Electron spin-labeling of the EutC subunit in B12-dependent ethanolamine ammonia-lyase reveals dynamics and a two-state conformational equilibrium in the N-terminal signal-sequence-associated domain

摘要

The B12 (adenosylcobalamin) -dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine untilization (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Toward elucidation of the molecular basis of EAL catalysis, and its intracellular trafficking and targeting to the Eut biomicrocompartment (BMC), we have applied electron spin-labeling and electron paramagnetic resonance spectroscopy to wild type (wt) EAL from S. typhimurium, by using the sulfhydryl-specific, 4-maleimido-TEMPO (4MT) spin label. One cysteine residue per active site displays exceptional reactivity with 4MT. This site is identified as βC37 on the EutC subunit, by using 4MT-labeling of site-specific cysteine-to-alanine mutants, enzyme kinetics, and accessible surface area calculations. EPR spectra of 4MT-labeled wt EAL are collected over 200–265 K in frozen, polycrystalline water-only and 1% v/v DMSO solvents. EPR simulations reveal two mobility components for each condition. Detectable spin probe reorientational motion of the two components occurs at 215 and 225 K with 1% v/v DMSO, relative to the water-only condition, consistent with formation of an aqueous-DMSO solvent mesodomain around EAL. Parallel trends in fast- and slow- reorientational correlation times and interconversion of the two populations with increasing temperature, indicate 4MT labeling of a single site (βc37). A two-state model is proposed, in which the fast and slow motional populations represent EAL-bound and free conformations of the EutC N-terminal domain. The approximately equal proportion of each state may represent a balance between EutC and EAL protein stability and efficient targeting to the BMC.