Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site.
Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site.
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DOI：
10.1186/s12934-023-02127-w
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发表时间：
2023-07-21
影响因子：
6.4
通讯作者：
中科院分区：
文献类型：
--
作者：
氢化酶（h2ase）是一种金属酶，能够将质子和电子可逆地转化为氢分子。利用h2ase独特的酶活性可以推动生物制氢和绿色能源生产的进程。在这里，我们创建了一个功能性的，优化的操纵子，用于快速和稳健地生产重组[NiFe]脱硫微生物杆状体氢化酶（Dmb H2ase）。[NiFeSe] Dmb H2ase在基因水平上通过定点诱变转化为[NiFe]型。天然dmb操纵子包括两个结构H2ase基因，编码大亚基和小亚基，以及一个额外的基因，编码特定的成熟酶（蛋白酶），这对酶的适当成熟至关重要。Dmb和所有h2ase一样，需要复杂的生物生产机制来整合其关键的无机配体和辅因子。严格的厌氧，硫酸盐还原菌D. baculatum细菌是不同的，在其生物学方面，从大肠杆菌。因此，我们在原生dmb基因中引入了一系列的改变。结果，构建了100多个元素，并进一步编译为32个操纵子变体。通过人工截断大Dmb亚基，省略了对特定成熟酶的初始要求。在体外和体内研究了产生的H2ase亚基变体的组装。该方法获得了4个重组[NiFe] Dmb酶变体，能够进行H2进化。本研究的目的是克服基因表达、蛋白质生物合成、成熟和配体装载等瓶颈，利用一种常见的大肠杆菌菌株，实现重组[NiFe] H2ase的简单、快速和经济地递送。优化的遗传结构以及开发的生长和纯化程序似乎为进一步研究具有全活性和耐氧性的重组[NiFeSe] Dmb H2ase提供了一个有希望的平台，类似于天然Dmb酶。这可能是通过在[NiFe] Dmb变体的活性位点内选择性地将半胱氨酸替换为硒代半胱氨酸来实现的。在线版本包含补充材料，可在10.1186/s12934-023-02127-w获得。
Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant. The online version contains supplementary material available at 10.1186/s12934-023-02127-w.
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影响因子：
5.2
作者：
通讯作者：
--
DOI：
10.1016/s0969-2126(99)80072-0
发表时间：
1999-05-01
期刊：
STRUCTURE WITH FOLDING & DESIGN
影响因子：
--
作者：
Garcin, E;Vernede, X;Fontecilla-Camps, JC
通讯作者：
Fontecilla-Camps, JC
影响因子：
3.2
作者：
Corless, Elliot, I;Mettert, Erin L.;Antony, Edwin
通讯作者：
Antony, Edwin
影响因子：
--
作者：
Green, Michael R;Sambrook, Joseph
通讯作者：
Sambrook, Joseph
影响因子：
14.9
作者：
Käll L;Krogh A;Sonnhammer EL
通讯作者：
Sonnhammer EL