Structure-Function Relationship study of HupZ

HupZ的结构-功能关系研究

• 批准号: 10613305
• 负责人: Ephrahime Traore
• 金额: $ 3.89万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613305
• 关键词: Active Sites; Affinity; Bilirubin; Biliverdin reductase; Biliverdine; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Copper; Coupled; Crystallography; Disease; Electron Spin Resonance Spectroscopy; Enzymatic Biochemistry; Enzymes; Flavin Mononucleotide; Flavins; Foundations; Goals; Heme; Histidine; Immune response; Infection; Iron; Kinetics; Length; Ligands; Mediating; Mediator; Metals; Molecular; Mycobacterium tuberculosis; NADP; Operon; Outcome; Outcome Study; Oxidoreductase; Oxygenases; Pathogenesis; Pathway interactions; Periplasmic Proteins; Porphyrins; Proteins; Publishing; Reaction; Recycling; Reducing Agents; Resolution; Role; Streptococcus; Streptococcus pyogenes; Structure; Structure-Activity Relationship; System; Work; analog; cofactor; combat; experimental study; heme a; his6 tag; human pathogen; innovation; new therapeutic target; novel; oxidation; pathogen; prevent; therapeutic target; uptake

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.

摘要 血红素利用蛋白操纵子hupYZ编码的周质蛋白最近已被确定，从 化脓性链球菌是一种重要的致病菌，与多种疾病有关。其中一个编码的 蛋白质HupZ最初被指定为血红素加氧酶。然而，我们的研究表明，这种微弱的活动 是由于多聚His标签诱导的与真正的血红素加氧酶活性位点的结构相似性。的 无标签HupZ不降解血红素;然而，我们最近发现无标签HupZ结合FMN。的 FMN-结合的HupZ晶体结构与最近发现的胆绿素还原酶（BVR）重叠， 结核分枝杆菌（Mtb）利用F420 H2辅因子。HupZ中FMN的黄素部分排列良好 与F420 H2辅因子的脱氮黄素部分。因此，我们认为HupZ是黄素/脱氮黄素 氧化还原酶（FDOR）依赖性BVR在链球菌血红素利用途径（Hup）中的作用。结构和 提出了功能表征，以进一步了解这种Hup蛋白的分子使用 生物化学、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，我们将确定参与胆绿素还原的关键残基。我们将 试图表征催化中间体并阐明催化机理。完成 提出的生物化学和结构研究将填补我们目前对血红素理解的空白 利用途径在一个重要的病原体。仅描述了少数FDOR依赖性酶， F420 H2依赖的BVR机制仍有待阐明;因此，这项工作也非常重要 机械酶学