Characterization of Putative N-acetylcysteine Deacetylase Enzymes Involved in S-(2-succino)-cysteine Breakdown
Spontaneous modification of free thiols by the TCA cycle intermediate fumarate– a process known as succination– can disrupt essential metabolic processes involved in the TCA cycle. Thiols on cysteine within the cell can succinate fumarate to form S-(2-succino)-cysteine (2SC). High concentrations of 2SC have been correlated to deleterious physiological effects in humans, such as obesity, cancer, and diabetes. To combat this buildup, an auxiliary pathway exists to break down 2SC and recover free cysteine and fumarate. In order to accomplish this, an acetyl group is added to the nitrogen on 2SC, which can then be broken down into N-acetylcysteine (NAC) and oxaloacetic acid. The NAC must then be enzymatically deacetylated in order to recover free cysteine. Four homologous genes (yxeP, ykuR, yhaA, and ytnL) have been identified in Bacillus subtilis with suspected deacetylase activity. Proteins encoded by these genes were heterologously expressed in E. coli and affinity purified, and assayed by HPLC methods to determine activity and kinetic parameters. YxeP, YhaA, and YtnL were found to show deacetylase activity, and YkuR was not. Redundancy of enzymes for this pathway suggests an acute biological relevance for the ability to add and remove N-acetyl groups in order to tailor reactivity.