AUTHOR=Araujo Jessica , Ottinger Sara , Venkat Sumana , Gan Qinglei , Fan Chenguang 

TITLE=Studying Acetylation of Aconitase Isozymes by Genetic Code Expansion

JOURNAL=Frontiers in Chemistry

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2022.862483

DOI=10.3389/fchem.2022.862483

ISSN=2296-2646

ABSTRACT=<p>Aconitase catalyzes the second reaction of the tricarboxylic acid cycle, the reversible conversion of citrate and isocitrate. <italic>Escherichia coli</italic> has two isoforms of aconitase, AcnA and AcnB. Acetylomic studies have identified acetylation at multiple lysine sites of both <italic>E. coli</italic> 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 <italic>in vitro</italic> 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.</p>