The nos gene cluster from gram-positive bacterium Geobacillus thermodenitrificans NG80-2 and functional characterization of the recombinant NosZ
The nos gene cluster from gram-positive bacterium Geobacillus thermodenitrificans NG80-2 and functional characterization of the recombinant NosZ
复制标题
革兰氏阳性菌热脱氮地芽孢杆菌NG80-2的nos基因簇及重组NosZ的功能表征
DOI：
10.1111/j.1574-6968.2008.01362.x
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发表时间：
2008-12-01
影响因子：
2.1
通讯作者：
Feng, Lu
中科院分区：
文献类型：
--
作者：
Liu, Xueqian;Gao, Chunxu;Feng, Lu
在革兰氏阴性菌中，编码反硝化途径最后一步的一氧化二氮还原酶(N2OR)活性的NOS基因簇已经得到了很好的研究。我们先前对耐热地杆菌NG80-2基因组的研究发现，在这株革兰氏阳性菌中存在nos基因簇。在后续研究中，进一步分析了嗜热嗜热菌NG80-2的nos基因簇，并与其他来源的nos基因簇进行了比较。在大肠杆菌中异源表达了nosZ结构基因，并将其纯化为组氨酸标记的融合蛋白。所制备的重组NosZ具有可检测到的N2OR活性，并且在Ar和铜化合物预孵育的情况下，该蛋白的活性被增强。重组的NOSZ每个二聚体含有2.5个铜原子，在可见光区表现出微弱的光谱特征，表明铜化合物自发地掺入到NG80-2 NOSZ中可以导致真实的NG80-2 N2OR的部分但不是全部的活性。并对该酶的酶学性质进行了研究。这是来自革兰氏阳性细菌的nosZ基因的第一个功能特征。本研究表明，革兰氏阴性菌和革兰氏阳性菌之间N2O还原的分子机制是保守的。
The nos gene cluster encoding the activity of nitrous oxide reductase (N2OR) for the final step of the denitrification pathway has been well studied in gram-negative bacteria. Our previous study on the genome of Geobacillus thermodenitrificans NG80-2 revealed the presence of the nos gene cluster in this gram-positive bacterium. In this follow-up study, the nos gene cluster of G. thermodenitrificans NG80-2 was further analyzed and compared with those of other origins. The structural gene nosZ was heterologously expressed in Escherichia coli and the product was purified as a His-tagged fusion protein. The recombinant NosZ as prepared showed detectable N2OR activity, and the activity was enhanced by preincubation of the protein under argon and with copper compounds. The recombinant NosZ contains 2.5 atoms of copper per dimer and exhibits weak spectral features in the visible range, indicating that spontaneous incorporation of copper compounds into the NG80-2 NosZ can result in some but not full activity of the authentic NG80-2 N2OR. The enzymatic properties of the NosZ were also investigated. This is the first functional characterization of nosZ gene from gram-positive bacteria. This study indicates that the molecular mechanism for N2O reduction is conserved between gram-negative and gram-positive bacteria.