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Structural Characterization of AdoMet Radical Enzyme-Catalyzed Posttranslational Modifications in Bacterial Anaerobic Metabolism

细菌厌氧代谢中 AdoMet 自由基酶催化的翻译后修饰的结构表征

基本信息

批准号：
10057221
负责人：
Emily Ulrich
金额：
$ 6.53万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
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：
2019-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10057221
关键词：
Acetyl Coenzyme A Acids Activase Active Sites Address Adopted Amino Acids Anabolism Anaerobic Bacteria Bacteria Binding Cell physiology Chemistry Coenzyme A Complex Cryoelectron Microscopy Crystallization DNA biosynthesis Drug Targeting Electron Microscopy Engineering Environment Enzyme Activation Enzymes Fermentation Formulation Future Goals Growth Homologous Gene Hydrocarbons Hydrogen Individual Infection Investigation Length Lyase Metabolism Methionine Methods Modification Molecular Conformation Molecular Sieve Chromatography Molecular Weight Multiple Bacterial Drug Resistance Natural Products Negative Staining Outcome Outcome Study Oxygen Pathogenicity Pathway interactions Peptides Positioning Attribute Post-Translational Protein Processing Process Production Proteins Pyruvate Reaction Ribosomes Roentgen Rays S-Adenosylmethionine Sampling Site Specific qualifier value Specificity Structure Substrate Specificity System X-Ray Crystallography bacterial metabolism bioactive natural products cytotoxic dimer enzyme mechanism enzyme substrate complex epimerase epimerization experimental study formate acetyltransferase activating enzyme host colonization insight member monomer novel novel therapeutics peptide drug prospective protein complex screening

项目摘要

Project Summary Enzymes that install posttranslational modifications (PTMs) on bacterial peptides and proteins are integral in cellular functions such as the formation of bioactive peptide natural products and the activation of enzymes important for bacterial adaptation to oxygen-limited environments. Understanding the process of PTM formation can inform on the engineering of novel peptide therapeutics and on the methods of bacterial colonization of host environments in infection. S-adenosyl-L-methionine (AdoMet) radical enzymes produce numerous PTMs that change the functionality of the targeted residue(s). AdoMet radical enzymes perform oxygen-sensitive, site-selective radical chemistry on macromolecular substrates, yet a structural understanding of how they accomplish this impressive chemistry has lagged behind in the analysis of the AdoMet radical enzyme superfamily, with no complete AdoMet radical enzyme-protein complex fully visualized. The aims of this proposal include structural characterization of two AdoMet radical enzymes that modify the Cα of specific amino acids within their large substrates: 1) an AdoMet radical epimerase with a peptide substrate and 2) pyruvate formate lyase activase (PFL-AE) in complex with its partner PFL. The epimerase irreversibly converts L-amino acids to D-amino acids within a ribosomal peptide, thus altering the final conformation and influencing its bioactivity. Determining how one epimerase positions substrate to perform multiple turnovers at specified residues will require structural insight. X-ray crystallography will be used to examine interactions of AdoMet radical epimerases with peptide substrates. PFL-AE forms the catalytically essential glycyl radical on PFL to make formate and acetyl-CoA from pyruvate and CoA. How PFL-AE contacts PFL and how the glycyl radical transitions from the PFL-AE active site to the buried PFL active site remain to be elucidated. X-ray crystallography and electron microscopy will be used to determine structures of PFL-AE in complex with PFL. Structural analysis of both systems will provide much needed insight into interactions required for construction of a protein complex that performs site-selective oxygen-sensitive radical-generating chemistry.

项目总结