Studying the Lysine Acetylation of Malate Dehydrogenase

Abstract

Protein acetylation plays important roles in many biological processes. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, has been identified to be acetylated in bacteria by proteomic studies, but no further characterization has been reported. One challenge for studying protein acetylation is to get purely acetylated proteins at specific positions. Here, we applied the genetic code expansion strategy to site-specifically incorporate N^ε-acetyllysine into MDH. The acetylation of lysine residues in MDH could enhance its enzyme activity. The Escherichia coli deacetylase CobB could deacetylate acetylated MDH, while the E. coli acetyltransferase YfiQ cannot acetylate MDH efficiently. Our results also demonstrated that acetyl-CoA or acetyl-phosphate could acetylate MDH chemically in vitro. Furthermore, the acetylation level of MDH was shown to be affected by carbon sources in the growth medium.

Keywords: acetyltransferase; deacetylase; genetic code expansion; post-translational modification; protein acetylation.

Figure 1. AcK incorporation into MDHs

A) SDS-PAGE and western blotting analyses of purified MDHs and their variants. Lane 1, wild-type eMDH; lane 2, eMDH 99AcK; lane 3, eMDH 140AcK; lane 4, eMDH 162AcK; lane 5; wild-type hMDH2; lane 6, hMDH2 185 AcK; lane 7, hMDH2 301AcK; lane 8, hMDH2 307AcK; lane 9, hMDH2 314AcK. The same amounts of proteins were loaded. B) The enzyme activities of MDHs and their acetylated variants. The activities of wild-type E. coli MDH and human MDH2 were set as 1, respectively. The mean values and standard errors were calculated based on three replicates.

Figure 2. CobB deacetylates MDHs

A) Western blotting of purified MDHs from TOP10 and TOP10 ΔcobB cells. Lane 1 and 4 were from wild-type TOP10 cells. Lane 2 and 5 were from TOP10 ΔcobB cells. Lane 3 was from TOP10 ΔcobB ΔyfiQ cells. The same amounts of proteins were loaded. B) The enzyme activities of purified MDHs from TOP10 and TOP10 ΔcobB cells. The activity of eMDH purified from wild-type TOP10 cells was set as 1. The mean values and standard errors were calculated based on three replicates. C) Western blotting of acetylated MDH variants treated with the CobB protein. The acetylation levels of MDH variants after 2-hour incubation were compared with those without CobB treatment. The same amounts of proteins were loaded.

Figure 3. Acetylation of E. coli MDH

A) The enzyme activities of purified eMDHs from TOP10, TOP10 ΔcobB, and TOP10 ΔcobB ΔyfiQ cells. The activity of eMDH purified from TOP10 cells was set as 1. The mean values and standard errors were calculated based on three replicates. B) Western blotting of purified eMDH from BL21(DE3) treated with the YfiQ protein and acetyl-CoA for 2 hours. The same amounts of proteins were loaded. C) B) Western blotting of purified eMDH from BL21(DE3) treated with acetyl-CoA (AcCoA) or acetyl-phosphate (AcP) for 2 hours and 12 hours, respectively. The same amounts of proteins were loaded.

Figure 4. Acetylation of E. coli MDH with different carbon sources

A) Western blotting of purified eMDHs from TOP10 cells grown with different carbon sources. LB medium, minimal medium with different glucose (Glu) concentrations (5%, 2%, 1%, or 0.5%) were used. The same amounts of proteins were loaded. B) The enzyme activities of purified eMDHs from TOP10 cells grown with different carbon sources. The activity of eMDH purified from TOP10 cells grown with LB medium was set as 1. The mean values and standard errors were calculated based on three replicates.

Figure 5. Mapping of acetylation sites on the crystal structures of MDHs

The crystal structures of eMDH (PDB ID: 1EMD) and hMDH2 (PDB ID: 2DFD) were demonstrated with the selected acetylation sites in this study labelled.