首页> 外文期刊>Journal of Molecular Biology >The NreA protein functions as a nitrate receptor in the staphylococcal nitrate regulation system.
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The NreA protein functions as a nitrate receptor in the staphylococcal nitrate regulation system.

机译:NreA蛋白在葡萄球菌硝酸盐调节系统中起硝酸盐受体的作用。

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摘要

Staphylococci are able to use nitrate as an alternative electron acceptor during anaerobic respiration. The regulation of energy metabolism is dependent on the presence of oxygen and nitrate. Under anaerobic conditions, staphylococci employ the nitrate regulatory element (Nre) for transcriptional activation of genes involved in reduction and transport of nitrate and nitrite. Of the three proteins that constitute the Nre system, NreB has been characterized as an oxygen sensor kinase and NreC has been characterized as its cognate response regulator. Here, we present structural and functional data that establish NreA as a new type of nitrate receptor. The structure of NreA with bound nitrate was solved at 2.35? resolution, revealing a GAF domain fold. Isothermal titration calorimetry experiments showed that NreA binds nitrate with low micromolar affinity (KD=22μM). Two crystal forms for NreA were obtained, with either bound nitrate or iodide. While the binding site is hydrophobic, two helix dipoles and polar interactions contribute to specific binding of the ions. The expression of nitrate reductase (NarGHI) was examined using a narG-lip (lipase) reporter gene assay in vivo. Expression was regulated by the presence of NreA and nitrate. Structure-guided mutations of NreA reduced its nitrate binding affinity and also affected the gene expression, thus providing support for the function of NreA as a nitrate receptor.
机译:葡萄球菌能够在厌氧呼吸期间使用硝酸盐作为替代电子受体。能量代谢的调节取决于氧和硝酸盐的存在。在厌氧条件下,葡萄球菌利用硝酸盐调节元件(Nre)激活涉及硝酸盐和亚硝酸盐还原和运输的基因的转录激活。在构成Nre系统的三种蛋白质中,NreB被表征为氧传感器激酶,NreC被表征为其关联反应调节剂。在这里,我们介绍将NreA建立为新型硝酸盐受体的结构和功能数据。与硝酸盐结合的NreA的结构在2.35?分辨率,揭示GAF域折叠。等温滴定热法实验表明,NreA以低微摩尔亲和力(KD =22μM)结合硝酸盐。获得了NreA的两种晶体形式,分别是结合硝酸盐或碘化物。虽然结合位点是疏水的,但两个螺旋偶极子和极性相互作用有助于离子的特异性结合。使用narG-lip(脂肪酶)报告基因在体内检测硝酸盐还原酶(NarGHI)的表达。 NreA和硝酸盐的存在调节表达。 NreA的结构指导突变降低了其硝酸盐结合亲和力,也影响了基因表达,从而为NreA作为硝酸盐受体的功能提供了支持。

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