Oxidative stress inactivates cobalamin -independent methionine synthase in <italic>Escherichia coli</italic>.

Abstract

In nature, <italic>Escherichia coli</italic> are exposed to harsh and non-ideal growth environments---nutrients may be limiting, and cells are often challenged by oxidative stress. For <italic>E. coli</italic> cells confronted by these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). <italic>E. coli</italic> cells subjected to transient oxidative stress during growth in minimal medium were found to develop a methionine auxotrophy, which could be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation and disruption of zinc binding. The unique site of oxidation was identified by selectively cleaving on the N-terminal side of each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Moreover, glutathionylation was found to induce a conformational change concomitant with enzyme inactivation. Direct evidence for high levels of MetE oxidation <italic>in vivo</italic> was obtained from thioltrapping experiments in two different <italic>E. coli </italic> strains that contain oxidizing cytoplasmic environments. In addition, MetE is completely oxidized in wild-type <italic>E. coli</italic> treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. Thus, for <italic>E. coli</italic> experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein may provide a strategy with which to modulate the <italic>in vivo</italic> activity of the enzyme while protecting the active site from more extensive damage. Although glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in <italic>E. coli</italic> are known to be modified in this manner. The extreme sensitivity of MetE to oxidation <italic> in vivo</italic> may have significant cellular consequences. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. This study suggests a simpler mechanism may be employed by <italic> E. coli</italic> to achieve the same effect.