Lysine Malonylome May Affect the Central Metabolism and

Article pubs.acs.org/jpr

Lysine Malonylome May Affect the Central Metabolism and Erythromycin Biosynthesis Pathway in Saccharopolyspora erythraea Jun-Yu Xu,† Zhen Xu,† Ying Zhou,† and Bang-Ce Ye*,†,‡ †

Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China ‡ School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang 832000, China S Supporting Information *

ABSTRACT: Lysine acylation is a dynamic, reversible post-translational modification that can regulate cellular and organismal metabolism in bacteria. Acetylome has been studied well in bacteria. However, to our knowledge, there are no proteomic data on the lysine malonylation in prokaryotes, especially in actinomycetes, which are the major producers of therapeutic antibiotics. In our study, the first malonylome of the erythromycin-producing Saccharopolyspora erythraea was described by using a high-resolution mass spectrometry-based proteomics approach and high-affinity antimalonyllysine antibodies. We identified 192 malonylated sites on 132 substrates. Malonylated proteins are enriched in many biological processes such as protein synthesis, glycolysis and gluconeogenesis, the TCA cycle, and the feeder metabolic pathways of erythromycin synthesis according to GO analysis and KEGG pathway analysis. A total of 238 S/T/Y/H-phosphorylated sites on 158 proteins were also identified in our study, which aimed to explore the potential cross-talk between acylation and phosphorylation. After that, site-specific mutations showed that malonylation is a negative regulatory modification on the enzymatic activity of the acetyl−CoA synthetase (Acs) and glutamine synthetase (Gs). Furthermore, we compared the malonylation levels of the two-growth state to explore the potential effect of malonylation on the erythromycin biosynthesis. These findings expand our current knowledge of the actinomycetes malonylome and supplement the acylproteome databases of the whole bacteria. KEYWORDS: protein acylation, malonylome, phosphorylome, Saccharopolyspora erythraea, post-translational modification, actinomycetes

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INTRODUCTION Cell-signaling networks that control various kinds of biological processes are reversibly regulated by protein post-translational modifications (PTM). Histone post-translation modification is used by cells to dynamically modulate chromatin structure and function and is well-studied.1,2 Modification of the ε-amino group on lysines can control almost all cellular functions and allow cells to rapidly respond to internal and external cues.3 Recent studies have found that there are more than 2000 acetylated proteins, including transcriptional factors, ribosomal proteins, and metabolic enzymes related to glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid and nitrogen metabolisms and secondary metabolisms. Acetylation is considered to be an important post-translational modification that regulates diverse cellular functions. The acetylation of lysine is catalyzed by acetyltransferases (KATs) and reversed by lysine deacetylases (KDACs), including three major families of acetyltransferases (GCN5, CBP/p300, and MYST) and two major families of deacetylases (HDAC1-11 and SIRT1-7). In eukaryotes, protein acetylation is important for its role in chromatin-associated processes within the nucleus and metabolisms within cytoplasm. The first nonhistone acetylation © XXXX American Chemical Society

target, tubulin, has been discovered 20 years after the modification was discovered on histone.4 In addition, protein acetylation has emerged as an important metabolic regulatory mechanism in bacteria since the discovery of acetylation of the Salmonella enterica acetyl−CoA synthetase in 2002 and the corresponding acetyltransferase in 2004.5,6 After that, several kinds of acetyltransferases have been identified in bacteria such as Bacillus subtilis, Escherichia coli, Mycobacterium tuberculosis, Rhodopseudomonas palustris, and Micromonospora aurantiaca and in Saccharopolyspora erythraea.7−12 Acetylation emerged on almost every enzyme in intermediary metabolism, which corresponds to different metabolic demands, and the acetylation of each enzyme may differentially affect enzymatic activity or protein stability.13 In recent years, the improvement of mass-spectrometry technologies subsequently enabled protein acetylation to be profiled on a global level in precise details, which was also applied in microbiology.14−18 In the past decade, lysine acetylome has been reported in E. coli extensively. A total of 85 acetylated proteins were identified Received: February 13, 2016

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DOI: 10.1021/acs.jproteome.6b00131 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research

Figure 1. Workflow for the global profiling of lysine malonylation in S. erythraea.

by Yu et al., of which 24 (28%) are involved in protein biosynthesis and 16 (19%) are involved in carbohydrate metabolism.19 Zhang et al. also found that over 70% of the 91 acetylated proteins in E. coli are linked with the metabolic process and translation regulatory process.20 After that, Zhang et al. increased the lysine acetylated proteins and acetylation sites to 3 times and 8 times in E. coli, respectively.21 In addition, the acetylome of other microorganisms has been reported, involving S. enterica, B. subtilis, Thermus thermophilus, Staphylococcus aureus, and Vibrio parahemolyticus.22−26 Acetyl−CoA can chemically acetylate lysine residues in vitro, and acetyl−CoA levels are correlated with acetylation levels in vivo, indicating that acetyl−CoA or acetyl−phosphate may acetylate proteins nonenzymatically in bacteria, which contributes to the extensive acetylation in the organisms.27 Thus, we can infer that kinds of acyl−CoA can catalyze the autoacylated reactions of substrate proteins, and acyl−CoA is also the cosubstrate of precursors for erythromycin. Intercellular acyl− CoA pools affecting the synthesis of secondary metabolites are related with the level of acylated proteins. Thus, it is important to describe the whole acylome in those erythromycinproducing actinomycetes. Our previous studies have analyzed the landscape of acetylome in S. erythraea, and 363 proteins are proven to be acetylated.28 Acetylated proteins are involved in many biological processes, such as protein synthesis, glycolysis and gluconeogenesis, the TCA cycle, fatty acid metabolism, secondary metabolism, and the feeder metabolic pathways of erythromycin synthesis. In addition, other acylations may also have influence on the protein synthesis process and metabolism process. In the process for producing erythromycin in S. erythraea, propionyl−CoA and methylmalonyl−CoA are the precursor metabolites of erythromycin production, which function as the starter unit and extender unit, respectively, and propionyl−CoA is derived from the dehydrogenation of malonyl−CoA. Thus, other acylomes are also deserving of study in S. erythraea. Up to now, several types of novel nonacetylation acylations have been identified, including crotonylation, succinylation, glutarylation, and formylation.29−33

