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

Abstract

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 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.

Keywords: Acetyl phosphate; bacteria; crystallography; deacetylase; mass spectrometry; posttranslational modification.

Figure 1

Schematic of N^ε-lysine acetylation and deacetylation. K, lysine; P_i, pyrophosphate; CoA, coenzyme A; acCoA, acetyl-coenzyme A; acP, acetyl phosphate, NAD⁺, nicotinamide adenine dinucleotide; NAM, nicotinamide. (i) The canonical enzymatic acetylation catalyzed by YfiQ and using acCoA as the acetyl donor (green); (ii) the nonenzymatic reaction using acP as the acetyl donor (blue); and (iii) CobB acting as deacetylase.

Figure 2

Schematic of E. coli ribosomal subunits and other protein synthesis-related proteins showing proteins with acetyllysines that are sensitive to CobB. Proteins identified as acetytated in cobB mutants are circled in red, proteins containing verified CobB-sensitive acetyllysines are circled in orange. In addition to ribosomal and protein synthesis-related proteins, other DNA and RNA pathways are also shown (see WikiPathways and (Papanastasiou et al. 2013). Note that Papanastasiou and coauthors did not detect ribosomal subunit S17 and thus it is not depicted in the figure.

Figure 3

SAMDI MS peptide specificity profiles and LOGO plots for recombinant CobB protein and WT lysates. Activity of recombinant E. coli CobB (A) and of lysate from wild-type E. coli (strain MG1655; B) was determined using a GXKacZGC peptide library and SAMDI mass spectrometry. All data are the average of three separate trials (Table S4). Shading is indicative of percent conversion to product as shown. We did not detect any deacetylase activity in the lysate of the cobB deletion mutant (strain AJW5037). WebLogo (Crooks et al. 2004) was used to generate consensus sequence logos showing the amino acid composition in positions −1 to +1 relative to the recombinant CobB SAMDI peptide substrate lysines (C), the WT lysate SAMDI peptide substrate lysines (D), 69 cobB-sensitive lysines on 51 proteins (E), the 736 lysines on the same 51 proteins that were not cobB-sensitive (F), and the substrate lysines in 3D space (not including sequences where the adjacent residue could not be identified; see Table S7). SAMDI, self-assembled monolayers with matrix-assisted laser desorption-ionization.

Figure 4

Correlation of CobB-sensitive acetyllysines to protein structure. (A) Mass spectrometric quantification of relative abundance of acetyllysine-containing peptides and their fold change between the cobB mutant (strain AJW5037) and its WT parent (strain MG1655) grown in TB7 supplemented with 0.4% glucose. Data were obtained from four independent biological replicates and three technical replicates. Bar graphs show the average fold change (cobB/WT) for individual acetyllysine sites in the proteins GadA, YihD, GreA, and RplL. Acetyllysine sites with the most significant CobB-sensitive upregulation are highlighted in red, sites with significant upregulation in orange, and sites with relatively no change in blue. (B) E. coli crystal structures of these proteins (from PDB) are visualized using Pymol, indicating the lysine residues whose acetylation was monitored by mass spectrometry and shown in (A). Crystal structures for each protein are shown in cyan (PDB ID: chain A of 1XEY (GadA), 2KO6 (YihD), 1GRJ (GreA), and 1CTF (RplL)). Acetyllysine sites with the most significant CobB-sensitive upregulation are highlighted in red, sites with significant upregulation in orange, and sites with relatively no change in blue. In GadA, a portion of the K4 residue is disordered and residues K453 and K464 are not present in structure. PDB, Protein Data Bank.

Figure 5

Antiacetyllysine Western immunoblot analyses. Cells were aerated at 37°C in TB7 (A, C, F), in TB7 supplemented with 0.4% glucose (B, D), or in TB7 supplemented with 25 μg/mL chloramphenicol (E) and harvested at three time points, when the OD₆₁₀ reached 0.5 or 1.0, and then at 8 h. (A and B) cobB ackA epistasis analysis: E. coli WT (strain AJW678) and isogenic mutants ackA (strain AJW2067), cobB (strain AJW4343), and ackA cobB (strain AJW5024). The white arrow points to an ∼70-kDa band that is sensitive to CobB and less prominent in glucose. The black arrows point to bands that are observed only in the ackA cobB double mutant. (C and D) cobB yfiQ epistasis analysis: WT (strain AJW678) and isogenic mutants cobB (strain AJW4343), yfiQ (strain AJW4344), and cobB yfiQ (strain AJW5121). The white arrow points to an ∼70-kDa band that is sensitive to YfiQ and CobB. The black arrows point to bands that are observed only in the cobB yfiQ double mutant. (E) YfiQ overexpression: WT (strain AJW678) transformed with the vector pCA24n (strain AJW5126), the yfiQ mutant (strain AJW4344) transformed with the vector pCA24n (strain AJW5130) or with the YfiQ expression plasmid pCA24n-yfiQ (strain AJW5131), the cobB mutant (strain AJW4343) transformed with the vector pCA24n (strain AJW5129) or with the YfiQ expression plasmid pCA24n-yfiQ (strain AJW5124), and the double cobB yfiQ mutant (strain AJW5121) transformed with the vector pCA24n (strain AJW5128) or with the YfiQ expression plasmid pCA24n-yfiQ (strain AJW5125). YfiQ expression was induced by IPTG (15 or 25 μmol/L). The white arrow points to an ∼70-kDa band that is sensitive to YfiQ and CobB. The black arrows point to 3 more YfiQ-sensitive bands that depend on CobB for deacetylation. The gray arrow points to one YfiQ-sensitive protein that is not sensitive to CobB. (F) acs cobB epistasis analysis: E. coli WT (strain AJW678), and isogenic mutants acs (strain AJW1781), cobB (strain AJW4343), and acs cobB (strain AJW5185). The white arrow points to an ∼70-kDa band that is sensitive to CobB and depends on Acs.