Abstract
Posttranslational regulation of protein function by acetylation is present throughout nature. Regulation of protein function by Sir2 protein (sirtuin) deacetylases is conserved in all domains of life. In the prokaryote Salmonella enterica, the metabolic enzyme acetyl-coenzyme A synthetase (Acs) is regulated by a Sir2-dependent protein acetylation/deacetylation system (SDPADS). The recent identification of the acetyltransferase enzyme responsible for the acetylation of Acs defined the SDPADS in prokaryotes. This report identifies one residue in Acs, Leu-641, which is critical for the acetylation of Acs by the protein acetyltransferase enzyme. In vivo and in vitro evidence shows that mutations at Leu-641 prevent the acetylation of Acs by protein acetyltransferase, maintain the Acs enzyme in its active state, and bypass the need for sirtuin deacetylase activity during growth on acetate.
Highlights
Sir2 proteins are biologically conserved NADϩ-dependent protein deacetylases involved in the posttranslational modification of a wide variety of protein substrates including histones (1–5), tumor suppressor protein p53 (6, 7), and microtubule protein ␣-tubulin (8)
The Variant AcsL641P Enzyme Is No Longer Posttranslationally Controlled by the Sir2-dependent protein acetylation/deacetylation system (SDPADS)—We used a genetic approach to identify derivatives of Acs that were no longer acetylated by protein acetyltransferase (Pat). cobB patϩ strains of S. enterica do not grow at low acetate concentrations (Յ10 mM), because in the absence of CobB sirtuin deacetylase Acs remains acetylated (37)
The effect of the L641P mutation on Acs activity or stability has not been further characterized. Results from these studies suggest that the structural contributions of residue Leu-641 of Acs are critical for its interaction with the Pat protein acetyltransferase
Summary
All bacterial strains used in this study were derivatives of the S. enterica serovar Typhimurium LT2. Bacterial strains were grown on nocarbon E minimal medium (NCE) (26), supplemented with potassium acetate as the source of carbon and energy, MgSO4 (1 mM), and Lmethionine (0.5 mM). Luria-Bertani broth (LB) was used as rich medium. Growth behavior was analyzed in 96-well microtiter dishes (BD Biosciences) using a computer-controlled BioTek EL808-I Ultra microplate reader (BioTek Instruments Inc.) with the incubation chamber set at 37 °C. A 2-l inoculum of an overnight culture of S. enterica was used to seed 198 l of freshly prepared minimal medium in each well; the medium was supplemented with 10 mM potassium acetate as the carbon and energy source.
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