Functional analyses of methylglyoxal sensing and scavenging proteins in Bacillus subtilis

Abstract

Methylglyoxal, a reactive carbonyl compound, is generated ubiquitously as a side-product of cellular metabolism. Methylglyoxal can damage cellular constituents through glycation reactions whereby reactive aldehydes modify amino groups of proteins, nucleic acids and basic phospholipids. Since the high concentration of methylglyoxal can cause cell death, sensing and detoxification of methylglyoxal is essential for all living organisms. In this study, I investigated the proteins involved in sensing and detoxification of methylglyoxal in low-GC Gram-positive Firmicutes, Bacillus subtilis. In Part I, it is discussed that how B. subtilis regulates the expression of defense enzymes against methylglyoxal using a MerR family transcriptional regulator, AdhR. In Part II, it is discussed that how B. subtilis detoxify methylglyoxal using a DJ-1 family glyoxalase III. It has been reported that AdhR, a MerR family regulator, regulates its own expression and the expression of an adhA-yraA operon, and that the Cys52 of AdhR is required for the methylglyoxal-dependent positive regulations of AdhR itself and adhA-yraA operon. In this study, I have found that the adhR mutant exhibited decreased resistance to methyglyoxal compared to wild type and the mutation in the conserved Cys52 also resulted in decreased resistance to methylglyoxal. Although it has been hypothesized that the Cys52 residue is modified via thiol-(S)-alkylation for transcriptional activation, I could not find any evidence of Cys52 modification under methylglyoxal stress conditions as judged by in vitro MS analysis. To further investigate the role of the Cys52, I have solved the crystal structure of Cys52 to Ser mutant AdhR. Methylglyoxal can be detoxified to D-lactate by the thiol-dependent glyoxalase I/II system or by the thiol-independent glyoxalase III system. It has recently been shown that B. subtilis utilizes bacillithiol (BSH), a low-molecular-weight (LMW) thiol, in glyoxalase I (GlxA) and II (GlxB) system. And several DJ-1/ ThiJ/ PfpI superfamily proteins (YdeA, YraA and YfkM) have been suggested as candidate for glyoxalase III enzymes, which can directly convert methylglyoxal to D-lactate in a BSH-independent manner. I investigated the glyoxalase III system using combinations of mutations in four DJ-1 superfamily proteins (YdeA, YraA, YfkM and YoaZ) in B. subtilis. In the presence of the BSH-dependent glyoxalase I/II system, no single mutation in ydeA, yraA, yfkM, or yoaZ led to a significant decrease in resistance to methylglyoxal. However, triple mutant strains containing yraA::tet exhibited significantly decreased resistance to methylglyoxal, but not triple mutant strain (ydeA::kan, yfkM::cat, yoaZ::em) which has intact yraA. Furthermore, in the absence of the functional BSH-dependent glyoxalase I/II system, mutation in only yraA significantly decreased resistance to methylglyoxal. All these results indicate that YraA plays an important role in thiol-independent detoxification of methylglyoxal in B. subtilis. Indeed, purified YraA protein after overexpression in E. coli exhibited thiol-independent glyoxalase activity, and mutation in putative active site abolished glyoxalase activity, indicating that YraA is a main enzyme in glyoxalase III system in B. subtilis.