Mechanisms, Detection, and Relevance of Protein Acetylation in Prokaryotes

Abstract

Posttranslational modification of a protein, either alone or in combination with other modifications, can control properties of that protein, such as enzymatic activity, localization, stability, or interactions with other molecules. N-ε-Lysine acetylation is one such modification that has gained attention in recent years, with a prevalence and significance that rival those of phosphorylation. This review will discuss the current state of the field in bacteria and some of the work in archaea, focusing on both mechanisms of N-ε-lysine acetylation and methods to identify, quantify, and characterize specific acetyllysines. Bacterial N-ε-lysine acetylation depends on both enzymatic and nonenzymatic mechanisms of acetylation, and recent work has shed light into the regulation of both mechanisms. Technological advances in mass spectrometry have allowed researchers to gain insight with greater biological context by both (i) analyzing samples either with stable isotope labeling workflows or using label-free protocols and (ii) determining the true extent of acetylation on a protein population through stoichiometry measurements. Identification of acetylated lysines through these methods has led to studies that probe the biological significance of acetylation. General and diverse approaches used to determine the effect of acetylation on a specific lysine will be covered.

Keywords: acetylation; acetylome; bacteria; lysine acetyltransferase; mass spectrometry; proteomics.

FIG 1

Types of chemical (nonenzymatic) and enzymatic protein acetylation in prokaryotes and comparison of superfamilies of lysine acetyltransferases (KATs) in eukaryotes and prokaryotes. (A) Chemical acetylation of an N-ε-lysine of a protein can occur with acetyl phosphate (AcP; yellow arrows) or acetyl coenzyme A (AcCoA; blue arrows), whereas enzymatic acetylation of an N-ε-lysine of a protein utilizes AcCoA and a KAT (purple arrows). The acetyl functional group is shown in red. (B) A variety of KATs are found in eukaryotes and prokaryotes, including members of the p300/CBP, MYST, GNAT, and YopJ effector superfamilies. To date, p300/CBP and MYST are found only in eukaryotes, whereas YopJ effectors are found only in prokaryotes. GNATs are found in both eukaryotes and prokaryotes.

FIG 2

Classification of GNAT KATs in prokaryotes. Prokaryotic KATs can be currently divided into three classes (I, II, and III) based on their size. These classes are further subdivided into types (I to V) based on the number of GNAT domains present in a particular sequence, the arrangement of domains, and type of domain (i.e., allosteric/regulatory or not). Characterized representatives are listed in colored boxes below each type.

FIG 3

Metabolic pathways that generate AcP. (A) The EMP pathway produces pyruvate, which is converted to AcCoA. AcCoA can be fermented as acetate via the Pta-AckA pathway, through which AcP is made as an intermediate. In E. coli and other bacteria, PoxB (pyruvate oxidase) is expressed in stationary phase and directly converts pyruvate to acetate. Alternatively, in Streptococcus pneumoniae and other bacteria, SpxB (pyruvate oxidase) instead converts pyruvate to AcP. (B) The heterolactic acid pathway found in some lactic acid bacteria uses a phosphoketolase (EC 4.1.29) that cleaves xylulose-5-phosphate into glyceraldehyde-3-phosphate and AcP, which can be converted to ethanol or acetate. (C) The Bifidobacterium shunt generates AcP in two separate reactions with the bifunctional phosphoketolase XFP.

FIG 4

Global acetylation is diminished in exponentially growing cells. Cells were grown overnight in M9 minimal medium supplemented with 0.4% glucose (lane 1) and subsequently diluted into fresh M9 supplemented with 0.4% glucose. Samples were harvested hourly, normalized for protein concentration, and analyzed by antiacetyllysine Western blot assay as described previously (49, 130).