Studying Acetylation of Aconitase Isozymes by Genetic Code Expansion

Aconitase catalyzes the second reaction of the tricarboxylic acid cycle, the reversible conversion of citrate and isocitrate. Escherichia coli has two isoforms of aconitase, AcnA and AcnB. Acetylomic studies have identified acetylation at multiple lysine sites of both E. coli aconitase isozymes, but the impacts of acetylation on aconitases are unknown. In this study, we applied the genetic code expansion approach to produce 14 site-specifically acetylated aconitase variants. Enzyme assays and kinetic analyses showed that acetylation of AcnA K684 decreased the enzyme activity, while acetylation of AcnB K567 increased the enzyme activity. Further in vitro acetylation and deacetylation assays were performed, which indicated that both aconitase isozymes could be acetylated by acetyl-phosphate chemically, and be deacetylated by the CobB deacetylase at most lysine sites. Through this study, we have demonstrated practical applications of genetic code expansion in acetylation studies.

Automated summary

By studying acetylation, we have used the genetic code expansion approach to produce aconitase variants with specific acetylation sites, and found that acetylation has different effects on the enzyme activity of the two aconitase isozymes. We also found that both isozymes can be chemically acetylated by acetyl-phosphate and deacetylated by the CobB deacetylase.