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Nickel-pincer nucleotide enzymes

镍钳核苷酸酶

基本信息

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
项目类别：
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

项目摘要

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/文摘