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. 2010 May;76(4):874-88.
doi: 10.1111/j.1365-2958.2010.07127.x. Epub 2010 Mar 16.

Reversible N epsilon-lysine acetylation regulates the activity of acyl-CoA synthetases involved in anaerobic benzoate catabolism in Rhodopseudomonas palustris

Heidi A Crosby  1 , Erin K Heiniger, Caroline S Harwood, Jorge C Escalante-Semerena
Affiliations

Reversible N epsilon-lysine acetylation regulates the activity of acyl-CoA synthetases involved in anaerobic benzoate catabolism in Rhodopseudomonas palustris

Heidi A Crosby et al. Mol Microbiol. 2010 May.

Abstract

Rhodopseudomonas palustris grows photoheterotrophically on aromatic compounds available in aquatic environments rich in plant-derived lignin. Benzoate degradation is regulated at the transcriptional level in R. palustris in response to anoxia and the presence of benzoate and/or benzoyl-CoA (Bz-CoA). Here, we report evidence that anaerobic benzoate catabolism in this bacterium is also regulated at the post-translational level. In this pathway, benzoate is activated to Bz-CoA by the AMP-forming Bz-CoA synthetase (BadA) enzyme. Mass spectrometry and mutational analysis data indicate that residue Lys512 is critical to BadA activity. Acetylation of Lys512 inactivated BadA; deacetylation reactivated BadA. Likewise, 4-hydroxybenzoyl-CoA (HbaA) and cyclohexanecarboxyl-CoA (AliA) synthetases were also reversibly acetylated. We identified one acetyltransferase that modified BadA, Hba and AliA in vitro. The acetyltransferase enzyme is homologous to the protein acetyltransferase (Pat) enzyme of Salmonella enterica sv Typhimurium LT2, thus we refer to it as RpPat. RpPat also modified acetyl-CoA (Ac-CoA) synthetase (Acs) from R. palustris. In vivo data indicate that at least two deacetylases reactivate BadA(Ac). One is SrtN (encoded by srtN, formerly rpa2524), a sirtuin-type NAD(+)-dependent deacetylase (O-acetyl-ADPribose-forming); the other deacetylase is LdaA (encoded by ldaA, for lysine deacetylase A; formerly rpa0954), an acetate-forming protein deacetylase. LdaA reactivated Hba(Ac) and AliA(Ac)in vitro.

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Figures

Figure 1
Figure 1. Anaerobic benzoate catabolism in R. palustris
A. Pathways for degradation of benzoate, 4-hydroxybenzoate, and cyclohexanecarboxylate in R. palustris. BadA, benzoyl-CoA synthetase; HbaA, 4-hydroxybenzoyl-CoA synthetase; AliA, cyclohexanecarboxyl-CoA synthetase. Adapted from (Egland & Harwood, 1999). B. Alignment of protein sequences near C-terminal end of acyl- and aryl-CoA synthetases (residues 504-523 of BadA). Arrow indicates lysine residue that is acylated in S. enterica Acs and PrpE. Rp, R. palustris; Se, S. enterica; Acs, Ac-CoA synthetase; PrpE, propionyl-CoA synthetase. Alignment generated with ClustalW (Thompson et al., 1994) and ESPript (Gouet et al., 1999).
Figure 2
Figure 2. Modeling the effect of Nε-Lys acetylation on the structure of benzoyl-CoA synthetase from B. xenovorans LB400
A.The crystal structure of Bz-CoA synthetase from B. xenovorans LB400 (PDB 2V7B) was reported with benzoate (shown in purple) in the active site (Bains & Boulanger, 2007). A close-up view of this region shows the hydrogen bonds formed between the oxygen atoms of benzoate (red) and the epsilon amino group of residue Lys520 (blue), with bond distances indicated. B. An acetylated Lys residue was modeled in lieu of Lys520 using Pymol (http://www.pymol.org). Acetylation of Lys520 suggests the loss of hydrogen bonding between the epsilon amino group of Lys520 and the carboxylic acid oxygen atoms of benzoate.
Figure 3
Figure 3. Photoheterotrophic growth of R. palustris on benzoate
(A) Growth behavior of the R. palustris ΔldaA ΔsrtN strain carrying a plasmid encoding a wild-type allele of ldaA or srtN. Data points are averages of three replicates, and error bars represent standard deviations. Open triangles, wild type (CGA009) harboring plasmid pBBR1MCS-2 as a vector control; filled triangles, strain JE11636 (ldaA srtN / pBBR1MCS-2); circles, strain JE11637 (ldaA srtN / pRpLDAA4); squares, strain JE11638 (ldaA srtN / pRpSRTN4). (B) Growth behavior of the R. palustris ΔldaA ΔsrtN Δpat strain (open circles) compared to the wild type (open triangles) and the ΔldaA ΔsrtN strain (filled triangles). Optical density was monitored during photosynthetic growth on 3 mM benzoate
Figure 4
Figure 4. Residue K512 of BadA is the site of acetylation
LC/MS-MS analysis of H10-BadA protein purified from R. palustris strain JE11639 (ldaA srtN / pRpBADA4) grown photosynthetically on benzoate (3 mM). MS/MS spectrum of the 915.52 amu tryptic peptide, where peaks in red indicate b series m/z (predicted fragment ion masses of TATGKAcIQR with the charge on the NH2-terminal amino acid), and blue peaks indicate y series m/z (predicted fragment ion masses of TATGKAcIQR with the charge on the COOH-terminal amino acid). These ion series confirm that the sequence of the 915.52 amu peptide as TATGKAcIQR, which corresponds to the predicted acetylation site of Lys512.
Figure 5
Figure 5. Acetylation of BadA, HbaA, AliA, and Acs proteins using [14C-1]-Ac-CoA
Either wild-type (WT) or Lys-to-Ala variants of CoA synthetases were incubated with [14C-1]-Ac-CoA with or without RpPat. Top panel shows SDS-PAGE of acetylation reactions; lower panel shows the phosphor image of the same gel.
Figure 6
Figure 6. Deacetylation of BadA, HbaA and AliA using LdaA in cell lysates
A.[14C]-BadAAc protein was incubated with cell lysates of E. coli harboring either plasmid pRpLDAA1 (triangles) or cloning vector (squares). Aliquots of the reaction were quenched at each time point, resolved using SDS-PAGE, and quantified by phosphor imaging. Data represent averages and standard deviations of three reactions. B. BadA, HbaA and AliA activity is restored upon deacetylation. Each acyl-CoA synthetase (or no-synthetase control) was pre-incubated with or without Pat and acetyl-CoA, and the acetylation reaction was stopped by buffer exchange. Cell-free extracts of strains JE11958 (empty vector) or JE11959 (pRpLDAA1) were added to the reaction, and acyl-CoA synthetase activity was measured in triplicate.
Figure 7
Figure 7. Proposed model for the posttranslational regulation of BadA
In this model, BadA protein is acetylated by RpPat (and possibly by other unknown acetyltransferases) at residue Lys512, rendering it inactive. BadA is reactivated by the deacetylases LdaA and SrtN. LdaA uses water to hydrolyze the acetyl group, releasing acetate, whereas SrtN uses NAD+ as a substrate, generating O-acetyl-ADP-ribose (O-AADPR) and nicotinamide (Nm). Regulation of HbaA and AliA is predicted to be similar to that for BadA. CoASH, coenzyme A; PPi, pyrophosphate.
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