Characterizing lysine acetylation of glucokinase

Abstract

Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose-6-phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site-specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl-phosphate without enzymes and deacetylated by CobB deacetylase.

Keywords: deacetylase; genetic code expansion; glucokinase; glycolysis; lysine acetylation.

FIGURE 1

The incorporation of acetyllysine (AcK) at individual lysine acetylation site of Escherichia coli glucokinase (GK). Sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting analyses of purified GK and its variants from BL21(DE3) cells. Lane 1, marker; Lane 2, wild‐type GK; Lanes 3–9, acetylated GK variant at individual lysine site. The same amounts of proteins were loaded for each lane. The full picture of western blotting is in Figure S1.

FIGURE 2

Relative enzyme activities of Escherichia coli GK and its variants. (a) The activity of WT E. coli GK was set as 1. Mean values and standard deviations were calculated based on three replicates. Two‐tailed p values were determined by the t‐test, and the significance level is 0.05. **p Value is less than 0.001. (b) The structures of acetyllysine, lysine, and glutamine.

FIGURE 3

Tests of YfiQ‐mediated acetylation of Escherichia coli glucokinase (GK). SDS–PAGE and western blotting of (a) purified native GK from the WT, ΔyfiQ, ΔcobB, or ΔyfiQ ΔcobB of BW25113 cells growth in 0.2% glucose media. The full picture of western blotting is in Figure S9. (b) Purified WT GK from BL21(DE3) cells incubated with purified YfiQ and 0.2 mM AcCoA for 1 h. YfiQ itself was slightly acetylated when purified from the A Complete Set of Escherichia coli K‐12 ORF Archive (ASKA) collection. The full picture of western blotting is in Figure S10. AcK, acetyllysine.

FIGURE 4

Acetylation of Escherichia coli glucokinase (GK) by acetyl‐phosphate (AcP). SDS–PAGE and western blotting of (a) purified GK from BL21(DE3) cells treated with 1 mM AcP at the start point, 30 and 60 min in vitro. The full picture of western blotting is in Figure S11. (b) Purified native GK from the WT, Δpta, or ΔackA of BW25113 cells growth in 0.2% glucose media. The full picture of western blotting is in Figure S18. AcK, acetyllysine.

FIGURE 5

In vitro deacetylation of Escherichia coli glucokinase (GK) by CobB. (a) SDS–PAGE and western blotting of site‐specifically acetylated GK variants incubated with purified CobB in vitro. Numbers above each lane are the positions of acetylated lysine residues. GK and CobB have similar molecular weights and merged in the SDS–PAGE gel. The full picture of western blotting is in Figure S19. (b) Mapping of lysine residues resistant to CobB in vitro on the GK crystal structure protein data bank (PDB ID: 1SZ2). (c) Mapping of lysine residues sensitive to CobB in vitro on the GK crystal structure (PDB ID: 1SZ2). Lysine residues are labeled in red and from the same monomer of the GK dimer. AcK, acetyllysine.

FIGURE 6

The enzyme activities of purified glucokinase (GK) from WT, ∆ackA, Δpta, or ∆cobB cells grown in 0.2% glucose media before and after 1‐h CobB treatment. The activity of GK purified from the WT strain was set as 1. Mean values and standard deviations were calculated based on three replicates. Two‐tailed p values were determined by the t‐test between the activities of GK from each strain before and after CobB treatment, and the significance level is 0.05. **p Value is less than 0.001.

FIGURE 7

Crystal structures of Escherichia coli glucokinase (GK) and human GK. The PDB ID for E. coli GK is 1SZ2, and the PDB ID for human GK is 4DCH. The acetylation sites are marked as red, and the substrate glucose (Glc) is marked as purple. Both GKs are shown as monomers.