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System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases.

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.

SUBMITTER: Crosby HA

PROVIDER: S-EPMC3346091 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The degradation of lignin-derived aromatic compounds such as benzoate has been extensively studied in Rhodopseudomonas palustris, and the chemistry underpinning the conversion of benzoate to acetyl coenzyme A (acetyl-CoA) is well understood. Here we characterize the last unknown gene, badL, of the bad (benzoic acid degradation) cluster. BadL function is required for growth under photoheterotrophic conditions with benzoate as the organic carbon source (i.e., light plus anoxia). On the basis of bioinformatics and in vivo and in vitro data, we show that BadL, a Gcn5-related N-acetyltransferase (GNAT) (PF00583), acetylates aminobenzoates to yield acetamidobenzoates. The latter relieved repression of the badDEFGAB operon by binding to BadM, triggering the synthesis of enzymes that activate and dearomatize the benzene ring. We also show that acetamidobenzoates are required for the expression of genes encoding the photosynthetic reaction center light-harvesting complexes through a BadM-independent mechanism. The effect of acetamidobenzoates on pigment synthesis is new and different than their effect on the catabolism of benzoate.IMPORTANCE This work shows that the BadL protein of Rhodopseudomonas palustris has N-acetyltransferase activity and that this activity is required for the catabolism of benzoate under photosynthetic conditions in this bacterium. R. palustris occupies lignin-rich habitats, making its benzoate-degrading capability critical for the recycling of this important, energy-rich biopolymer. This work identifies the product of the BadL enzyme as acetamidobenzoates, which were needed to derepress genes encoding benzoate-degrading enzymes and proteins of the photosynthetic apparatus responsible for the generation of the proton motive force under anoxia in the presence of light. In short, acetamidobenzoates potentially coordinate the use of benzoate as a source of reducing power and carbon with the generation of a light-driven proton motive force that fuels ATP synthesis, motility, transport, and many other processes in the metabolically versatile bacterium R. palustris.

Project description:Rhodopseudomonas palustris, a nonsulphur purple photosynthetic bacteria, has been extensively investigated for its metabolic versatility including ability to produce hydrogen gas from sunlight and biomass. The availability of the finished genome sequences of six R. palustris strains (BisA53, BisB18, BisB5, CGA009, HaA2 and TIE-1) combined with online bioinformatics software for integrated analysis presents new opportunities to determine the genomic basis of metabolic versatility and ecological lifestyles of the bacteria species. The purpose of this investigation was to compare the functional annotations available for multiple R. palustris genomes to identify annotations that can be further investigated for strain-specific or uniquely shared phenotypic characteristics. A total of 2,355 protein family Pfam domain annotations were clustered based on presence or absence in the six genomes. The clustering process identified groups of functional annotations including those that could be verified as strain-specific or uniquely shared phenotypes. For example, genes encoding water/glycerol transport were present in the genome sequences of strains CGA009 and BisB5, but absent in strains BisA53, BisB18, HaA2 and TIE-1. Protein structural homology modeling predicted that the two orthologous 240 aa R. palustris aquaporins have water-specific transport function. Based on observations in other microbes, the presence of aquaporin in R. palustris strains may improve freeze tolerance in natural conditions of rapid freezing such as nitrogen fixation at low temperatures where access to liquid water is a limiting factor for nitrogenase activation. In the case of adaptive loss of aquaporin genes, strains may be better adapted to survive in conditions of high-sugar content such as fermentation of biomass for biohydrogen production. Finally, web-based resources were developed to allow for interactive, user-defined selection of the relationship between protein family annotations and the R. palustris genomes.

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System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases.

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