Oxidation of cysteine 645 of cobalamin-independent methionine synthase causes a methionine limitation in Escherichia coli.

Cobalamin-independent methionine synthase (MetE) catalyzes the final step in Escherichia coli methionine biosynthesis but is inactivated under oxidative conditions, triggering a methionine deficiency. This study demonstrates that the mutation of MetE cysteine 645 to alanine completely eliminates the methionine auxotrophy imposed by diamide treatment, suggesting that modulation of MetE activity via cysteine 645 oxidation has significant physiological consequences for oxidatively stressed cells.

Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL.

In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.

Methionine.

This review focuses on the steps unique to methionine biosynthesis, namely the conversion of homoserine to methionine. The past decade has provided a wealth of information concerning the details of methionine metabolism and the review focuses on providing a comprehensive overview of the field, emphasizing more recent findings. Details of methionine biosynthesis are addressed along with key cellular aspects, including regulation, uptake, utilization, AdoMet, the methyl cycle, and growing evidence that inhibition of methionine biosynthesis occurs under stressful cellular conditions. The first unique step in methionine biosynthesis is catalyzed by the metA gene product, homoserine transsuccinylase (HTS, or homoserine O-succinyltransferase). Recent experiments suggest that transcription of these genes is indeed regulated by MetJ, although the repressor-binding sites have not yet been verified. Methionine also serves as the precursor of S-adenosylmethionine, which is an essential molecule employed in numerous biological processes. S-adenosylhomocysteine is produced as a consequence of the numerous AdoMet-dependent methyl transfer reactions that occur within the cell. In E. coli and Salmonella, this molecule is recycled in two discrete steps to complete the methyl cycle. Cultures challenged by oxidative stress appear to experience a growth limitation that depends on methionine levels. E. coli that are deficient for the manganese and iron superoxide dismutases (the sodA and sodB gene products, respectively) require the addition of methionine or cysteine for aerobic growth. Modulation of methionine levels in response to stressful conditions further increases the complexity of its regulation.

Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli.

In nature, Escherichia coli are exposed to harsh and non-ideal growth environments-nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting 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). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can 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. The unique site of oxidation was identified by selectively cleaving N-terminally to 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. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper (Leichert and Jakob 2004), which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect.