Mechanism for reactivation of inactivated holoenzymes of coenzyme B12-dependent ethanolamine ammonia-lyase by the reactivating chaperone EutA.

Abstract

Adenosylcobalamin-dependent ethanolamine ammonia-lyase (EAL) undergoes irreversible inactivation when incubated in the absence of substrate or in the presence of certain substrates or pseudosubstrates. We have previously identified Escherichia coli EutA as an EAL-reactivase (or reactivating factor). Herein, untagged and tagged EutAs were purified to homogeneity. It showed a tendency to form multimers, but its monomer was mainly responsible for reactivation. It showed extremely low but distinct ATPase activity. In the presence of ATP, Mg²⁺, and free adenosylcobalamin, EutA reactivated holoEALs that had been inactivated in the absence of substrate or suicidally inactivated by 2-aminopropanol, ethylene glycol, or glycolaldehyde. It also activated inactive complexes of EAL with inactive cobalamins, such as cyanocobalamin, hydroxocobalamin, and methylcobalamin, but not the complex with adeninylpentylcobalamin. EutA-mediated cofactor exchange by facilitating the removal of damaged cofactors in the presence of ATP and Mg²⁺. Here, we postulate a mechanism of action of EutA, which is similar to that of diol dehydratase-reactivase (DD-R). However, contrary to DD-R, its ATP- or 5'-adenylyl imidodiphosphate (AMP-PNP)-bound and ADP-bound forms were suggested to be high-affinity and low-affinity forms for EAL, respectively.