Malonylation is a newly found acylation that has been proved by both pan anti-Kmal antibody and synthetic peptides.34 Malonyl−CoA is a vital intermediate in the pathways of cell metabolism and can be synthesized by acetyl−coenzyme A carboxylase (ACC), the carboxylation of acetyl−CoA catalyzed by propionyl−CoA carboxylase and the β-oxidation of oldchain-length dicarboxylic acids. Colak et al. identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts, which has been the largest malonylome until now. 35 Malonylation is thus important, but the knowledge of the malonylome in bacteria and the effect of malonylation on protein substrates is limited. In our study, we combined the high-affinity antimalonyllysine pan antibodies and highly sensitive mass spectrometry together with bioinformatics tools to describe a profiling of whole malonylome in S. erythraea, a Gram-positive bacterium that is related with produce of erythromycin (Figure 1). We identified 192 malonylation sites on 132 malonylated proteins and characterized the malonylated substrates on diverse cellular functions according to gene ontology (GO) and KEGG pathways. The identified malonylated proteins are involved in diverse pathways, especially in ribosomes and central metabolic enzymes. A total of three motifs were found, and protein− protein interaction networks and high-score clusters were analyzed by using bioinformatics tools. Moreover, we present a phosphoproteomic study of S. erythraea by using TiO2 enrichment and LC−ESI−MS/MS analysis; a total of 238 phosphorylation sites on 158 proteins were identified. We explored the potential relationship between acylations and phosporylation through analysis of the functional domains and the modified sites nearby. We further dissected the regulatory role of malonylation on acetyl−CoA synthetase (Acs) and glutamine synthetase (Gs) via site-specific mutagenesis analysis, and enzyme activity analysis showed that reversible lysine malonylation could inhibit the activity of Acs and Gs. After that, a quantitative analysis of malonylome was used to compare the malonylation levels of the exponential phase and stationary phase in S. erythraea. We identified 158 dynamic sites on 100 substrates, and there is no obvious difference between the malonylome in that two-growth state. However, it was B


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each sample were dried by speed Vac and stored at −80 °C for further use.

interesting that all six sites in methylmalonate-semialdehyde dehydrogenase (mmsA2), the enzyme that controls the production of propionyl CoA, were malonylated higher in the exponential phase. It may reflect the effect of malonylation on the erythromycin biosynthesis. Together, this is the first report about the rough description of malonylome in prokaryotes, and a more profound understanding of the specific acylated modification is further studied in the future.

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Enrichment of Malonylpeptides

All four fractions of each sample underwent immunoprecipitation by antimalonyllysine agarose beads (catalog no. PTM904; PTM Biolabs, Hangzhou, China), as previously reported.36 After gentle shaking at 4 °C for 12 h incubating with peptides, the beads were washed four times with NETN buffer (50 mM Tris−Cl, pH 8.0, 100 mM NaCl, 17 mM EDTA, and 0.5% NP40), twice with ETN buffer (50 mM Tris−Cl, pH 8.0, 100 mM NaCl, and 17 mM EDTA) and twice with water (pH 8.0). We used 0.1% trifluoroacetic acid to elute the peptides with malonylation modification three times from the beads. The peptides were dried by Speed Vac and were cleaned by with C18 ZipTips (Millipore, MA) for nano-HPLC−mass spectrometric analysis.

MATERIALS AND METHODS

Media and Culture Conditions

S. erythraea NRRL23338 was grown in tryptone soya broth with yeast (TSBY) medium (30 g of tryptone soya broth and 5 g of yeast extract per L of distilled H2O; purchased from Oxoid, Basingstoke, UK) at 30 °C and 200 rpm for 24 h. We added 1 mL cultured cells to new medium with or without 100 mM sodium malonate dibasic at 30 °C and 200 rpm for further culturing. During the exponential growth phase, cultured cells were harvested by centrifugation and cleaned by using PBS buffer. After centrifugation, cells were resuspend in lysis buffer (8 M urea supplemented with protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) and 20 mM nicotinamide) and then were under sonication. After being incubated on ice for 15 min, cell debris was removed by centrifugation at 6000g for 20 min. Protein concentration was monitored by the BCA method using lysis buffer as control.

