Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

Abstract

Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue's charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation.IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.

Keywords: acetylphosphate; central metabolism; enolase; protein acetylation; proteomics.

FIG 1

Analysis of pathways enriched in lysine-acetylated proteins. For each organism, we determined whether the set of acetylated proteins was enriched in a given KEGG pathway, calculating the enrichment with a Fisher exact test. The graph shows the average enrichment P values across the 48 different bacterial strains.

FIG 2

Substrate-binding lysine residues in metabolic enzymes are evolutionarily conserved and acetylated in different organisms. In many glycolytic and TCA cycle catalytic binding sites, lysine is present and universally conserved. The binding pockets of four enzymes are shown with their native substrate or cofactor. Catalytically required lysine residues are shown and outlined with spheres to show their proximity to the substrate or cofactor. (A) Succinyl-CoA synthetase from E. coli, PDB 1CQI, shown with the ADP cofactor and lysine 46. (B) Phosphotransacetylase from Methanosarcina thermophila, PDB 2AF4, shown with coenzyme A and two lysine residues 257 and 283. (C) Triosephosphate isomerase from Staphylococcus aureus, PDB 3UWU, shown with glycerol-3-phosphate and lysine 12. Catalytically essential histidine and glutamic acid residues are also shown. (D) Phosphoglycerate kinase from Francisella tularensis, PDB 4FEY, shown with the ADP cofactor and lysine residues 193 and 197.

FIG 3

Lysine conservation in enolase across bacteria. A multiple-sequence alignment of enolase showing residues N334 to T349 and G387 to N404 (B. subtilis; P37869). Phylogenetic clusters are shown in color to highlight the prominent taxonomic divisions as follows: pink for Alphaproteobacteria; blue for Gamma- and Betaproteobacteria; orange for Bacteroidetes; purple for Actinobacteria; green for Firmicutes. The lysine residues boxed in red are invariant across all organisms.

FIG 4

Acetylation of substrate/cofactor-binding lysine residues across taxa. The phyloproteomic data identify acetylated lysines from 48 organisms. Enzymes from glycolysis are listed along with universally conserved lysine residues, numbered according to the numbering system for Bacillus subtilis (except for GpmA from L. casei gene S6CBB3). Catalytically essential sites as described in the text are shown in boldface type. Nonboldfaced sites are listed if they are universally conserved across bacteria but are not known to be involved in substrate/cofactor binding. Observed acetylations are indicated with solid blue circles. The phylogenetic tree of organisms is based on RplB sequence alignment with the major taxonomic groups colored: Alphaproteobacteria, Gamma- or Betaproteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. Protein name abbreviations: Pgi, phosphoglucose isomerase; Fba, fructose-bisphosphate aldolase; Tpi, triose-phosphate isomerase; Pgk, phosphoglycerate kinase; GmpA, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; Eno, enolase; Pyk, pyruvate kinase.

FIG 5

Structural change of enolase acetylation. (A) The catalytic binding site of Synechococcus elongatus enolase, PDB 5J04. The boxed region in panel A is enlarged in panels B and C. (B) The binding pocket utilizes two lysine residues (shown in blue at the top and bottom left of the pocket) to bind and stabilize phosphoenolpyruvate (shown as a stick diagram). (C) Acetylating the two active site lysines disrupts both the electrostatic binding potential and the geometry of the binding site, precluding substrate binding.

FIG 6

Enolase activity regulation by acetylation. (A) Western blot analysis of enolase treated with acetylphosphate (AcP). From right to left, serial 10 × dilutions of enolase incubated in the presence (+) or absence (−) of 15 µM acetylphosphate. MWM, molecular weight markers. (B) Colorimetric assay of serial 10× dilutions of enolase incubated in the presence or absence of 15 µM acetylphosphate. (C) Enzymatic activity assay for E. coli enolase with and without addition of acetyl phosphate. Acetyl phosphate was added at concentrations ranging from 1.5 nM to 150 μM. Acetylation of E. coli enolase by acetyl phosphate resulted in a dose-dependent inhibition of E. coli enolase activity. (D) Enzymatic activity assay showing that acetylation inhibits enolase catalytic activity. The assay was performed in triplicate. Enolase activity was measured in the presence or absence of 1.5 μM acetylphosphate. Along with wild-type E. coli and B. subtilis enzymes, four E. coli mutant strains were tested. In the 7xKtoQ mutant and 7xKtoR mutant, the seven lysine residues that are not conserved or at the active site were replaced with either glutamine (K→Q) or arginine (K→R) (see Materials and Methods). In the K393Q mutant and K342Q mutant, either active site lysine 393 or active site lysine 342 were mutated to glutamine (see Materials and Methods). wt, wild type.