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.

生物技术旨在开发的蛋白质中有很大一部分需要金属。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，为工业化合物的可持续生产而设计生物体）必须考虑如何也设计足够的金属辅因子供应，因为如此多的生物催化是由金属驱动的。关于细胞如何帮助蛋白质获得正确的金属的假设已经在播客中向普通观众介绍，该播客可以从链接到评论文章（Nature（2009）460，823-830）的Nature网站下载（搜索“nature podcast pick your partners”）。本研究将验证这些假设。蛋白质有与不合适的金属形成伙伴关系的倾向。据认为，细胞将每种金属维持在最佳缓冲浓度以克服这一挑战。假设最具竞争力的金属保持在最低缓冲浓度。在这种制度下，蛋白质与其他蛋白质竞争竞争金属，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部分蛋白质的金属辅因子的保真度至关重要。它提出了缓冲金属水平可能从细胞到细胞变化的可能性，对代谢具有广泛的影响。此外，该模型提出了一个机会，工程师的最佳金属水平，以协助辅助辅助因子的有用的蛋白质，例如，合成biology.Metal传感器蛋白检测的波动缓冲浓度的金属在胞质溶胶。我们之前的BBSRC资助的研究已经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。重要的是，假设这种传感器对检测到的金属的亲和力与细胞内缓冲金属浓度之间存在直接关系。将对该假设进行镍检测检验。本研究的目的是设计一种与生物能源相关的酶的镍供应，这种酶是一组对绿色技术具有重要意义的生物体的代表。氢化酶是少数需要镍的酶之一（生物圈中可能不到20种）。在正确的条件下，这种酶可以产生二氢气体，因此可能有助于氢经济。光合生物，包括蓝细菌，可以利用来自阳光的能量来产生有用的化合物，包括能量载体，如氢，因此有潜力创造可持续的过程。然而，氢化酶的活性是有限的，在蓝藻镍供应不足。我们最近（在BBSRC的支持下）在蓝细菌的胞质溶胶中发现了镍传感器（Journal of Biological Chemistry（2012）287，12142-12151）。这一发现创造了一个机会，工程金属稳态的目的是优化镍供应氢化酶在蓝藻。

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 specificity of cyanobacterial nickel-responsive repressor InrS: cells maintain zinc and copper below the detection threshold for InrS.

• DOI: 10.1111/mmi.12594
• 发表时间: 2014-05
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Foster AW;Pernil R;Patterson CJ;Robinson NJ
• 通讯作者: Robinson NJ