Enrichment of Phosphopeptides

Proteins extract (4 mg) were digested by trypsin as mentioned above. We divided it to two fractions (2 mg/fraction); each was incubated three times with TiO2 (GL Sciences Inc., Japan) at a peptide-to-bead ratio of 1:2.5 in 750 μL of binding buffer (70% ACN, 5% TFA, 8% lactic acid). Each fraction was gently rotated for 40 min and then briefly centrifuged (2000g × 2 min). Phosphopeptides bound to the beads were washed in 150 μL of binding buffer five times followed by an additional wash with washing buffer I (30% ACN, 0.5% TFA) for one time and with washing buffer II (80% ACN, 0.5% TFA) for two times. The enriched phosphorylated peptides were eluted with elution buffer containing 15% ammonia and 80% ACN for two times and then dried using a vacuum centrifuge. Phosphopeptides were loaded onto C18 stage tip with a gradient elution (15% ammonia and 0%, 2%, 5%, 8%, 10%, and 40% acetonitrile for one fraction and 0%, 1%, 2%, 4%, 6%, and 60% for another). After combination, six fractions were ready for nano-HPLC− mass spectrometric analysis.

Western Blotting Analysis

Proteins (10 μg) cultured with or without sodium malonate dibasic were electrophoresed on SDS−polyacrylamide gels (12% acrylamide, 1.2% bis-acrylamide) and transferred to a nitrocellulose filter membrane for 90 min at 100 V. The membrane was blocked in 5% BSA blocking buffer for 60 min. Then, the membrane was incubated with antimalonyllysine antibody (1:1000, catalog no. PTM-901; PTM Biolabs, Hangzhou, China) in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 8.0, and 0.1% Tween 20) with 5% BSA for 2 h at room temperature; after the membrane was washed with PBST three times for 10 min each, it was incubated with horseradish-peroxidase-conjugated antirabbit IgG (1:5000) in PBST at room temperature for 1 h. After the membrane was washed with PBST three times for 10 min each, an ECL kit was used for signal detection.

Nano-HPLC and MS/MS Analyses

The malonylated peptides were dissolved in 4 μL of solvent A (0.1% (v/v) formic acid and 2% acetonitrile in water) and the solution were injected onto a manually packed reverse-phase C18 column (10 cm length × 75 μm inner diameter; C18 resin with 3 μm particle size; 90 Å pore diameter; Dikma Technologies Inc., Lake Forest, CA) coupled to an EASYnLC 1000 system (Thermo Fisher Scientific, Waltham, MA) and eluted by 60 min gradient with 8%−32% solvent B (0.1% formic acid and 10% water in acetonitrile) for 51 min, 32%− 48% solvent B for 5 min, 48%−80% solvent B for 1 min, and 80% solvent B for 3 min at a flow rate of 300 nL/min. The HPLC elute was directly electrosprayed into a LTQ orbitrap Elite mass spectrometer (Thermo Fisher Scientific) using a nanospray source. The mass spectrometric analysis was carried out in a data-dependent analysis with an automatic switch between a full MS scan using FTMS in the Orbitrap and an MS/MS scan using collision-induced dissociation (CID) in the dual linear ion trap. Full MS spectra with an m/z range of 350 to 1700 with a mass resolution of 240 000 at m/z 200 were acquired. The 15 most intense ions above a threshold ion count of 500 in each full MS spectrum were sequentially isolated for MS/MS fragmentation with a normalized collision energy of 35%. The isolation window (m/z) was set as 2. Ions with either a single charge or more than four charges were excluded from MS/MS fragmentation. The electrospray voltage was 2 kV.

In-Solution Tryptic Digestion and Fractioned by Sep-Pak Column

Protein extracts (10 mg) were precipitated with 20% volume trichloroacetic acid followed by centrifugation at 4000g for 15 min. After being washed twice with cold acetone, each resulting pellet was suspended in 100 mM NH4HCO3 (pH 8.0). Trypsin (an enzyme-to-substrate ratio of 1:50) was added to the suspension incubated at 37 °C for 12 h. Then, the peptides acquired were reduced by 5 mM DTT at 55 °C for 30 min and alkylated by 15 mM iodoacetamide in darkness for 0.5 h at room temperature. After that, 30 mM cysteine was added to the solution to terminate the reaction for 30 min at room temperature. Additional trypsin (an enzyme-to-substrate ratio of 1:100) was added for a 4 h complete digestion. The peptides were dried by Speed Vac. The tryptic peptides were separated by Sep-Pak Vac 6 cm3 (1 g) tC18 Cartridges (Waters, MA) with a gradient elution (5%, 20%, 40%, and 80% acetonitrile). A total of four fractions of C


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malonylation on Lys with phosphorylation on serine, threonine, tyrosine, and histidine residues. Phosphopeptides were considered as high-confidence phosphorylation events and taken for further analysis only if they had a localization probability of >0.75 and had a posterior error probability (PEP) score of 0.4) in the STRING database.40 We used the MCODE plug-in toolkit to identify highly connected clusters, and the interaction network was visualized by Cytoscape software (version 2.8.2).

Automatic gain control (AGC) was used to prevent overfilling of orbitrap and ion trap (3E4 for ion trap and 1E6 for orbitrap). The dynamic exclusion duration was set as 40 s. The phosphorylated peptides were handled in the same way and eluted by a 90 min gradient with 2%−8% solvent B for 13 min, 8%−18% solvent B for 34 min, 18%−35% solvent B for 37 min, 35%−80% solvent B for 3 min, and 80% solvent B for 3 min at a flow rate of 300 nL/min. Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) was used for analysis. Intact peptides were detected in the Orbitrap at a resolution of 120 000 at m/z 200. The electrospray voltage was 2.2 kV. Automatic gain control was used by setting the ion count at 5E5 for orbitrap. For MS scans, the m/z scan range was 300 to 1400 m/z. The isolation window (m/z) was set as 1, and precursor ions with intensity greater than 5000 are used for MS2 analysis. MS/MS acquisition was performed in top-speed mode with a 3 s cycle time. Ions with charge states 2+ to 6+ were fragmented in the ion trap by higher energy collisional dissociation (HCD) with normalized collision energy (NCE) of 32%. The dynamic exclusion duration was set as 65 s. Protein-Sequence Database Search and Bioinformatic Analysis

