Structure-Function Relationship study of HupZ

摘要 编码周质蛋白的血红素利用蛋白操纵子hupYZ最近从 化脓性链球菌是一种重要的致病菌，涉及多种疾病。其中一个编码的 蛋白质HupZ最初被认为是一种血红素加氧酶。然而，我们的研究表明，这种微弱的活动 这是由于多组氨酸标签诱导的与真正的血红素加氧酶活性部位的结构相似。这个 无标签的HupZ不降解血红素；然而，我们最近发现无标签的HupZ与FMN结合。这个 FMN结合的HupZ晶体结构与最近发现的胆绿素还原酶(BVR)重叠 利用F420H2辅因子的结核分枝杆菌(Mtb)。HupZ中FMN的黄素部分对齐良好 与F420H2辅因子的去氮黄素部分结合。因此，我们认为HupZ是黄素/去氮黄素 氧化还原酶(FDOR)在链球菌血红素利用途径(Hup)中的依赖BVR结构和 提出了功能表征，以进一步了解该Hup蛋白的分子水平 生化、光谱和结构方法。我们将询问无标签HupZ如何与 它的辅因子和底物，我们将确定胆绿素还原的关键残基。我们会 尝试对催化中间体进行表征并阐明其催化机理。完成 拟议的生物化学和结构研究将填补我们目前对血红素了解的空白 在重要病原体中的利用途径。只有几种依赖FDOR的酶被描述，并且 F420H2依赖的BVR机制仍有待阐明，因此，这项工作也具有重要意义 到机械酶学。

Abstract A heme utilization protein operon hupYZ encoding periplasmic proteins has recently been identified from Streptococcus pyogenes, a significant pathogen involved in a wide range of diseases. One of the encoded proteins, HupZ, was initially assigned as a heme oxygenase. However, our study revealed that this weak activity was due to a poly‐His‐tag induced structural similarity with the active site of genuine heme oxygenases. The tag‐free HupZ does not degrade heme; however, we recently discovered that tag‐free HupZ binds FMN. The FMN‐bound HupZ crystal structure superimposes with the biliverdin reductase (BVR) recently found in Mycobacterium tuberculosis (Mtb) that utilizes an F420H2 cofactor. The flavin moiety of FMN in HupZ aligns well with the deazaflavin moiety of the F420H2 cofactor. Therefore, we propose that HupZ is a flavin/deazaflavin oxidoreductase (FDOR) dependent BVR in the heme utilization pathway (Hup) of Streptococcus. Structural and functional characterizations are proposed to further the molecular understanding of this Hup protein using biochemical, spectroscopic, and structural approaches. We will interrogate how tag‐free HupZ interacts with its cofactor and substrate, and we will determine the critical residues involved in biliverdin reduction. We will attempt to characterize catalytic intermediates and elucidate the catalytic mechanism. Completing the proposed biochemical and structural studies will fill the gaps in our current understanding of the heme utilization pathway in a significant pathogen. Only a few FDOR‐dependent enzymes have been described, and the F420H2‐dependent BVR mechanism remains to be elucidated; therefore, this work is also highly significant to mechanistic enzymology.