Nickel-pincer nucleotide enzymes

项目总结/摘要 镍钳核苷酸（NPN）辅因子是一种新发现的乳酸消旋酶辅酶 （LarA）来自植物乳杆菌（Lactobacillus plantarum）。活性酶的合成需要三种辅助酶的参与 依次作用的蛋白质：LarB羧酸化吡啶环并水解 在烟酸腺嘌呤二核苷酸中，LarE将两个吡啶鎓环羧酸盐转化为硫代羧酸盐，并且 LarC在新型辅因子的合成过程中插入镍（形成两个S-Ni键和一个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 和人类病原体之间的关系。本文所述努力的长期目标是大幅度推进 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）表征NPN生物合成系统的组成部分 和（2）鉴定NPN辅因子在乳酸消旋酶和其它酶中的作用。LarB的研究 将定义该吡啶鎓环羧化酶/磷酸酐水解酶的结构和机制。研究 多半胱氨酸和可能含有[4Fe 4S]形式的LarE将确定它是否通过催化作用起作用。 硫转移机理，与L.单活性中心半胱氨酸植物蛋白酶 转化为脱氢丙氨酸CTP依赖型嵌镍LarC的结构与机理分析 将阐明这种蛋白质如何将镍安装到辅因子中。乳酸与L. plantarum LarA将被定义，该酶使用的底物的全部范围将被建立，并且底物将被确定。 确定替代LarA样蛋白。将鉴定共价结合NPN辅因子的蛋白质， 其特征在于使用使辅酶与荧光标记反应的创新化学。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，然后进行质量分析。 光谱，生物信息学和生物化学研究，以确定这些新酶的功能。的 通过这些努力获得的发现将大大增加镍的合成和利用的知识， 细菌中的钳形辅因子，包括那些对人类健康重要的辅因子， 潜在的抗微生物药物靶点。

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.