Lysine deacetylases
(KDACs) are enzymes that catalyze the hydrolysis
of acyl groups from acyl-lysine residues. The recent identification
of thousands of putative acylation sites, including specific acetylation
sites, created an urgent need for biochemical methodologies aimed
at better characterizing KDAC-substrate specificity and evaluating
KDACs activity. To address this need, we utilized genetic code expansion
technology to coexpress site-specifically acylated substrates with
mammalian KDACs, and study substrate recognition and deacylase activity
in live Escherichia coli. In this system the bacterial
cell serves as a “biological test tube” in which the
incubation of a single mammalian KDAC and a potential peptide or full-length
acylated substrate transpires. We report novel deacetylation activities
of Zn2+-dependent deacetylases and sirtuins in bacteria.
We also measure the deacylation of propionyl-, butyryl-, and crotonyl-lysine,
as well as novel deacetylation of Lys310-acetylated RelA by SIRT3,
SIRT5, SIRT6, and HDAC8. This study highlights the importance of native
interactions to KDAC-substrate recognition and deacylase activity.
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