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DOI	10.1073/pnas.2100298118
	Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity
	Nguyen T.T.; Ghirlando R.; Roche J.; Venditti V.
发表日期	2021
ISSN	0027-8424
卷号	118 期号:20
英文摘要	Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer-dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI's monomeric state have yet been hampered by the dimer's high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI's monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Carbon metabolism; Enzyme regulation; High pressure; Phosphotransferase system; Solution NMR
语种	英语
scopus关键词	enzyme I; phosphoenolpyruvate; phosphotransferase; unclassified drug; allosterism; Article; controlled study; dimerization; dissociation; enzyme active site; enzyme activity; enzyme conformation; enzyme engineering; enzyme regulation; enzyme stability; enzyme structure; gene mutation; hydrostatic pressure; ligand binding; nonhuman; oligomerization; protein domain; structure analysis; Thermoanaerobacter; Thermoanaerobacter tengcongensis; thermodynamics
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/238566
作者单位	Department of Chemistry, Iowa State University, Ames, IA 50011, United States; Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, United States; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, United States
推荐引用方式 GB/T 7714	Nguyen T.T.,Ghirlando R.,Roche J.,et al. Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity[J],2021,118(20).
APA	Nguyen T.T.,Ghirlando R.,Roche J.,&Venditti V..(2021).Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity.Proceedings of the National Academy of Sciences of the United States of America,118(20).
MLA	Nguyen T.T.,et al."Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity".Proceedings of the National Academy of Sciences of the United States of America 118.20(2021).