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.

抽象的 最近从编码周质蛋白的血红素利用蛋白操纵子 hupYZ 中鉴定出 化脓性链球菌是一种与多种疾病有关的重要病原体。编码之一 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，这种微弱的活性 是由于多组氨酸标签诱导的与真正血红素加氧酶活性位点的结构相似性。这 无标签 HupZ 不会降解血红素；然而，我们最近发现无标签的 HupZ 结合 FMN。这 FMN 结合的 HupZ 晶体结构与最近发现的胆绿素还原酶 (BVR) 叠加 利用 F420H2 辅因子的结核分枝杆菌 (Mtb)。 HupZ 中 FMN 的黄素部分排列良好 与 F420H2 辅因子的去氮黄素部分。因此，我们认为 HupZ 是黄素/去氮黄素 链球菌血红素利用途径 (Hup) 中氧化还原酶 (FDOR) 依赖性 BVR。结构和 提出功能表征以进一步加深对该 Hup 蛋白的分子理解 生物化学、光谱和结构方法。我们将探究无标签 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留物。我们将 尝试表征催化中间体并阐明催化机制。完成 拟议的生化和结构研究将填补我们目前对血红素理解的空白 重要病原体的利用途径。仅描述了少数 FDOR 依赖性酶，并且 F420H2依赖的BVR机制仍有待阐明；因此，这项工作也是非常有意义的 机械酶学。