Schilling - Wolfe_KAT_2018

Identification of novel protein lysine acetyltransferases in Escherichia coli
  • Organism: Escherichia coli
  • Instrument: TripleTOF 6600
  • SpikeIn: No
Post-translational modifications, such as Nε -lysine acetylation, regulate protein function. Nε-lysine acetylation can occur either non-enzymatically or enzymatically. The non-enzymatic mechanism uses acetyl phosphate (AcP) or acetyl coenzyme A (AcCoA) as acetyl donors to modify an Nε-lysine residue of a protein. The enzymatic mechanism uses Nε-lysine acetyltransferases (KATs) to specifically transfer an acetyl group from AcCoA to Nε-lysine residues on proteins. To date, only one KAT (YfiQ, also known as Pka and PatZ) has been identified in E. coli. Here, we demonstrate the existence of 4 additional E. coli KATs: RimI, YiaC, YjaB, and PhnO. In a genetic background devoid of all known acetylation mechanisms (most notably AcP and YfiQ) and one deacetylase (CobB), overexpression of these putative KATs elicited unique patterns of protein acetylation. We mutated key active site residues and found that most of them eliminated enzymatic acetylation activity. We used mass spectrometry to identify and quantify the specificity of YfiQ and the four novel KATs. Surprisingly, our analysis revealed a high degree of substrate specificity. The overlap between KAT-dependent and AcP-dependent acetylation was extremely limited, supporting the hypothesis that these two acetylation mechanisms play distinct roles in the post-translational modification of bacterial proteins. We further showed that these novel KATs are conserved across broad swaths of bacterial phylogeny. Finally, we determined that one of the novel KATs (YiaC) and the known KAT (YfiQ) can negatively regulate bacterial migration in soft agar. Together, these results emphasize distinct and specific non-enzymatic and enzymatic protein acetylation mechanisms present in bacteria.
Experiment Description
We processed isolated frozen bacterial pellets from the gutted strains carrying vector control (AJW5426) or one of the 5 KAT candidates (i) YjgM (AJW5496), (ii) RimI (AJW5499), (iii) YiaC (AJW5501), (iv) YjaB (AJW5504), (v) PhnO (AJW5513), as well as the known KAT YfiQ (AJW5497). Each of the strains was processed as 3 biological replicates.
Sample Description
Cell pellets of the indicated strains were suspended in 6 mL of PBS and centrifuged at 4°C, 15,000 g for 20 min. The firm cell pellet was collected and re-suspended and denatured in a final solution of 6 M urea, 100 mM Tris, 75 mM NaCl, and the deacetylase inhibitors tricostatin A (1 mM) and nicotinamide (3 mM). Samples were sonicated on ice (5× each for 15 sec), cellular debris removed, and the supernatants were processed for proteolytic digestion. Lysate containing 1.5 mg of protein was reduced with 20 mM DTT (37°C for 1 h), and subsequently alkylated with 40 mM iodoacetamide (30 min at RT in the dark). Samples were diluted 10-fold with 100 mM Tris (pH 8.0) and incubated overnight at 37°C with sequencing grade trypsin (Promega) added at a 1:50 enzyme:substrate ratio (wt/wt). In parallel, separate 1.5 mg protein aliquots were digested using endoproteinase Glu-C (Roche, Indianapolis, IN) by adding Glu-C at a 1:50 protease to substrate protein ratio (wt:wt), and incubating overnight at 37 °C. Subsequently, samples were acidified with formic acid and desalted using HLB Oasis SPE cartridges (Waters) (Keshishian et al., 2007). Proteolytic peptides were eluted, concentrated to near dryness by vacuum centrifugation, and re-suspended in NET buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). A small aliquot of each protein digestion (∼ 10 μg) was saved for protein-level identification and quantification. The remaining proteolytic peptide samples were used for affinity purification of acetylated peptides (Kac). Acetylated peptides were enriched using 1/4 tube of anti-acetyl lysine antibody-bead conjugated ‘PTMScan Acetyl-Lysine Motif [Ac-K]’ Kit (Cell Signaling Technologies) for each of the 1 mg protein lysate samples according to the manufacturer’s instructions. Prior to mass spectrometric analysis, the acetylated peptide enrichment samples were concentrated and desalted using C-18 zip-tips (Millipore, Billerica, MA).
Created on 7/5/18, 9:17 AM
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