Heterologous Expression of Halomonas halodenitrificans Nitric Oxide Reductase and its N-Terminally Truncated NorC Subunit in Escherichia coli
Heterologous Expression of Halomonas halodenitrificans Nitric Oxide Reductase and its N-Terminally Truncated NorC Subunit in Escherichia coli
复制标题
卤代硝化盐单胞菌一氧化氮还原酶及其 N 端截短的 NorC 亚基在大肠杆菌中的异源表达
DOI：
10.1016/j.jinorgbio.2017.01.006
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发表时间：
2017
影响因子：
3.9
通讯作者：
Takeshi Sakurai
中科院分区：
文献类型：
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作者：
Nobuhiko Sakurai;Kunishige Kataoka;Noriko Sugaya;Takaki Shimodaira;Mie Iwamoto;Munehiro Shoda;Hajime Horiuchi;Miyuki Kiyono;Yasuke Ohta;Banbang Triwiyono;Daisuke Seo;Takeshi Sakurai
嗜盐盐单胞菌一氧化氮还原酶（NOR）是由NorC和NorB组成的异源二聚体复合物，NorC含有一个低自旋血红素中心，NorB含有一个低自旋血红素中心、一个高自旋血红素中心和一个非血红素中心。利用缺失norC基因5′端84个核苷酸的表达质粒，在大肠杆菌中异源表达了NorC的可溶性结构域NorC *（Δ Met 1-Val 37）。当使用完整的C基因时，作为膜锚的N-末端螺旋发生类似的断裂。NorC* 表现出典型的低自旋血红素的光谱。此外，NorC* 还能接受从H.卤化镁和小的还原剂。NorC* 的氧化还原电位发生了变化。40 mV的负方向从NorC。与NorC不同，重组NorB不异源表达。重组NOR（rNOR）在大肠杆菌中表达。大肠杆菌通过使用含有所有的基因的质粒norCBQDX，从其中删除了三个发夹环（mRNA），并通过使用用于C型血红素成熟的cm基因。rNOR具有与NOR相同的光谱特性和对NO和O2的反应性，但其对NO的酶活性显著降低。这些关于rNOR和NorC* 表达的结果将使我们能够更深入地研究该嗜盐细菌的四个Fe中心的性质和NOR的反应机制。
Halomonas halodenitrificansnitric oxide reductase (NOR) is the membrane-bound heterodimer complex of NorC, which contains a low-spin hemeccenter, and NorB, which contains a low-spin hemebcenter, a high-spin hemeb3center, and a non-heme FeBcenter. The soluble domain of NorC, NorC* (ΔMet1–Val37) was heterologously expressed inEscherichia coliusing expression plasmids harboring the truncatednorCgene deleted of its 84 5′-terminal nucleotides. Analogous scission of the N-terminal helix as the membrane anchor took place when the wholenorCgene was used. NorC* exhibited spectra typical of a low-spin hemec. In addition, NorC* functioned as the acceptor of an electron from a cytochromecisolated from the periplasm ofH. halodenitrificansand small reducing reagents. The redox potential of NorC* shifted ca. 40 mV in the negative direction from that of NorC. Unlike NorC, recombinant NorB was not heterologously expressed. However, recombinant NOR (rNOR) could be expressed inE. coliby using a plasmid harboring all genes in thenoroperon,norCBQDX, from which the three hairpin loops (mRNA) were deleted, and by using theccmgenes for the maturation of C-type heme. rNOR exhibited the same spectroscopic properties and reactivity to NO and O2as NOR, although its enzymatic activity toward NO was considerably decreased. These results on the expression of rNOR and NorC* will allow us to develop more profound studies on the properties of the four Fe centers and the reaction mechanism of NOR from this halophilic denitrifying bacterium.