Zymographic differentiation of [NiFe]-hydrogenases 1, 2 and 3 of Escherichia coli K-12.
Zymographic differentiation of [NiFe]-hydrogenases 1, 2 and 3 of Escherichia coli K-12.
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DOI：
10.1186/1471-2180-12-134
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发表时间：
2012-07-06
期刊：
影响因子：
4.2
通讯作者：
Sawers G
中科院分区：
文献类型：
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作者：
Pinske C;Jaroschinsky M;Sargent F;Sawers G
当在厌氧条件下生长时，大肠杆菌K-12能够合成三种活性[NiFe]-氢化酶（Hyd 1 -3）。这些氢化酶中的两种是催化氢氧化的呼吸酶，其中Hyd-1是耐氧的，而Hyd-2被认为是标准的氧敏感性氢化酶。Hyd-3与甲酸脱氢酶H（Fdh-H）一起形成甲酸氢解酶（FHL）复合物，其负责完整细胞的H2进化。可以使用苄基紫精（BV; Eo′ = -360 mV）作为人工电子受体来测定所有三种氢化酶的氢氧化活性;然而，将活性归因于特定的同工酶并不是微不足道的。以前，凝胶内测定可以区分Hyd-1和Hyd-2，而Hyd-3长期以来被认为太不稳定而不能在这种天然凝胶上可视化。这项研究确定了条件，允许区分所有三种酶，使用简单的凝胶内酶谱分析。使用一种改进的凝胶内分析，氢依赖性BV还原催化的Hyd-3已被首次描述。高浓度的氢促进了Hyd-3活性的可视化。该活性是膜相关的，尽管对于Hyd-3的可视化不是必需的，但在功能性Fdh-H酶存在下活性最大。此外，通过使用氮蓝四唑（NBT; Eo′ = -80 mV），证明了Hyd-1以氢依赖性方式还原该氧化还原染料，而Hyd-2和Hyd-3都不能将氢氧化与NBT还原偶联。NBT的氢依赖性还原也被Hyd-1的氧敏感变体催化，该变体在小亚基的位置19处具有多余的半胱氨酸残基取代甘氨酸。这一发现表明，对氧的耐受性不是决定电子捐赠给更多氧化还原阳性电子受体（如NBT）的主要决定因素。在不同的氢浓度和氧化还原电位下利用特定的电子受体与相应氢化酶的已知生理功能相关。快速区分耐氧和标准[NiFe]-氢化酶的能力为发现新酶提供了一个简单的新筛选。一个可靠的测定Hyd-3将重振研究的特点，氢演变FHL复杂。
When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize three active [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymes catalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered a standard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H (Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2 evolution by intact cells. Hydrogen oxidation activity can be assayed for all three hydrogenases using benzyl viologen (BV; Eo′ = -360 mV) as an artificial electron acceptor; however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assay could differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable to be visualized on such native gels. This study identifies conditions allowing differentiation of all three enzymes using simple in-gel zymographic assays. Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 has been described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential for visualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-H enzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo′ = -80 mV) it was demonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, while neither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogen-dependent reduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 that had a supernumerary cysteine residue at position 19 of the small subunit substituted for glycine. This finding suggests that tolerance toward oxygen is not the main determinant that governs electron donation to more redox-positive electron acceptors such as NBT. The utilization of particular electron acceptors at different hydrogen concentrations and redox potentials correlates with the known physiological functions of the respective hydrogenase. The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenases provides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3 will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex.
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影响因子：
3.5
作者：
CHEREPANOV, PP;WACKERNAGEL, W
通讯作者：
WACKERNAGEL, W
影响因子：
2.1
作者：
BEGG, YA;WHYTE, JN;HADDOCK, BA
通讯作者：
HADDOCK, BA
影响因子：
56.9
作者：
Boyington, JC;Gladyshev, VN;Sun, PD
通讯作者：
Sun, PD
DOI：
10.1111/j.1432-1033.1986.tb09578.x
发表时间：
1986-04-15
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
BALLANTINE, SP;BOXER, DH
通讯作者：
BOXER, DH
大肠杆菌K-12的构造框架，单基因敲除突变体：Keio Collection。
影响因子：
9.9
作者：
通讯作者：
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