Identification of novel protein lysine acetyltransferases inEscherichia coli

Abstract

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. Together, these results emphasize distinct and specific non-enzymatic and enzymatic protein acetylation mechanisms present in bacteria.ImportanceNε-lysine acetylation is one of the most abundant and important post-translational modifications across all domains of life. One of the best-studied effects of acetylation occurs in eukaryotes, where acetylation of histone tails activates gene transcription. Although bacteria do not have true histones, Nε-lysine acetylation is prevalent; however, the role of these modifications is mostly unknown. We constructed an E. coli strain that lacked both known acetylation mechanisms to identify four new Nε-lysine acetyltransferases (RimI, YiaC, YjaB, and PhnO). We used mass spectrometry to determine the substrate specificity of these acetyltransferases. Structural analysis of selected substrate proteins revealed site-specific preferences for enzymatic acetylation that had little overlap with the preferences of the previously reported acetyl-phosphate non-enzymatic acetylation mechanism. Finally, YiaC and YfiQ appear to regulate flagellar-based motility, a phenotype critical for pathogenesis of many organisms. These acetyltransferases are highly conserved and reveal deeper and more complex roles for bacterial post-translational modification.

Abundances of transcripts, proteins, and metabolites in the cell cycle of budding yeast reveal coordinate control of lipid metabolism

“…Briefly, samples were analyzed by reverse-phase HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) with a cHiPLC system (Eksigent), which was directly connected to a quadrupole time-of-flight (QqTOF) TripleTOF 6600 mass spectrometer (SCIEX, Concord, Canada) ( Christensen et al , 2018 ). After injection, peptide mixtures were loaded onto a C18 precolumn chip (200 µm × 0.4 mm ChromXP C18-CL chip, 3 µm, 120 Å, SCIEX) and washed at 2 µl/min for 10 min with the loading solvent (H 2 O/0.1% formic acid) for desalting.…”

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions

“…This phenomenon is not exclusive to glucose. Lactate, fructose, and xylose can also potently induce acetylation in E. coli as assessed by anti-acetyllysine western blot (Schilling et al, 2015, 2019; Christensen, 2019). Mass spectrometric analysis has determined the acetylomes of cells grown in xylose, a pentose sugar, and cells grown in glucose, a hexose sugar (Schilling et al, 2019).…”

Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation

“…In E. coli two distinct mechanisms are responsible for Nε‐lysine acetylation. The Nε‐lysine acetyltransferases, such as the best characterized YfiQ (also known as PatZ or Pka) and the recently discovered RimI, YiaC, YjaB and PhnO catalyze acetylation of specific lysines using acetyl‐CoA as the acetyl donor (Christensen et al , ). An alternative non‐enzymatic mechanism involves acetyl phosphate and is a dominant form of acetylation in E. coli.…”