The identification of protein and malonylation site was performed by MaxQuant with an integrated Andromeda search engine (v. 1.4.1.2). Tandem mass spectra were searched against Uniprot Saccharopolyspora erythraea database (7154 sequences; release date, September 2015) concatenated with reverse decoy database and protein sequences of common contaminants. Trypsin/P was specified as cleavage enzyme allowing up to two missing cleavages, three modifications per peptide, and four charges. Main mass tolerance of precursor ion was 4.5 ppm, and mass error was set to 20 ppm for precursor ions and 0.5 Da for fragmentions. Carbamidomethylation on Cys was specified as the fixed modification, and oxidation on Met, malonylation on Lys, and acetylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for the protein, peptide, and modification site were specified at 0.01. Minimum peptide length was set at 7, and the minimum score for modified peptides is 40. The minimum peak length is 2, and the maximum peptide mass is 4600 Da. In the protein quantification, the minimum ratio count is 2. Lys malonylation site identifications with localization probability of less than 0.75 or from reverse or contaminant protein sequences were removed. After that, we also used a Thermo Proteome Discoverer v1.4.0.288 (Thermo Fisher Scientific) to generate .mgf files that were subsequently searched against the S. erythraea database with a Mascot search engine (v2.3.01, Matrix Science). The search parameters were: enzyme, trypsin−P; missed cleavage, 2; fixed modification, carbamidomethy-C; variable modification, acetyl (protein Nterm), oxidation-M, and malonylation-K; peptide mass tolerance, 10 ppm; fragment mass tolerance, 0.5 Da for .mgf files analyzed by Orbitrap Elite; and selected charge states, + 2, + 3, and +4. Malonylated peptides with mascot ion scores of >25 were used for further selection, and we used manually verification to improve the identification of the malonylated peptides.37 For phosphorylation identification, the maxquant software package version 1.4.1.2 with integrated Andromeda search engine was used to analyze the raw MS spectra against Uniprot S. erythraea database (7154 sequences). All other parameters in MaxQuant were set as mentioned except that we replaced

Cloning and Mutagenesis of Sace_0337 and Sace_1623 Protein

The list of primers used for PCR and mutagenesis are provided in Table S-10. The Sace_0337 and Sace_1623 gene was amplified by using primers 0337F, 0337R, 1623F, and 1623R, with the genomic DNA of S. erythraea as the template. The product was digested with EcoRI and HindIII and cloned into similarly digested pET28 to generate pET28-Sace_0337 and pET28-Sace_1623. The clone was confirmed by sequencing. Point mutations in the two reconstructed plasmids were generated by site-directed mutagenesis. Using primers 0337F, 0337R, 1623F, 1623R, 0337K620EF, 0337K620ER, 1623K357EF, and 1623K357ER, we obtained the mutation genes through overlap extension PCR. The mutation gene was cloned into pET28 for sequencing. Expression and Purification of the Two Enzymes and the Two Mutants

The proteins were expressed using the E. coli BL21 (DE3) strain. A single colony was selected to begin a 5 mL overnight culture, which was then used for inoculation in 50 mL of Luria−Bertani medium that was supplemented with 0.1% kanamycin. The cells were grown at 37 °C and then induced with 0.7 mM isopropyl-β-D-thiogalactoside at 20 °C overnight. Cells were harvested by centrifugation and resuspended in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and pH 7.4) and incubated on ice for 15 min. The cells were sonicated in PBS buffer, and cell debris was removed by centrifugation at 6000g for 20 min. The supernatant was purified with a nickel−nitrilotriacetic acid− agarose column (Merck). After discarding the flow through, the column was washed with 10 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH 8.0) to D


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dimethyl labeled cells of the two groups. We made technology triplicates, and data were taken by MaxQuant with the integrated Andromeda search engine (v.1.4.1.2). We used the similar parameters for MaxQuant as mentioned above and included the isotope dimethyl labeling pair search, in which the peptide N-terminal and lysine dimethylation (+28 Da; 2CH2) are set for light and (+32 Da; 2CD2) are set for heavy. Falsediscovery-rate thresholds for the protein, peptide, and modification site were fixed at 0.01. The ratios of malonylated peptides were normalized to the ratios of their corresponding proteins level. Normalized ratios of the peptides were used for further analysis.

remove the hybrid proteins, and bound proteins were eluted using a linear gradient from 20 to 250 mM imidazole in 50 mM NaH2PO4 and 300 mM NaCl, pH 8.0. The fractions were pooled and dialyzed against buffer P (37 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 5% glycerol, and pH 7.9). Protein concentration was monitored by the BCA method using buffer P as the control, and the amount of protein after concentration was analyzed by SDS-PAGE. In Vitro Acetyl−CoA Synthetase Assays

The activity of acetyl−CoA synthetase (Acs) was determined at 37 °C using a microplate reader (BioTek Instruments, Winooski, VT) in a transparent 384 well microplate at 340 nm. The standard reaction mixture contained 100 mM Tris− HCl (pH 7.7), 10 mM malate (pH 7.7), 0.2 mM coenzyme A, 8 mM ATP (pH 7.5), 1 mM NAD, 10 mM MgCl2, three units of malate dehydrogenase, 0.4 units of citrate synthase, and 10 μg of enzyme. The reaction was started with 100 mM potassium acetate. A single unit was defined as the amount of enzyme catalyzing the acetate-dependent formation of 1 mmol of NADH/min in the coupled assay.

