Novel hydrogenases from deep-sea hydrothermal vent metagenomes identified by a recently developed activity-based screen
Novel hydrogenases from deep-sea hydrothermal vent metagenomes identified by a recently developed activity-based screen
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DOI：
10.1038/s41396-017-0040-6
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发表时间：
2018-05-01
期刊：
影响因子：
11
通讯作者：
Perner, Mirjam
中科院分区：
文献类型：
--
作者：
Adam, Nicole;Perner, Mirjam
氢是地球上最常见的元素之一。把氢分子转化为质子和电子的酶是氢化酶。氢化酶普遍存在于生命的所有三个领域，它们在细胞代谢中起着核心作用。到目前为止，氢化酶的恢复仅限于依赖于培养和基于序列的方法。我们最近开发了唯一一种基于活性的筛选方法，用于从宏基因组中寻找h -2摄取酶，而无需依赖于氢氧化微生物的富集和分离或先前的宏基因组测序。利用该方法对3个热液喷口元基因组的14400个fosmid克隆进行筛选，鉴定出4个具有H-2摄取活性的克隆，其部分纯化膜组分的比活性高达258 +/- 19 nmol H-2/min/mg蛋白。相应的宏基因组片段与公共数据库中的序列大多表现出非常低的相似性或没有相似性。用隐马尔可夫模型对不同的氢化酶组进行了搜索，结果显示，四种宏基因组插入物中有三种没有找到，这表明它们不编码经典的氢化酶。我们的基于活性的筛选是发现（新型）氢化酶的有力工具，这些酶是目前可用技术无法识别的。该筛选可以理想地与基于培养和序列的方法相结合，以研究环境中巨大的氢转化潜力。
Hydrogen is one of the most common elements on Earth. The enzymes converting molecular hydrogen into protons and electrons are the hydrogenases. Hydrogenases are ubiquitously distributed in all three domains of life where they play a central role in cell metabolism. So far, the recovery of hydrogenases has been restricted to culture-dependent and sequence-based approaches. We have recently developed the only activity-based screen for seeking H-2-uptake enzymes from metagenomes without having to rely on enrichment and isolation of hydrogen-oxidizing microorganisms or prior metagenomic sequencing. When screening 14,400 fosmid clones from three hydrothermal vent metagenomes using this solely activity-based approach, four clones with H-2-uptake activity were identified with specific activities of up to 258 +/- 19 nmol H-2/min/mg protein of partially purified membrane fractions. The respective metagenomic fragments exhibited mostly very low or no similarities to sequences in the public databases. A search with hidden Markov models for different hydrogenase groups showed no hits for three of the four metagenomic inserts, indicating that they do not encode for classical hydrogenases. Our activity-based screen serves as a powerful tool for the discovery of (novel) hydrogenases which would not have been identified by the currently available techniques. This screen can be ideally combined with culture- and sequence-based approaches to investigate the tremendous hydrogen-converting potential in the environment.