Dataset Information

The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites.

ABSTRACT: N(?) -lysine acetylation is an abundant posttranslational modification of thousands of proteins involved in diverse cellular processes. In the model bacterium Escherichia coli, the ?-amino group of a lysine residue can be acetylated either catalytically by acetyl-coenzyme A (acCoA) and lysine acetyltransferases, or nonenzymatically by acetyl phosphate (acP). It is well known that catalytic acCoA-dependent N(?) -lysine acetylation can be reversed by deacetylases. Here, we provide genetic, mass spectrometric, structural and immunological evidence that CobB, a deacetylase of the sirtuin family of NAD(+) -dependent deacetylases, can reverse acetylation regardless of acetyl donor or acetylation mechanism. We analyzed 69 lysines on 51 proteins that we had previously detected as robustly, reproducibly, and significantly more acetylated in a cobB mutant than in its wild-type parent. Functional and pathway enrichment analyses supported the hypothesis that CobB regulates protein function in diverse and often essential cellular processes, most notably translation. Combined mass spectrometry, bioinformatics, and protein structural data provided evidence that the accessibility and three-dimensional microenvironment of the target acetyllysine help determine CobB specificity. Finally, we provide evidence that CobB is the predominate deacetylase in E. coli.

SUBMITTER: AbouElfetouh A

PROVIDER: S-EPMC4335977 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites.

AbouElfetouh Alaa A Kuhn Misty L ML Hu Linda I LI Scholle Michael D MD Sorensen Dylan J DJ Sahu Alexandria K AK Becher Dörte D Antelmann Haike H Mrksich Milan M Anderson Wayne F WF Gibson Bradford W BW Schilling Birgit B Wolfe Alan J AJ

MicrobiologyOpen 20141122 1

N(ε) -lysine acetylation is an abundant posttranslational modification of thousands of proteins involved in diverse cellular processes. In the model bacterium Escherichia coli, the ε-amino group of a lysine residue can be acetylated either catalytically by acetyl-coenzyme A (acCoA) and lysine acetyltransferases, or nonenzymatically by acetyl phosphate (acP). It is well known that catalytic acCoA-dependent N(ε) -lysine acetylation can be reversed by deacetylases. Here, we provide genetic, mass sp ...[more]

PMID: 25417765

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Project description:Lysine acetylation is a protein posttranslational modification (PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometry on a proteome-wide scale remains challenging. Most methods employ a comparison of chemically acetylated peptides to native acetylated peptides, however, the potentially large differences in abundance between these peptides presents a challenge for accurate quantification. Stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry (MS) is one of the most widely used quantitative proteomic methods. Here we show that serial dilution of SILAC-labeled peptides (SD-SILAC) can be used to identify accurately quantified peptides and to estimate the quantification error rate. We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild-type and Sirtuin deacetylase CobB-deficient cells. To further analyze CobB-regulated sites under conditions of globally increased or decreased acetylation, we measured stoichiometry in phophotransacetylase (pta?) and acetate kinase (ackA?) mutant strains in the presence and absence of the Sirtuin inhibitor nicotinamide. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurred at a low stoichiometry. Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such as stationary-phase arrest and deletion of ackA, resulted in globally increased acetylation stoichiometry. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. Similar to Sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry in WT, pta?, and ackA? cells. Together, our results provide a detailed view of acetylation stoichiometry in E. coli and suggest an evolutionarily conserved function of Sirtuin deacetylases in suppressing low stoichiometry acetylation.

Dataset Information

The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites.

Publications

The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites.

Similar Datasets

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