Campus Units

Chemistry, Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of

Document Type

Article

Publication Version

Published Version

Publication Date

5-18-2021

Journal or Book Title

Proceedings of the National Academy of Sciences

Volume

118

Issue

20

First Page

e2100298118

DOI

10.1073/pnas.2100298118

Abstract

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.

Comments

This article is published as Nguyen, Trang T., Rodolfo Ghirlando, Julien Roche, and Vincenzo Venditti. "Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity." Proceedings of the National Academy of Sciences 118, no. 20 (2021): e2100298118. DOI: 10.1073/pnas.2100298118.

Rights

Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.

Language

en

File Format

application/pdf

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