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RESULTS AND DISCUSSION

Global Profiling of Lysine Malonylome in S. erythraea

Lysine malonylation is evolutionarily conserved PTM observed in mammalian cells and bacterial cells, which was first reported by mass spectrometry in 2011.34One year later, one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells were identified, respectively.42 In 2015, Nishida et al. reported 1137 sites of in vivo lysine malonylation from multiple cellular compartments, cataloging lysine malonylation’s regulation by SIRT5, which suggested the correlation between malonylation and glycolysis.43 However, to date, study about the global malonylome in prokaryotes has not been reported yet. We have known that malonate can be used in turn by Hela cells for lysine malonylation, and we suggested that a similar fashion can also be found in S. erythraea.34 We cultured S. erythraea with 100 mM malonate, and the lysine malonylation status was then increased in whole-cell lysate by Western blot analysis (Figure 2). To determine the global malonylated proteins of S.

In Vitro Glutamine Synthetase Assays

The assay for in vitro glutamine synthetase (GS) was carried out at 37 °C for 30 min (reaction mixture consisted of Tris, 100 mM; MgSO4, 80 mM; cysteine, 20 mM; L-glutamate, 20 mM; ATP, 40 mM; EGTA, 2 mM; protein sample, 50 μg; and hydroxylamine, 80 mM in the reaction volume of 300 μL (PH 7.4)). The reaction was terminated by adding 100 μL of color reagent (0.2 M TCA, 0.4 M FeCl3, and 5% (v/v) concentrated hydrochloric acid). The absorbance was determined at 540 nm by using a microplate reader (BioTek Instruments). The enzyme activity was represented by the formation of γglutamylhydroxamate per protein amount of time. Quantitative Analysis of Lysine Malonylome in S. erythraea by Using Stable Isotope Dimethyl Labeling

We cultured the S. erythraea as mentioned above and collected the cells both in the latter exponential phase and the middle stationary phase. In each time course, we cultured S. erythraea triplicately in parallel and combined all three parallel groups to one group for the sequent experiment. A total of 3 mg of proteins of each group were digested by trypsin and desalted by Sep-pak cartridges. The stable isotope dimethyl labeling method we used was based on the previously reported protocal with slight modification.41 In brief, 3 mg of peptides from each group were reconstituted in 300 μL of triethylammonium bicarbonate buffer (100 mM). Then, 15 μL of 20% CH2O or CD2O was added to the sample to be light- or heavy-labeled, respectively, and 15 μL of 3 M NaBH3 CN was added to both samples subsequently (light for the cells in the exponential phase and heavy for the cells in the stationary phase). The mixture was incubated for 1 h at room temperature. Labeling efficiency were checked for the ensuring of the data quality before mixed. After that, 15 μL of ammonia (20%) and 10 μL of TFA were added to the mixture to neutralize the excess labeling reagents and acidify for the solid-phase extraction. A gradient elution (9%, 15%, 20%, 25%, 30%, and 80%) was used for separating the sample, and three fractions are combined for subsequent antimalonyllysine pan antibodies enrichment. The details for antibody enrichment and MS analysis by Orbitrap Fusion mass spectrometer are described mentioned above. In parallel, we also quantified changes of protein expression using whole-cell lysates derived from a mixture of stable isotope

Figure 2. Western blotting analysis of malonylation levels in protein lysates from S. erythraea cells with a pan antimalonyllysine antibody (PTM-901) cultured with (right) or without (left) sodium malonate dibasic. Coomassie staining was used for the loading controls.

erythraea, we combined highly specific immune-affinity enrichment and LC−ESI−MS/MS analysis; tandem mass spectra were searched against the Uniprot S. erythraea protein database (7154 sequences; release date: September 2015) concatenated with the reverse decoy database and protein sequences of common contaminants. With FDR thresholds below 1% for peptides through the Mascot search engine and MaxQuant software, we identified all total malonylated 192 sites from 132 proteins in S. erythraea (Table S-1). All 192 spectra are presented in the Figure S-6. Malonylated peptides bear a unique mass signature in MS/MS spectra, in which satellite peak with a mass loss of 44 Da is specific feature because of the E


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Figure 3. Representative MS/MS spectra of malonylpeptides from 60 kDa chaperonin 1 (groEL), L-lysine-ε aminotransferase (lat), methylmalonate−semialdehyde dehydrogenase (mmsA2) and dihydrolipoamide succinyltransferase (sucB). (A) MS/MS spectra of a tryptic peptide ion FDK (ma) GYISPYFVTDSER. (B) MS/MS spectra of a tryptic peptide ion SGYTLSLTNTDPSK (ma) IR. (C) MS/MS spectra of a tryptic peptide ion TVADAK (ma) GDIQR. (D) MS/MS spectra of a tryptic peptide ion AK (ma) QNFEAAEGVK. The neutral loss of the carbon dioxide (CO2) is designated as “−44”. In the sequence of peptide, the site of modification is indicated as (ma) in red color. In both the sequence of peptide and the MS/MS spectra, blue color and red color represent the b ions and y ions, respectively. The yellow color represent the loss of H2O or NH3. Asterisks represent the neutral loss and the precursor m/z; charge state and mass error are shown for each spectrum.

