Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May 4;287(19):15590-601.
doi: 10.1074/jbc.M112.352104. Epub 2012 Mar 13.

System-wide Studies of N-lysine Acetylation in Rhodopseudomonas Palustris Reveal Substrate Specificity of Protein Acetyltransferases

Affiliations
Free PMC article

System-wide Studies of N-lysine Acetylation in Rhodopseudomonas Palustris Reveal Substrate Specificity of Protein Acetyltransferases

Heidi A Crosby et al. J Biol Chem. .
Free PMC article

Abstract

N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.

Figures

FIGURE 1.
FIGURE 1.
Identification of RpKatA, a new acyl-CoA synthetase acetyltransferase in R. palustris. A, purified RpKatA acetylated cyclohexanecarboxyl-CoA synthetase (AliA) with 14C-labeled acetyl-CoA. Residue Lys-532 of AliA was required for acetylation by RpKatA. The top panel shows the SDS-PAGE gel, and the bottom panel is the phosphorimage of the same gel. An alignment of RpKatA with select protein acetyltransferases is presented in supplemental Fig. S1. B, the N-Lys acylation/deacylation system that controls the activities of acyl-CoA synthetases in R. palustris. ACS represents a generic acyl-CoA synthetase that activates an organic acid to an acyl-CoA thioester. R. palustris has two acetyltransferases, RpPat and RpKatA, and two deacetylases, RpSrtN and RpLdaA, which can act on acyl-CoA synthetases. RpSrtN is a sirtuin-type deacetylase that uses NAD+ as a co-substrate and produces O-acetyl-ADP-ribose (O-AADPR) and nicotinamide (Nm), whereas RpLdaA produces free acetate upon deacetylation of acyl-CoA synthetases.
FIGURE 2.
FIGURE 2.
Newly identified acyl-CoA synthetases. The activities of six putative acyl-CoA synthetases were tested in vitro with a variety of organic acids. Mono- and dicarboxylic acids are indicated as CX and DCX, respectively, where X specifies the carbon chain length. Compound names are listed under “Experimental Procedures.” Data are averages from 6 replicates; standard deviations were calculated from the same data sets.
FIGURE 3.
FIGURE 3.
Acetylation of newly identified acyl-CoA synthetases. Top, acyl-CoA synthetases were incubated with [1-14C]acetyl-CoA and without (lane A) or with (lane B) RpPat. In lane C, the lysine residue homologous to K512A in BadA was changed to alanine, and the variant protein was incubated with RpPat. The top panel shows SDS-PAGE of acetylation reactions, and the bottom panel shows the phosphorimage of the same gel. Bottom, acetylation of the same set of proteins with RpKatA.
FIGURE 4.
FIGURE 4.
Deacetylation of newly identified acyl-CoA synthetases using cell-free extracts enriched for RpLdaA. 14C-Acetylated acyl-CoA synthetases were incubated with cell lysates of E. coli harboring either plasmid pRpLDAA1 or cloning vector. Deacetylated reactions were resolved using SDS-PAGE, and the specificity of bacterial protein acetyltransferases was quantified by phosphorimaging and normalized to the reaction mixture devoid of cell-free extract. Data represent averages and S.D. of three independent experiments.
FIGURE 5.
FIGURE 5.
Probing acetylation of other proteins reported to be substrates of Pat. A, R. palustris proteins BadA (positive control), CbbG, and Rpa1177 were incubated with [1-14C]acetyl-CoA and with or without RpPat or RpKatA. B, S. enterica proteins Acs (positive control), AceA, AceK, GapA, PckA, Pgk, and SodB were incubated with [1-14C]acetyl-CoA and with or without SePat. Molecular mass standards in kDa are shown on the left side of the gel. Top panels show SDS-PAGE of acetylation reactions, and the bottom panels show the phosphorimage of the same gels.
FIGURE 6.
FIGURE 6.
Alignment of RpPat substrates centered on acetylated lysine residue. A, the acyl-CoA synthetases that are acetylated by RpPat were aligned using ClustalW (57), and the figure was generated using ESPript (58). B, MEME motif generated from the alignment in A (E-value = 8.9 × 10−35) (59). The height of each letter corresponds to the frequency of that amino acid residue in that position. Arrows indicate the acetylated lysine residue (Lys-512 in BadA).

Similar articles

See all similar articles

Cited by 35 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback