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 镍钳核苷酸(Npn)辅酶是最近发现的一种存在于乳酸外消旋酶中的辅酶。 (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：LLAB羧化、C5上的吡啶环和水解物 烟酸腺嘌呤二核苷酸磷酸酐(NAAD)，LARE是一种依赖于ATP的酶，它 通过牺牲蛋白质半胱氨酸基，将两个吡啶环羧酸盐转化为硫代羧酸盐 或通过涉及[4Fe-4S]团簇和非核心硫化物的机制，并且LaRC插入镍 (形成C-Ni键和S-镍键)在CTP依赖的反应中。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群有关的微生物和人类病原体中。这个 这里描述的工作的长期目标是显著提高我们对如何 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生物合成途径的组成部分将在结构上和 机械表征和(2)NPN辅因子的新作用将被表征超越其在 乳酸消旋酶。对LAB的研究将通过以下方式确定NaAD和CO2结合的变体的结构 用停流紫外-可见吸收光谱确证了共价半胱氨基-吡啶中间体的形成 可见分光光度法。体内分析将测试细胞逆转半胱氨酸硫牺牲损失的能力 从一种乳牛中分离出来。含[4Fe-4S]簇型LARE的结构特征如下 结晶学。第三种含过硫化物的LARE的假说将会被评估。该结构 而完整的LARC的动力学将通过低温电子显微镜来解决。或者，的N端结构域 LARC将由结晶学定义(补充已知的C-末端结构)，以更好地描绘 酶的单一周转，依赖CTP的镍插入机制。其他Lara同系物将是 在结构上和功能上都有定义。将使用紧密结合较大衬底的示例来探查打开 关于NPN依赖的酶反应机制的问题。其他的例子被预测包含 NPN和一个铁-硫团簇，其功能将被研究。此外，非LARA样蛋白 将确定和表征BIND NPN。通过这些努力获得的结果将大大增加。 细菌中镍钳核苷酸辅因子的合成和利用的知识，包括那些 对人类健康很重要，对确定潜在的抗微生物药物靶标具有影响。