cleoprotein complex (9.6%), and proton-transporting ATP synthase complex (2.6%) (Figure 4C), from which the proteins in ribosome are well-malonylated, which is in accordance with the acetylation. In agreement with these observations, InterProt domain analysis revealed that lysine-malonylated substrates were abundant with 2-oxoacid dehydrogenase acyltransferase, which is an important enzyme in cellular metabolism (Figure S1 and Table S-7). To explore the general function of lysine malonylation in S. erythraea, we analyzed the malonylproteome of S. erythraea through KEGG pathway analysis. The malonylated proteins involved in metabolism were further categorized into several subclasses as shown in Figure 4D and Table S-6. The results showed that seven major pathways were enriched, including the citrate cycle (10.4%); ribosome (9.6%); Valine, leucine, and isoleucine degradation (9.6%); glycolysis and gluconeogenesis (8.7%); aminoacyl−tRNA biosynthesis (5.2%); RNA degradation (3.5%); and pyruvate metabolism (6.9%). The malonylated proteins enriched in TCA cycle and glycolysis indicates that malonylation may be an important modification involved in the central metabolic pathway, and the ribosome pathway was also enriched in a high degree, which was consistent with other groups’ study that 36 out of total 54 proteins from the ribosome are found to be acetylated in S. aureus.25 Overall, the pervious study have proved that acetylation frequently occurs on highly conserved proteins such as metabolic enzymes, ribosomes, and chaperones, and it is tempting to suggest that the cellular metabolism may be functionally regulated by malonylation and that malonylated proteins involved in S. erythraea may affect the regulation of protein-synthesis machinery.

thermal decarboxylation that readily occurs among derivatives of malonic acid. In Figure 3, we have shown that some newly identified MS/MS spectra of malonylation peptides, such as FDK (ma) GYISPYFVTDSER in 60 kDa chaperonin 1 (groEL), SGYTLSLTNTDPSK (ma) IR in L-lysine-epsilon aminotransferase (lat), TVADAK (ma) GDIQR in methylmalonate-semialdehyde dehydrogenase (mmsA2), and AK (ma) QNFEAAEGVK in dihydrolipoamide succinyltransferase (sucB) in S. erythraea have the specific mass signature of a mass loss of 44 Da. GO Annotation Analysis of Lysine Malonylation in S. erythraea

To functionally characterize the list of malonylated proteins in S. erythraea, we performed gene ontology (GO) analysis based on the David web site (Table S-5). All malonylated proteins are classified into groups according to cell component, molecular function, and biological process. The classification results for both biological process and molecular function showed that the largest group of malonylated proteins are associated with translation and metabolism. In biological process, proteins associated with translation (FDR 1.30 × 10−11) and cellular metabolic process (FDR 8.75 × 10−06) account for 20% and 62% the total malonyl proteins, respectively. Moreover, the process related with tRNA aminoacylation, the hexose catabolic process, the alcohol catabolic process, and the macromolecule biosynthetic process were found to be highly enriched (Figure 4A). In molecular function, protein with structural constituent of ribosome has a higher tendency to be malonylated, and other processes such as RNA binding, structural molecule activity, aminoacyl−tRNA ligase activity, and purine nucleotide binding are also significantly enriched (Figure 4B). The cellular component analysis of malonylated substrates showed that the majority of the malonylated proteins were located in the nonmembrane-bounded organelle (11.3%), macromolecular complex (14.8%), ribosome (9.6%), ribonu-

Pattern Analysis of Malonylated Peptides in S. erythraea

Several studies on both eukaryotes and prokaryotes have proved preferences for amino-acid residues at particular positions surrounding the acetylated lysine and succinylated F


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Figure 4. Functional annotation of lysine malonylome: gene ontology annotations of Kmal sites for (A) biological process (top panel), (B) molecular function (upper middle panel), and (C) cellular component (lower-middle-bottom panel). (D) KEGG pathway analysis for lysine malonylated proteins (bottom panel).

lysine.15,20,24,44,45 To test if there are specific amino acids adjacent to malonylated lysines, we examined the amino-acid sequences surrounding malonylated lysines from −7 to +7 position in all 192 sites (Figure 5A−C and Table S-8). We have

removed the background by uploading the data of uniprot proteomic data of S. erythraea, and we found three similar motifs, defined as K(X6)Kmal, Kmal(X6)K, and K(X5)Kmal. That motif contains a conserved positively charged lysine G


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Figure 5. Malonylation motifs and conservation of malonylation sites. (A) The frequency of each amino-acid residue in that position is reflected by the −7 to +7 position of each letter and the central K represent the acetylated lysine. (B) The percentage of each motif presented in the pie chart. (C) The number of identified peptides containing the malonylated lysine in each motif.

