Nickel-pincer nucleotide enzymes

镍钳核苷酸酶

• 批准号: 10656600
• 负责人: ROBERT P HAUSINGER
• 金额: $ 34.51万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656600
• 关键词: Acids; Aerococcus; Anabolism; Bacteria; Bacterial Genome; Binding; Binding Proteins; Biochemical; Bioinformatics; Biological; C-terminal; Carbon; Carbon Dioxide; Catalysis; Cell Wall; Cells; Coenzyme A; Coenzymes; Complement; Complex; Cryoelectron Microscopy; Crystallography; Drug Targeting; Enzymes; Gene Cluster; Genes; Health; Homologous Gene; Human; Human Microbiome; Hydrolysis; Hydroxy Acids; In Vitro; Investigation; Iron; Knowledge; Lactic acid; Lactobacillus plantarum; Ligand Binding; Link; Metabolic; Metabolism; N-terminal; Nickel; Nucleotides; Ornithine; Pathogenicity; Pathway interactions; Property; Protein Binding Domain; Proteins; Public Health; Racemases; Reaction; Research; Role; Site; Specificity; Spectrophotometry; Spectrum Analysis; Structure; Sulfides; Sulfur; System; Testing; Thermotoga maritima; Thioamides; Vancomycin Resistance; Variant; Work; Zinc; adduct; analog; antimicrobial; antimicrobial drug; carboxylate; carboxylation; cofactor; cysteine desulfurase; dehydroalanine; enantiomer; enzyme structure; epimerase; human pathogen; in vivo; interest; microbiome; microorganism; nicotinic acid adenine dinucleotide; novel; pathogen; pathogenic bacteria; persulfides; racemization; skills

Project Summary/Abstract The nickel-pincer nucleotide (NPN) cofactor is a recently identified coenzyme discovered in lactate racemase (LarA) but found more widely in 2-hydroxyacid racemases and epimerases. Synthesis of the cofactor requires the sequential action of three accessory proteins: LarB carboxylates the pyridinium ring at C5 and hydrolyzes the phosphoanhydride of nicotinic acid adenine dinucleotide (NaAD), LarE is an ATP-dependent enzyme that converts the two pyridinium ring carboxylates into thiocarboxylates either by sacrificial loss of a protein cysteinyl sulfur atom or by a mechanism involving a [4Fe-4S] cluster with a non-core sulfide, and LarC inserts nickel (forming C-Ni and S-Ni bonds) in a CTP-dependent reaction. Gene homologs encoding these four proteins are widely distributed in microorganisms associated with the human microbiome and among human pathogens. The long-term objective of the effort described here is to significantly advance our understanding of how microorganisms, including pathogenic species, make and utilize the NPN cofactor. Two specific aims will achieve this objective: (1) the components of the NPN biosynthetic pathways will be structurally and mechanistically characterized and (2) new roles of the NPN cofactor will be characterized beyond its function in lactate racemase. Investigations of LarB will define the structure of an NaAD- and CO2-bound variant by crystallography and confirm the formation of a covalent cysteinyl-pyridinium intermediate by stopped-flow UV- vis spectrophotometry. In vivo analyses will test the cellular ability to reverse the sacrificial loss of cysteinyl sulfur from one type of LarE. The [4Fe-4S] cluster-containing version of LarE will be structurally characterized by crystallography. The hypothesis for a third, persulfide-containing form of LarE will be evaluated. The structure and dynamics of intact LarC will be solved by cryo-electron microscopy. Alternatively, the N-terminal domain of LarC will be defined by crystallography (complementing the known C-terminal structure) to better delineate the enzyme’s single-turnover, CTP-dependent mechanism of nickel insertion. Additional LarA homologs will be structurally and functionally defined. Examples that tightly bind larger substrates will be used to probe open questions regarding the NPN-dependent enzyme reaction mechanism. Other examples are predicted to contain both NPN and an iron-sulfur cluster whose function will be investigated. In addition, non-LarA-like proteins that bind NPN will be identified and characterized. The findings obtained through these efforts will greatly increase knowledge of the synthesis and utilization of nickel-pincer nucleotide cofactors in bacteria, including those important to human health, with implications for identifying potential antimicrobial drug targets.

项目摘要/摘要 镍胶状核苷酸（NPN）辅助因子是在鞋底种族酶中发现的最近鉴定的辅酶 （lara），但在2-羟基酸的种族赛和情节中发现了更广泛的发现。辅助因子的合成需要 三种辅助蛋白的顺序作用：larb羧酸盐在C5处的吡啶环和水解 烟酸腺苷二核苷酸（NAAD）的磷酸甘蔗氢化物是一种依赖ATP的酶 将两个吡啶环羧酸盐转化为硫代苯二甲酸酯，要么通过牺牲蛋白质的胱天因此而丧失 硫原子或通过一种机制涉及一个具有非核硫化物的[4FE-4S]簇，LARC插入镍 （形成C-Ni和S-Ni键）在CTP依赖性反应中。编码这四种蛋白质的基因同源物是 广泛分布在与人类微生物组和人类病原体有关的微生物中。 此处描述的努力的长期目标是显着提高我们对如何的理解 微生物（包括致病物种）使和利用NPN辅助因子。两个具体的目标将 实现此目标：（1）NPN生物合成途径的组成部分将在结构上和 机械性表征和（2）NPN辅因子的新作用将被表征在其功能之外 种族主义者。对LARB的研究将通过 晶体学并确认形成了通过停止流量的紫外线形成共价cysteinyl-pyridinium Vis分光光度法。体内分析将测试细胞的能力，以逆转胱天成蛋白酶硫的牺牲损失 从一种类型的Lare。 [4fe-4s]含聚类的Lare的版本将在结构上以结构为特征 晶体学。将评估三分之一的含有硫化物的LARE的假设。结构 完整的LARC的动力学将通过冷冻电子显微镜解决。另外， LARC将通过晶体学（补充已知的C末端结构）来定义，以更好地描述 酶的镍插入的CTP依赖机制。其他Lara同源物将是 结构和功能定义。紧密绑定较大基材的示例将用于探测打开 有关NPN依赖性酶反应机制的问题。预计其他示例包含 NPN和铁硫簇都将被研究。另外，非拉拉蛋白 将识别和表征NPN。通过这些努力获得的发现将大大增加 了解细菌中镍胶状核苷酸辅因子的合成和利用，包括 对人类健康很重要，对识别潜在的抗菌药物靶标有影响。