Bacillus subtilis encodes three N-acetylcysteine deacetylase enzymes that can catalyze the final step in S-(2-succino)cysteine breakdown.

Succination occurs when the TCA cycle intermediate fumarate reacts with cellular thiols, such as cysteine, yielding the damaged metabolite S-(2-succino)cysteine (2SC). Increased fumarate levels result in global succination of thiol-containing macromolecules and metabolites, which has been implicated in many human diseases. 2SC is a chemically stable molecule; however, enzymatic breakdown pathways have been identified in prokaryotes that involve N-acetylation of 2SC followed by a breakdown step that results in the release of the succino-moiety and N-acetylcysteine (NAC). NAC must be metabolized to cysteine to be assimilated, but enzymes catalyzing NAC deacetylation had hitherto not been thoroughly characterized. Here, we describe three enzymes in Bacillus subtilis, ScmP, YhaA, and YtnL, that all possess high NAC deacetylase activity in vitro. All three enzymes are metal-dependent hydrolases that are most active with cobalt and show remarkable specificity to NAC compared to structurally related acetylated small molecules. Growth assays demonstrated that these genes are functionally redundant in B. subtilis, and growth on NAC is only severely compromised when all three genes are knocked out of the genome. Together, our biochemical and genetic studies complete the functional characterization of the three-step 2SC degradation pathway in B. subtilis.