unique protein phosphatase identified the systematic perturbations that occurs both in the phosphorylation and the acetylation network. Reciprocally, several protein-specific effects can also be found on phosphorylation by the deletion of the two putative N-acetyltransferases. In 2014, Cuauhtemoc et al. studied the phosphorylome of S. erythraea during six timed courses, and a total of 109 phosphopeptides from 88 proteins have been identified.48 In our study, we performed serine, threonine, tyrosine, and histidine phosphorylation through TiO2 enrichment and LC− ESI−MS/MS analysis (Figure S-5A) and identified 238 sites (220 peptides) on 158 proteins detected in the same set of growth conditions and time courses as acetylation and malonylation in S. erythraea (Table S-2); we have presented all 220 phosphorylated peptides in Figure S-6. The size of the phosphoproteome is larger than the malonylome but is a little smaller than the acetylome, and threonine phosphorylation is the majority (Figure S-5B−C). In Figure 7A, only 16 proteins have the three PTMs at the same time, including ATP synthase (atpA), which catalyze the biosynthesis of ATP, acetyl− coenzyme A synthetase (AcsA), which converts the acetate to acetyl−CoA, and succinyl−CoA ligase (SACE_6668), which acts as a vital role in synthesizing the erythromycin precursor methylmalonyl−CoA. Besides, 52 proteins from S. erythraea were shared between lysine acetylation and serine, threonine, tyrosine, and histidine phosphorylation, and 25 proteins were shared for lysine malonylation and serine, threonine, tyrosine, and histidine phosphorylation. In addition, we compared the same sites on proteins between the two acylation modifications and found that 87 proteins and 51 sites from S. erythraea were shared between lysine acetylation and lysine malonylation (Figure. 8B). We also learned from data that the average number of malonylation sites occurring in an individual protein was about 1.5. Of all 132 malonylated protein substrates, approximately 24% are malonylated at two or more sites, involving only 4% that possess no less than four malonylated lysine sites (Figure 8D), which is smaller than both acetylation (38% for more than two sites) and phosphorylation (27% for more than two sites) (Figure 8C,E). Heavily malonylated proteins include 60 kDa chaperonin 1 (eight sites), histone-like

located nearby to the modified lysine, which is similar to the acetylated motif (Kac(X4)K) proven in S. erythraea.28 In addition to that three obvious motifs present here, a large amount of malonylated proteins (45%) contain no obvious motifs, which is probably because of the diversity of acyltransferases and the effect of nonenzymatic acylation.46 Protein Interaction Network of Lysine Malonylation in S. erythraea

Protein PTMs are vital for regulating the protein−protein interactions, leading to the change of functions in cellular physiology by providing a docking site to recruit binding partners. Acyl−lysine modifications can regulate protein− protein interactions (for example, bromodomain-containing proteins have been shown to bind acetylated lysines, especially in histone tails). We used the STRING database to acquire the whole protein−protein interaction network by submitting the data of malonylated proteins to the Web site (Figure 6 and Figure S-2). After that, we also analyzed the clusters of highly interconnected networks by using the MCODE plugin tool in Cytoscape according to the STRING database (Figure S-3). In total, the protein−protein interaction network has 109 malonylated proteins as nodes and 3893 identified direct physical connections among them, including four clusters of highly interconnected networks shown in Figure 7A−D and Figure S-4. The cluster with the highest degrees (cluster1 score: 38.7) are the ribosome-related proteins, which is aligned with the results of GO analysis and KEGG pathway analysis. In addition, proteins involved in the citrate cycle (cluster2 score: 15.7); valine, leucine, and isoleucine degradation (cluster3 score: 9.8); and glycolysis/gluconeogenesis (cluster4 score: 6.7) are also clustered with higher degree. The scores and forms of all four clusters are also presented in Table S-9. Multiple PTMs Occurrence upon the Same Proteins

PTMs represent important regulatory states that when combined have been hypothesized to set up the cross-talk between each other. Previous study has explored the relativity between serine, threonine, tyrosine, and histidine phosphorylation and lysine acetylation in the bacterium Mycoplasma pneumoniae.47 Deletion of the only two protein kinases and its H


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Figure 6. Complete protein−protein interaction network modulated by malonylation was constructed from the identified malonylated proteins.

DNA binding protein (seven sites), Elongation factor Ts (six sites), 50S ribosomal protein L7/L12 (six sites), methylmalonate−semialdehyde dehydrogenase (four sites), and malate synthase (four sites) (Figure 8F). The 60 kDa chaperonin 1 is also highly succinylated in M. tuberculosis H37Rv, which possesses 11 sites.49 The reason may be that malonyl and succinyl are structurally similar to each other. From the data, about 62% of the proteins are acetylated in only one site, which is less than that of the malonylated proteins. Furthermore, we have analyzed the repeated sites and verified that some proteins shared same sites that were both acetylated and malonylated, such as six sites for 60 kDa chaperonin 1, four sites for 50S ribosomal protein L7/L12, and three sites for elongation factor Ts (Figure 8G). To further study the effect of malonylation on the functions of protein substrates and the potential cross-talk between two acylation and phosphorylation, we have summarized the modified sites nearby the functional domains or binding domains by choosing 5 representative proteins. Several novel malonylated sites tend to affect the function of the proteins

together with other PTMs such as acetylation and phosphorylation. For instance, in Figure 9A−E, K3 is malonylated in glutamine synthetase (sace_1623), which may affect the acetylation level of K18. In Acetyl-coenzyme A synthetase (acsA), the acetylation of K588 and phosphorylation of S644 may have effects in metal binding, and the active site (K620) of the vital enzyme in Glycolysis/Gluconeogenesis is malonylated. The mitochondrial translation factor elongation factor Ts (tsf) has been found to be malonylated (K97) and acetylated (K36 and K97) near the regions involved in Mg(2+) ion dislocation from EF-Tu. Moreover, ATP synthase (atpA) bears Nucleotide binding domains with malonylated and acetylated sites (K200) nearby and T256 is phosphorylated, which may interact with the malonylated site K273 and the active site in Succinyl-CoA ligase (sace_6668). The major difference between malonylation and acetylation is that malonylation has a negatively charged carboxyl group, but both of the two acylations can eliminate the positive charge from the ε-amino group of lysine. Through our analysis about site information, we found that malonylation may share the I


