Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208.
Department of Chemistry, Northwestern University, Evanston, IL 60208.
Proc Natl Acad Sci U S A. 2021 Jun 8;118(23). doi: 10.1073/pnas.2100680118.
Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome peroxidase (bCP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a Cu primary-sphere ligand, being positioned ∼2.9 Å away from the Cu ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.
一些甲烷氧化细菌利用核糖体合成、翻译后修饰的天然产物甲烷菌素（Mbn）来获取铜，以用于其主要代谢酶——颗粒状甲烷单加氧酶。编码合成和运输 Mbns 的操纵子通常包括两种蛋白质的基因，MbnH 和 MbnP，这两种蛋白质在其他与铜稳态相关的基因组环境中也作为一对存在。虽然 MbnH 蛋白是细菌二血红素细胞色素过氧化物酶（bCP）/MauG 超家族的成员，但 MbnP 的结构和功能、两者之间的关系以及它们在铜稳态中的作用仍不清楚。现在，对甲烷氧化菌 OB3b 的 MbnP 的生化特征进行了研究，揭示了 MbnP 能够以高亲和力结合一个处于+1 氧化态的单个铜离子。MbnP 体内铜结合依赖于保守的 WxW 基序中的第一个色氨酸氧化为犬尿氨酸，这种转化是通过 MbnH 与 MbnP 的相互作用发生的。MbnP 的 2.04-Å 分辨率晶体结构揭示了一个独特的折叠和一个不寻常的铜结合位点，涉及一个组氨酸、一个蛋氨酸、一个溶剂配体和犬尿氨酸。尽管犬尿氨酸残基可能不作为 Cu 初级配体，与 Cu 离子的距离约为 2.9 Å，但它的存在是铜结合所必需的。基因组邻域分析表明，MbnP 蛋白，以及由此产生的含犬尿氨酸的铜结合位点，在微生物铜稳态中广泛存在，可能发挥多种作用。
