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.

项目总结/摘要 镍钳核苷酸（NPN）辅因子是最近在乳酸消旋酶中发现的辅酶 （LarA），但更广泛地存在于2-羟基酸消旋酶和差向异构酶中。辅因子的合成需要 三种辅助蛋白的顺序作用：LarB在C5处羧酸化吡啶环并水解 烟酸腺嘌呤二核苷酸磷酸酐（NaAD），LarE是一种ATP依赖性酶， 将两个吡啶鎓环羧酸盐转化为硫代羧酸盐， 硫原子或通过涉及具有非核心硫化物的[4Fe-4S]簇的机制，并且LarC插入镍 （形成C-Ni和S-Ni键）。编码这四种蛋白质的基因同源物是 广泛分布于与人类微生物组相关的微生物中和人类病原体中。的 这里所描述的工作的长期目标是大大提高我们对如何 微生物，包括致病物种，制造和利用NPN辅因子。两个具体目标将 实现这一目标：（1）NPN生物合成途径的组分将在结构上和 机械特征和（2）NPN辅因子的新作用将被描述为超越其功能， 乳酸消旋酶LarB的研究将通过以下方式定义NaAD和CO2结合变体的结构： 晶体学，并通过停流UV-120确认共价半胱氨酰-吡啶鎓中间体的形成。 维斯分光光度法体内分析将测试细胞逆转半胱氨酰硫的牺牲性损失的能力 一种LarE含[4Fe-4S]团簇的LarE的结构特征为： 结晶学第三，含过硫化物形式的LarE的假设将进行评估。结构 完整LarC的动力学将通过冷冻电子显微镜来解决。或者，N-末端结构域 LarC将通过晶体学定义（补充已知的C-末端结构），以更好地描绘 酶的单周转，CTP依赖的镍插入机制。其他LarA同系物将 结构和功能定义。将使用紧密结合较大基底的实例来探测打开的探针。 关于NPN依赖性酶反应机制的问题。其他例子预计将包含 NPN和铁硫簇，其功能将被研究。此外，非LarA样蛋白， 将识别和表征结合NPN。通过这些努力获得的结果将大大增加 了解细菌中镍钳核苷酸辅因子的合成和利用，包括那些 这对人类健康很重要，并对确定潜在的抗菌药物靶点有影响。