Departments of Molecular Biosciences and Chemistry, Northwestern University, Evanston, Illinois, USA.
Department of Biochemistry and the Rappaport Institute for Medical Sciences, Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.
J Biol Chem. 2022 Jan;298(1):101445. doi: 10.1016/j.jbc.2021.101445. Epub 2021 Nov 23.
The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this study, we present the first biochemical and functional studies of the AZY proteins. Biochemical characterization and structural modeling indicate that YobA binds a single Cu ion with high affinity. Bioinformatics analysis shows that YebY is widespread and encoded either in AZY operons or in other genetic contexts unrelated to copper homeostasis. We also determined the 1.8 Å resolution crystal structure of E. coli YebY, which closely resembles that of the lantibiotic self-resistance protein MlbQ. Two strictly conserved cysteine residues form a disulfide bond, consistent with the observed periplasmic localization of YebY. Upon treatment with reductants, YebY binds Cu and Cu with low affinity, as demonstrated by metal-binding analysis and tryptophan fluorescence. Finally, genetic manipulations show that the AZY operon is not involved in copper tolerance or antioxidant defense. Instead, YebY and YobA are required for the activity of the copper-related NADH dehydrogenase II. These results are consistent with a potential role of the AZY operon in copper delivery to membrane proteins.
大肠杆菌 yobA-yebZ-yebY（AZY）操纵子编码 YobA、YebZ 和 YebY 蛋白。YobA 和 YebZ 分别是 CopC 周质铜结合蛋白和 CopD 假定铜输入蛋白的同源物，而 YebY 属于未鉴定的功能未知域 2511 家族。尽管对大肠杆菌铜稳态和 AZY 操纵子在多种细菌中的存在进行了大量研究，但该操纵子的蛋白质及其功能作用尚未得到探索。在这项研究中，我们首次对 AZY 蛋白进行了生化和功能研究。生化特性和结构建模表明，YobA 能与单个 Cu 离子高亲和力结合。生物信息学分析表明，YebY 广泛存在，编码于 AZY 操纵子或与铜稳态无关的其他遗传背景中。我们还确定了大肠杆菌 YebY 的 1.8Å 分辨率晶体结构，它与类细菌素自我抗性蛋白 MlbQ 非常相似。两个严格保守的半胱氨酸残基形成二硫键，与 YebY 观察到的周质定位一致。用还原剂处理后，YebY 与 Cu 和 Cu 低亲和力结合，这可通过金属结合分析和色氨酸荧光实验证明。最后，遗传操作表明，AZY 操纵子不参与铜耐受或抗氧化防御。相反，YebY 和 YobA 是铜相关 NADH 脱氢酶 II 活性所必需的。这些结果与 AZY 操纵子在将铜递送到膜蛋白中的潜在作用一致。
