(The) mechanism for carbonyl stress response in escherichia coli

Abstract

Glyoxal (GO) and methylglyoxal (MG) are α-oxoaldehydes produced by diverse mechanisms in cells. As the compounds have two reactive carbonyl groups, the intracellular proteins and DNA are disrupted by the compounds. As a result, there are diverse detoxification mechanisms of carbonyl stress. In this study, two types of detoxification processes were suggested. One is the YqhD system, the NADPH-dependent aldehyde reducing mechanism, and the other is the cofactor-free glyoxalase III system, the DJ-1 homolog proteins, YajL, YhbO, and ElbB, mechanisms. The reason for focusing on the YqhD GO-detoxification system is due to the isolated GO-resistant $(GO^R)$ mutants. Fifteen GOR mutants were isolated from 10 mM GO plate and we analyzed the loci of mutations in the mutants. From these data, we found that the mutations were in yqhC gene N-termini sites and, therefore, YqhD protein was over-expressed in the mutants. Purified YqhC was bound to the predicted SoxS binding sites near the yqhD promoter regions, and was tested by EMSA and DNase I footprinting assay. Also, YqhC activated the transcription of yqhD in vivo proved by the reporter assay, using several different lengths of yqhD promoter-inserted reporter vectors. From these data, we concluded that YqhC activates the transcription level of yqhD. In order to explain the detail mechanism of YqhC activator, YqhC mutants were used. As the binding affinity of the mutant YqhC decreased compared with that of wild type YqhC, we measured the structural difference by fluorescence emission scanning. The N-terminal structure of mutant (V80E) showed higher fluorescent intensity compared with that of wild type. Therefore, we suggested that the N-terminal domain structure may be the key factor of unusual yqhD induction, but the exact mechanism is still unknown. Previous report suggested that Hsp31, the DJ-1 homolog protein in Escherichia coil, is the glyoxalase III, converting α-oxoaldehydes to carboxylic acids in cofactor-free condition. Therefore, we researched whether the other three Escherichia coli genes, yajL, yhbO and elbB also have glyoxalase III activities or not. Purified YajL, YhbO and ElbB exhibited the glyoxalase activity with different substrate specificities, optimal pHs, and metal effects. In case of metal ions, the DJ-1 homolog proteins are commonly inhibited by intracellular concentrated zinc ion. Although the DJ-1 homolog gene deficient mutants did not show the susceptibilities to GOs, over expressions of the homologs enhance cellular protection from exogenously added glyoxals and reduce the glyoxals-dependent increase in intracellular advanced glycation end products (AGEs). Based on their expression primarily during the stationary phase, we speculate that their roles in cell as glyoxalases are manifested during the stationary phase.