TY - JOUR
T1 - Reconstitution of Mammalian Enzymatic Deacylation Reactions in Live Bacteria Using Native Acylated Substrates
AU - Avrahami, Emanuel M.
AU - Levi, Shahar
AU - Zajfman, Eyal
AU - Regev, Clil
AU - Ben-David, Oshrit
AU - Arbely, Eyal
N1 - Funding Information:
This work was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation programme, grant agreement 678461 (to E.A.) and by the Israel Science Foundation (grant 807/15 to E.A.). We thank Inbar Segal for assistance with statistical analysis.
Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/10/19
Y1 - 2018/10/19
N2 - 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.
AB - 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.
KW - KDAC
KW - genetic code expansion
KW - histone deacetylase
KW - lysine acetylation
KW - sirtuin
UR - http://www.scopus.com/inward/record.url?scp=85053698437&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.8b00314
DO - 10.1021/acssynbio.8b00314
M3 - Article
AN - SCOPUS:85053698437
SN - 2161-5063
VL - 7
SP - 2348
EP - 2354
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 10
ER -