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.

项目摘要/摘要：拟议的研究将研究其机制和结构-功能 新型一氧化氮(NO)依赖的酶活性与蛋白质硝化的关系 天然产物的生物合成，以及对病原菌的亚硝化应激保护。细胞暴露于NO 导致细胞损伤，包括酪氨酸(Tyr)和色氨酸(Trp)等蛋白质残基的硝化。 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。 有些蛋白质的硝化反应可以被金属离子催化。金属介导的硝化途径的机理 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(Cyps)的硝化作用，包括 TxtE酶的活性，它是一种CYP同系物，催化色氨酸的区域特异性和NO依赖的硝化反应 4-硝基色氨酸(4-NO2-Trp)。观察到的区域特异性表明，可扩散的RN不在 路径。此外，CYP金属氧代中间体也得到了很好的表征。因此，对TxtE的研究 有很大的潜力提供关于硝化作用的明确的机械数据。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的配位特性是促进底物硝化和避免底物羟化所必需的 后者是典型的CYP活性。确定影响TxtE反应性的外球相互作用将 为设计捕获硝化中间体的策略提供结构-功能洞察力。为了避免细胞损伤， 不，致病分枝杆菌表达酶来清除NO。研究方向2将研究活动 以及金属酶参与分枝杆菌毒力的机制，包括杂氰菊酯类蛋白 (HLP)在致病分枝杆菌中发现，包括结核分枝杆菌。我们关于HLP的初步数据显示， 它的二铁氧化态可以通过还原亚硝化反应将NO氧化为亚硝酸盐(NO2-)。这种反应性是很好的 已知含有血红素酶，但从未报道过非血红素蛋白。在O2、HLP存在的情况下 与NO反应生成硝酸盐(NO3-)和一种具有350 nm吸收特征的金属产品。这 金属产物可通过分离的HLP与过氧亚硝酸根(ONOO-)反应而独立生成。 HLP与RNS的新反应表明它在亚硝化应激保护中发挥作用。一种不同寻常的轮胎 与HLP非血红素二铁结合的配基可能促进这些不寻常的反应。的五年目标 研究方向2是捕捉和表征RNS与HLP反应的中间体，确定 HLP的催化活性，并确定Tyr配体的作用。我们计划的愿景是发现新的 与天然产物生物合成和人类健康相关的氮素生物化学及其工程学 生物催化剂，并建立世界级的机械金属酶和蛋白质培训计划 西班牙裔服务机构UCF的工程和天然产品生物合成。