Nickel-pincer nucleotide enzymes

镍钳核苷酸酶

基本信息

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
项目类别：
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

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

展开

项目摘要

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/摘要镍胶状核苷酸（NPN）辅因子是在乳酸种族酶中发现的新鉴定的辅酶（Lara）来自乳杆菌的植物。活性酶的合成需要三个附件的参与作用顺序起作用的蛋白质：Larb羧化吡啶环并水解磷酸甘氨酸烟酸腺嘌呤二核苷酸，LARE将两个吡啶环羧酸盐转化为硫羧酸盐，和 LARC在合成新型辅因子过程中插入镍（形成两个S-Ni和一个C-Ni键）。基因编码这四种蛋白质广泛分布在与人类微生物组相关的微生物中在人类病原体中。这里描述的努力的长期目标是显着促进我们对微生物（包括致病物种）如何制造和利用NPN辅助因子的理解。两个具体目标将实现此目标：（1）表征NPN生物合成系统的组成部分（2）确定NPN辅因子在乳酸种族酶和其他酶中的作用。 LARB的调查将定义此吡啶环羧化酶/磷酸氢化盐水解酶的结构和机制。研究多半胱氨酸和可能的[4FE4S]含有lare的形式将确定其是否通过催化作用硫 - 转移机制，与植物的牺牲性l. 这转化为脱氢丙氨酸。 CTP依赖性镍插入LARC的结构和机械分析将阐明该蛋白如何将镍安装到辅因子中。乳酸与植物长乳杆菌的几何形状将定义LARA，将确定该酶使用的全范围底物，底物将是用于替代LARA样蛋白的鉴定。将确定共价结合NPN辅因子的蛋白使用创新的化学反应的辅酶与荧光标签反应的特征。放射性镍（63Ni）和14C-NICOTINIC还将用于标记新的NPN辅助剂结合蛋白，然后是质量光谱，生物信息学和生化研究以鉴定这些新酶的功能。这通过这些努力获得的发现将大大增加对镍合成和利用的了解细菌中的钳子辅因子，包括对人类健康重要的辅因子，对鉴定的影响潜在的抗菌药物靶标。