Identifying key residues in the active site of 2SL (2-succino lyase)

Abstract

Succination is a chemical reaction that occurs spontaneously at a rate directly related to fumarate, an intermediate in the citric acid cycle. It has been observed that high fumarate levels, and consequently increased succination, in humans contributes directly to the pathogenesis of diabetes, obesity, and certain cancers through deleterious effects on proteins. The stable adduct S-(2-succino)cysteine (2SC) is commonly formed by succination of fumarate with cysteinyl residues of proteins; this 2SC adduct is an oncometabolite—a product of metabolism that accumulates in cancers—as well as a biomarker for other human diseases. The first breakdown pathway for 2SC was discovered in Bacillus subtilis. This pathway contains an enzyme that exhibits 2-succino lyase ability (2SL) in Enterococcus italicus and Dickeya dadantii. These enzymes are all members of the large ‘lyase I-like’ superfamily of lyase enzymes, many of which cannot be functionally differentiated based on sequence alone. My project aimed to determine whether certain residues in the 2SL active site are important for 2SC lyase activity. We modeled the 2SL enzyme using https://swissmodel.expasy.org/ and PyMOL. By comparing 2SL to related enzymes, we made predictions about which residues are functional significant to 2SL (including residue N13). Using primers with a mismatch at position 13, we introduced a mutation converting polar asparagine to non-polar isoleucine (N13I). We were able to express and purify both mutant and WT 2SL proteins to near homogeneity. The HPLC assay of the reverse reaction seems to suggest that the mutant N13I enzyme is more efficient (product peak area = 9251.5) than the WT (product peak area = 6998.4). The disparity between the product peaks in the forward reaction was smaller. Upon a second HPLC assay, the results of the forward reaction were not replicated. Only one run of the HPLC assay is shown, more assays will need to be done in the forward direction to determine whether the results are conclusive. The understanding of 2SL active sites will contribute insight to the ongoing efforts to identify more 2SC catabolic pathways and, more broadly, understand and treat diseases such as diabetes and cancer.