Nickel-pincer nucleotide enzymes

镍钳核苷酸酶

• 批准号: 9816044
• 负责人: ROBERT P HAUSINGER
• 金额: $ 33.52万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9816044
• 关键词: Acids; Active Sites; Anabolism; Bacteria; Bacterial Genome; Binding; Binding Proteins; Biochemical; Bioinformatics; Biological; Carbon; Cell Wall; Chemicals; Chemistry; Coenzymes; Complex; Couples; Crystallization; Cysteine; Data Set; Drug Targeting; Enzymes; Exhibits; Genes; Geometry; Health; Human; Human Microbiome; Hydrolase; Hydrolysis; Hydroxy Acids; Investigation; Knowledge; Label; Lactic acid; Lactobacillus plantarum; Ligation; Link; Lysine; Mass Spectrum Analysis; Metabolic; Metabolism; Metals; Nickel; Nicotinic Acids; Nucleotides; Organism; Pathogenicity; Procedures; Property; Protein Binding Domain; Proteins; Public Health; Pyruvate; Racemases; Radioactive; Reaction; Research; Resolution; Role; Side; Structure; Sulfur; System; Testing; Thioamides; Vancomycin Resistance; Work; adduct; antimicrobial; antimicrobial drug; carboxylate; carboxylation; cofactor; dehydroalanine; enantiomer; enzyme mechanism; enzyme structure; human pathogen; innovation; interest; microorganism; nicotinic acid adenine dinucleotide; novel; pathogen; pathogenic bacteria; preference; racemization; radiotracer; skills; three dimensional structure

Project Summary/Abstract The nickel-pincer nucleotide (NPN) cofactor is a newly identified coenzyme discovered in lactate racemase (LarA) from Lactobacillus plantarum. Synthesis of the active enzyme requires the participation of three accessory proteins that act in sequence: LarB carboxylates the pyridinium ring and hydrolyzes the phosphoanhydride of nicotinic acid adenine dinucleotide, LarE converts the two pyridinium ring carboxylates to thiocarboxylates, and LarC inserts nickel (forming two S-Ni and one C-Ni bonds) during synthesis of the novel cofactor. Genes 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 advance significantly our understanding of how microorganisms, including pathogenic species, make and utilize the NPN cofactor. Two specific aims will achieve this objective: (1) characterize the components of the NPN biosynthetic systems and (2) identify the roles of the NPN cofactor in lactate racemase and additional enzymes. Investigations of LarB will define the structure and mechanism of this pyridinium ring carboxylase/phosphoanhydride hydrolase. Studies of a multi-cysteine and probable [4Fe4S]-containing form of LarE will establish whether it operates by a catalytic sulfur-transfer mechanism, in contrast to the sacrificial LarE of L. plantarum with its single active site cysteine that converts to dehydroalanine. Structural and mechanistic analysis of the CTP-dependent nickel-inserting LarC will elucidate how this protein installs nickel into the cofactor. The geometry of lactate binding to L. plantarum LarA will be defined, the full range of substrates used by this enzyme will be established, and substrates will be identified for alternative LarA-like proteins. Proteins that covalently bind the NPN cofactor will be identified and characterized using innovative chemistry that reacts the coenzyme with a fluorescent tag. Radioactive nickel (63Ni) and 14C-nicotinic acid also will be used to label new NPN cofactor-binding proteins, followed by mass spectrometry, bioinformatics, and biochemical studies to identify the functions of these novel enzymes. The findings obtained through these efforts will greatly increase knowledge of the synthesis and utilization of nickel- pincer cofactors in bacteria, including those important to human health, with implications for identification of potential antimicrobial drug targets.

项目摘要/摘要 镍钳核苷酸(Npn)辅酶是在乳酸消旋酶中发现的一种新的辅酶。 (Lara)从植物乳杆菌中分离得到。活性酶的合成需要三个辅酶的参与 按顺序起作用的蛋白质：LLAB使吡啶环上的羧基发生羧化，并使磷酸二氢酸酐水解。 烟酸腺嘌呤二核苷酸，LARE将两个吡啶环羧酸盐转化为硫代羧酸盐，以及 在合成新型辅因子的过程中，LARC插入镍(形成两个S-镍键和一个C-镍键)。基因 编码这四种蛋白质的蛋白质广泛分布于与人类微生物群有关的微生物中 在人类病原体中。这里所描述的努力的长期目标是取得显著进展 我们对微生物，包括致病物种，如何制造和利用NPN辅因子的理解。 两个具体目标将实现这一目标：(1)确定NPN生物合成系统的组成 以及(2)确定NPN辅助因子在乳酸消旋酶和其他酶中的作用。关于幼虫的调查 将确定这个吡啶环羧化酶/磷酸酸酐水解酶的结构和作用机制。研究 多个半胱氨酸和可能含有[4Fe4S]形式的LARE将确定它是否通过催化作用 硫的转移机制，与植物乳杆菌与其单一活性部位半胱氨酸的牺牲作用相反 会转化为脱氢丙氨酸。基于CTP的插镍LARC的结构和力学分析 将阐明这种蛋白质如何将镍安装到辅因子中。乳酸与植物乳杆菌结合的几何构型 LARA将被定义，这种酶所使用的所有底物范围将被确定，底物将被 确定了替代的LARA类蛋白质。将鉴定与NPN辅因子共价结合的蛋白质，并 用创新的化学反应辅酶和荧光标签来表征的。放射性镍 (~(63)Ni)和~(14)C-烟酸也将用于标记新的NPN辅因子结合蛋白，然后是MASS 光谱学、生物信息学和生化研究，以确定这些新型酶的功能。这个 通过这些努力获得的发现将极大地增加对镍的合成和利用的了解。 细菌中的钳状辅因子，包括那些对人类健康重要的因子，对鉴定 潜在的抗菌药物靶点。