Identification of NuoX and NuoY Ligand Binding Specificity in theCampylobacter Jejuni Complex I

L. I. Calderon-Gomez, C. Day, L. Hartley-Tassell, Jennifer C. Wilson, G. Mendz, V. Korolik
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引用次数: 1

Abstract

The components of the proton pump NADH:ubiquinone (Complex I) of the respiration pathway have been identified in the C. jejuni genome. However, the paradigm genes nuoE and nuoF encoding subunits of the NADH dehydrogenase module of Complex I are absent. Instead the genes cj1575c and cj1574c encoding NuoX and NuoY are present in the loci corresponding to nuoE and nuoF, respectively. Bioinformatics analyses showed the presence of nuoX and nuoY homologues in all sequenced strains of C. jejuni and in other Campylobacter species, as well as the presence of orthologues in other E›-Proteobacteria. To understand the involvement of the NuoX and NuoY proteins in the respiration of C. jejuni and to characterize their ligand binding specificity and affinity, a tricarboxylic acid cycle array was developed as a tool to identify proteins that can bind to intermediates of this cycle as well as other metabolites. This array showed that NuoX bound FAD2+, and NuoY bound FAD2+ and the electron donors malate and lactate. Saturation Transfer Difference Nuclear Magnetic Resonance studies confirmed the NuoY binding ligands, and suggested that the flavin moiety of FAD2+ interacted more strongly with NuoY than the adenine moiety. Affinity data generated by Surface Plasmon Resonance indicated that NuoY bound to FAD2+ with a KD of 337 nM; NuoX and NuoY had an affinity for NADH of a KD of 403 nM and 478 nM, respectively, and a ten-fold lower affinity for both NAD+ and FAD2+. The data suggested that the flavin-adenine dinucletoide could be bound preferentially to the NAD in the Complex I of C. jejuni.
空肠弯曲杆菌NuoX和NuoY配体结合特异性的鉴定
在空肠梭菌基因组中已鉴定出呼吸途径的质子泵NADH:泛醌(复合体I)的组分。然而,编码复合体I的NADH脱氢酶模块亚基的范例基因nuoE和nuoF缺失。相反,编码NuoX和nuy的基因cj1575c和cj1574c分别存在于nuoE和nuoF对应的位点上。生物信息学分析表明,在所有测序的空肠c菌和其他弯曲菌属中均存在nuoX和nuoY同源物,在其他E -变形菌属中也存在同源物。为了了解NuoX和NuoY蛋白在空肠梭菌呼吸中的作用,并表征它们的配体结合特异性和亲和力,研究人员开发了一个三羧酸循环阵列,作为鉴定可以结合该循环中间体和其他代谢产物的蛋白质的工具。该阵列显示,NuoX结合FAD2+, NuoY结合FAD2+和苹果酸盐和乳酸盐。饱和转移差核磁共振研究证实了NuoY的结合配体,并表明FAD2+的黄素部分比腺嘌呤部分与NuoY的相互作用更强。表面等离子体共振产生的亲和力数据表明,NuoY与FAD2+结合,KD为337 nM;NuoX和NuoY对NADH的亲和力分别为403 nM和478 nM,对NAD+和FAD2+的亲和力低10倍。结果表明,黄素-腺嘌呤二核苷可优先结合空肠梭菌复合体I中的NAD。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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