals in the cytosol. Our previous BBSRC-funded research has discovered multiple metal-sensors and has identified factors that influence which metals bind and trigger their sensing-mechanisms. Critically, it is hypothesised that there is a direct relationship between the affinity of such sensors for the detected metal and the intracellular buffered metal concentration. This hypothesis will be tested for the detection of nickel. This research aims to engineer the supply of nickel to an enzyme relevant to bioenergy in a representative of a group of organisms significant for green technology.Hydrogenase is one of a small number (probably less than twenty types in the biosphere) of enzymes that require nickel. Under the correct conditions this enzyme can generate di-hydrogen gas and hence might contribute to a hydrogen economy. Photosynthetic organisms, including cyanobacteria, can be exploited to use energy from sunlight to produce useful compounds including carriers of energy such as hydrogen, and thus have potential to create sustainable processes. However, hydrogenase activity is limited by poor nickel supply in cyanobacteria. We have recently (with support from BBSRC) discovered a nickel sensor in the cytosol of a cyanobacterium (Journal of Biological Chemistry (2012) 287, 12142-12151). This discovery creates an opportunity to engineer metal-homeostasis with the aim of optimising nickel supply to hydrogenase in a cyanobacterium.

生物技术所要开发的大部分蛋白质都需要金属。这些蛋白质以某种方式在细胞内获得正确的金属。合成生物学的努力（例如，为工业用化合物的可持续生产而设计生物体）必须考虑如何同时设计金属辅助因子的充足供应，因为很多生物催化都是由金属驱动的。关于细胞如何帮助蛋白质获得正确金属的假设已经在一个播客中介绍给普通观众，这个播客可以从自然网站下载（搜索“自然播客选择你的伙伴”），链接到评论文章（自然（2009）460,823 -830）。这项研究将检验这些假设。蛋白质有与不合适的金属形成伙伴关系的倾向。人们认为细胞将每种金属维持在最佳缓冲浓度以克服这一挑战。据推测，最具竞争力的金属保持在最低的缓冲浓度。在这种制度下，蛋白质与其他蛋白质竞争竞争性金属，而不是金属与其他金属竞争有限的蛋白质池。如果这个模型是正确的，那么维持每种金属的正确缓冲浓度的机制对于大量蛋白质的金属协同分解的保真度至关重要。它提出了一种可能性，即缓冲金属水平可能在细胞之间变化，对新陈代谢有广泛的影响。此外，该模型还提供了一个设计最佳金属水平的机会，以协助合成生物学中有用蛋白质的协同分解。金属传感器蛋白检测细胞质中缓冲金属浓度的波动。我们之前由bbsrc资助的研究发现了多种金属传感器，并确定了影响金属结合和触发其传感机制的因素。关键的是，假设这种传感器对被检测金属的亲和力与细胞内缓冲金属浓度之间存在直接关系。这一假设将在镍的检测中得到验证。本研究旨在设计一种与生物能相关的酶的镍供应，这种酶是一组对绿色技术具有重要意义的生物的代表。氢化酶是需要镍的少数几种酶（生物圈中可能不到20种）之一。在适当的条件下，这种酶可以产生二氢气体，因此可能有助于氢经济。包括蓝藻在内的光合生物可以利用来自阳光的能量来生产有用的化合物，包括氢等能量载体，因此有可能创造可持续的过程。然而，氢化酶活性受到蓝藻中镍供应不足的限制。我们最近（在BBSRC的支持下）在一种蓝藻的细胞质中发现了一种镍传感器（Journal of Biological Chemistry(2012) 287, 12142-12151）。这一发现为设计金属稳态创造了机会，其目的是优化蓝藻中氢化酶的镍供应。

项目成果

A silk purse from a sow's ear-bioinspired materials based on a-helical coiled coils.

一款丝绸钱包，采用母猪耳朵仿生材料制成，基于非螺旋线圈。

• DOI: 10.1016/j.ceb.2014.12.010
• 发表时间: 2015
• 期刊: Current opinion in cell biology
• 影响因子: 7.5
• 作者: Quinlan RA

A tight tunable range for Ni(II) sensing and buffering in cells.

• DOI: 10.1038/nchembio.2310
• 发表时间: 2017-04
• 期刊: Nature chemical biology
• 影响因子: 14.8
• 作者: Foster AW;Pernil R;Patterson CJ;Scott AJP;Pålsson LO;Pal R;Cummins I;Chivers PT;Pohl E;Robinson NJ
• 通讯作者: Robinson NJ

A unique ferredoxin acts as a player in the low-iron response of photosynthetic organisms.

• DOI: 10.1073/pnas.1810379115
• 发表时间: 2018-12-18
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Schorsch M;Kramer M;Goss T;Eisenhut M;Robinson N;Osman D;Wilde A;Sadaf S;Brückler H;Walder L;Scheibe R;Hase T;Hanke GT
• 通讯作者: Hanke GT

An XAS investigation of the nickel site structure in the transcriptional regulator InrS.

• DOI: 10.1016/j.jinorgbio.2017.08.003
• 发表时间: 2017-12
• 期刊: Journal of inorganic biochemistry
• 影响因子: 3.9
• 作者: Carr CE;Foster AW;Maroney MJ
• 通讯作者: Maroney MJ

Metal preferences and metallation.

• DOI: 10.1074/jbc.r114.588145
• 发表时间: 2014-10-10
• 期刊: The Journal of biological chemistry
• 作者: Foster AW;Osman D;Robinson NJ
• 通讯作者: Robinson NJ