Mechanisms of novel biological nitrogen chemistries

新型生物氮化学机制

• 批准号: 10668474
• 负责人: Jonathan D Caranto
• 金额: $ 36.46万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668474
• 关键词: Anabolism; Antibiotics; Bacteria; Biochemical Reaction; Biochemistry; Biological; Biological Markers; Cells; Chemistry; Cytochrome P450; Cytochrome a; Data; Engineering; Environment; Enzymes; Genus Mycobacterium; Goals; Health; Heme; Hemerythrin; Hispanic-serving Institution; Homologous Gene; Human; Hydroxylation; Immune response; Ions; Ligand Binding; Ligands; Mediating; Metals; Mycobacterium tuberculosis; Natural Products; Neurodegenerative Disorders; Nitrates; Nitric Oxide; Nitrites; Nitrogen; Pathogenicity; Pathway interactions; Peroxonitrite; Protein Engineering; Proteins; Reaction; Reporting; Research; Role; Site; Structure; Structure-Activity Relationship; Therapeutic; Training Programs; Tryptophan; Tyrosine; Virulence; Vision; Work; cell injury; cost; insight; metalloenzyme; mycobacterial; nitration; nitrosative stress; novel; oxidation; pathogenic bacteria; programs; protein function

Project Summary/Abstract: The proposed research will study the mechanisms and structure-function relationships of novel nitric oxide (NO)-dependent enzymatic activities with relevance to protein nitration, natural product biosynthesis, and nitrosative stress protection in pathogenic bacteria. Exposure of cells to NO results in cell damage including nitration of protein residues such as tyrosine (Tyr) and tryptophan (Trp). Protein nitration can modulate protein function and are biomarkers of some neurodegenerative diseases. Some protein nitration can be catalyzed by metal ions. The mechanisms of metal-mediated nitration pathways are unclear; therefore, Research Direction 1 is to study the mechanisms of metal-catalyzed protein nitration. To better understand these mechanisms, we will study the nitration by cytochromes P450 (CYPs), including that of the enzyme TxtE, a CYP homolog that catalyzes the regiospecific and NO-dependent nitration of Trp to 4-nitrotryptophan (4-NO2-Trp). The observed regiospecificity suggests that diffusible RNS are not produced on pathway. Furthermore, CYP metal-oxo intermediates have been well characterized. Therefore, studies of TxtE have great potential to provide clear mechanistic data on nitration. One challenge is that TxtE nitration intermediates are elusive. Therefore, the 5-year goal of Research Direction 1 is to determine the outer sphere coordination features of TxtE needed to promote substrate nitration and avoid substrate hydroxylation, the latter being canonical CYP activity. Identifying outer sphere interactions that influence TxtE reactivity will provide structure-function insight to devise strategies to trap nitration intermediates. To avoid cell damage from NO, pathogenic Mycobacteria express enzymes to scavenge NO. Research Direction 2 will study activities and mechanisms of metalloenzymes involved in Mycobacterial virulence, including hemerythrin-like proteins (HLPs) found in pathogenic Mycobacteria, including M. tuberculosis. Our preliminary data on HLPs shows that its diferric oxidation state can oxidize NO to nitrite (NO2–) by reductive nitrosylation. Such reactivity is well known for heme enzymes but has never been reported for a non-heme protein. In the presence of O2, HLP reacts with NO to form nitrate (NO3–) and a metalloproduct with a 350-nm absorbance feature. This metalloproduct can be independently generated by reacting the as isolated HLP with peroxynitrite (ONOO–). The novel reactivities of HLP with RNS suggest a role for it in nitrosative stress protection. An uncommon Tyr ligand bound to the HLP non-heme diiron site may facilitate these unusual reactivities. The 5-year goals of Research Direction 2 are to trap and characterize intermediates of RNS reactions with HLP, determine the catalytic activity of HLP, and identify the role of the Tyr ligand. The vision of our program is to discover novel nitrogen biochemistries related to natural product biosynthesis and human health and to pursue engineering biocatalysts and to establish a world-class training program in mechanistic metalloenzymology and protein engineering, and natural product biosynthesis at UCF, a Hispanic-serving institution.

项目概要/摘要：本研究将研究其作用机制和结构功能 新型一氧化氮（NO）依赖性酶活性与蛋白质硝化的关系， 天然产物的生物合成和病原菌的亚硝化胁迫保护。细胞暴露于NO 导致细胞损伤，包括蛋白质残基如酪氨酸（Tyr）和色氨酸（Trp）的硝化。 蛋白质硝化可以调节蛋白质的功能，是一些神经退行性疾病的生物标志物。 某些蛋白质的硝化反应可以被金属离子催化。金属离子介导的硝化反应机理 因此，研究方向1是研究金属催化蛋白质硝化的机理。 为了更好地理解这些机制，我们将研究细胞色素P450（CYP）的硝化作用，包括 TxtE是一种催化色氨酸的区域特异性和NO依赖性硝化， 4-硝基色氨酸（4-NO2-Trp）。所观察到的区域特异性表明， 通路此外，金属氧代中间体已得到很好的表征。因此，TxtE的研究 有很大的潜力提供明确的硝化机理数据。一个挑战是TxtE硝化 中间体是难以捉摸的。因此，研究方向1的5年目标是确定外层空间 TxtE的配位特征需要促进底物硝化和避免底物羟基化， 后者是典型的活动。识别影响TxtE反应性的外层相互作用将 提供结构-功能洞察力，以设计策略来捕获硝化中间体。为了避免细胞损伤， NO，致病性分枝杆菌表达酶以产生NO。研究方向2将研究活性 与分枝杆菌毒力有关的金属酶的机制，包括hemerythrin样蛋白 （HLP）在致病性分枝杆菌中发现，包括M.结核我们对HLP的初步数据显示， 其二铁氧化态可通过还原亚硝基化将NO氧化为亚硝酸根（NO2-）。这种反应性是好的。 已知用于血红素酶，但从未报道用于非血红素蛋白。在O2存在下，HLP 与NO反应生成硝酸根（NO3-）和具有350 nm吸收特征的金属产物。这 金属产物可以通过使分离的HLP与过氧亚硝酸根（ONOO-）反应独立地产生。 HLP与RNS的新反应性表明其在亚硝化应激保护中的作用。一个不寻常的轮胎 与HLP非血红素二铁位点结合的配体可能促进这些不寻常的反应性。五年目标 研究方向2是用HLP捕获和表征RNS反应的中间体， HLP的催化活性，并确定Tyr配体的作用。我们节目的愿景是发现新的 与天然产物生物合成和人类健康有关的氮生物化学， 生物催化剂，并建立一个世界级的机械金属酶学和蛋白质的培训计划， 工程，和天然产物生物合成在UCF，西班牙裔服务机构。