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Figure 7. Interaction network of lysine-malonylated proteins (listed in gene names) were analyzed by using Cytoscape software version 2.8.2. The lysine-malonylated proteins in the top one to four clusters were shown. (A) Cluster 1: MCODE score = 38.696, nodes = 23, and edges = 890. (B) Cluster 2: MCODE score = 15.682, nodes = 22, and edges = 345. (C) Cluster 3: MCODE score = 9.846, nodes = 13, and edges = 128. (D) Cluster 4: MCODE score = 6.75, nodes = 4, and edges = 27.

acetylome analysis have been reported, such as T. thermophilus and S. enteric.22,24 For instance, the key rate-limiting enzymes in glycolysis pathway, pyruvate kinase (pyk3) are found to be malonylated at K52 and K57, respectively. Moreover, other enzymes involved in that pathway, such as glucose-6-phosphate isomerase (pgiA), phosphoglycerate kinase (pgk), enolase (eno) and phosphoenolpyruvate carboxykinase (pckA) are also found to be malonylated at one or more sites. In TCA cycle, another two key rate-limiting enzymes, succinyl-CoA synthetase (sace_6668) and isocitrate dehydrogenase (sace_6636) are malonylated at two sites, respectively. Citrate synthase (sace_0649), which catalyzes the synthesis of citrate from oxaloacetate and aconitate hydratase (acn), which hydrolyases the citrate to isocitrate, are also malonylated. So the high appearance of malonylation in the glycolysis pathway and TCA cycle indicates that malonylation have a trend to affect the energy metabolism. In addition, the enzymes, such as dihydrolipoamide dehydrogenase (dldH2), dihydrolipoyl dehydrogenase (pdhD), flavoprotein disulfide reductase (lpdA) and pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase (Sace_5674), which are involved in pyruvate metabolic pathway, are also highly malonylated according to that figure. Malonylation also has an influence on the secondary metabolism, which can be inferred from Figure 10B.

same location with other PTMs or locate near other PTMs. In the phosphoproteome research, S. erythraea was cultured under the same condition and harvested at the same growth stage as the two acylomic studies to ensure the reliability of the three PTM analysis. In conclusion, it is highly possible that the malonylation may have a key role in affecting various proteins’ functions, such as the interaction between proteins, the enzyme activity of the proteins and the protein localization in cells. Lysine Malonylated Protein Involvement in Central Metabolism and Secondary Metabolism in S. erythraea

It has been proved that high levels of acetyl-CoA or acetylphosphate may lead to an increase in the acetylation of protein substrates in E. coli and lysine acetylation may have an import role in modulating metabolic pathway.50 Similar to acetylation, we inferred that malonylation can regulate the vital enzymes in metabolism pathway. We have conducted KEGG pathway analysis in Figure 4D and we realized that enzymes involved in central metabolism, such as glycolysis/gluconeogenesis and citrate cycle are highly malonylated. In Figure 10A, the central metabolic pathway may also be regulated by the malonylation of important enzyme among them. In glycolysis pathway and TCA cycle, we acquired malonylated enzymes in almost all the steps among them and our finding is similar to the results of other prokaryotes whose J


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Figure 8. Characterization of the lysine-malonylated, the lysine-acetylated, and the serine-, threonine-, tyrosine-, and histidine-phosphorylated S. erythraea proteins. (A) The Venn diagram illustrating the lysine-malonylated, lysine-acetylated, and the serine-, threonine-, tyrosine-, and histidinephosphorylated proteins. (B) Venn diagram showing the number of Kmal only, Kac only, and overlapping sites and proteins between the two modifications. (C) Pie chart showing the total number of lysine-acetylated sites per protein. (D) Pie chart showing the total number of lysinemalonylated sites per protein. (E) Pie chart showing the total number of serine-, threonine-, tyrosine-, and histidine-phosphorylated sites per protein. (F) The comparison between the lysine-acetylated sites, the lysine-malonylated sites, and the serine-, threonine-, tyrosine-, and histidinephosphorylated sites per protein. (G) The same sites shared in lysine-acetylated proteome and lysine-malonylated proteome per protein.

Figure 9. Annotation of lysine acetylation, malonylation sites, and S/T/Y/H-phosphorylated sites in identified proteins.

Erythromycin is supposed to be an important secondary metabolite in S. erythraea, which can treat the disease infected with Gram-positive bacteria, such as Staphylococcus aureus, Bordetella pertussis, Enterococcus faecalis. Propionyl-CoA and methylmalonyl-CoA are the precursor metabolites of erythromycin production as the starter unit and extender unit, respectively. In Figure 9, methymalonyl-CoA can be derived from the carboxylation of propionyl-CoA and rearrangement of

succinyl-CoA. Propionyl-CoA can be derived from the hydration and dehydrogenation of malonyl-CoA. In the biosynthesis of the extender unit methylmalonyl-CoA, succinyl−CoA ligase (sace_6668 and sucC), which controls the synthetic of succinyl−CoA, is highly acetylated and malonylated in multiple sites, indicating that the two acylated modifaications may affect the precursor metabolites synthesis. In addition, various enzymes in the pathway from malonyl− K


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Figure 10. Malonylated enzymes involved in major metabolic pathways. (A) Schematic illustration of lysine-malonylation events identified in central metabolic pathways of S. erythraea proteins. (B) Schematic illustration of lysine-malonylation events identified in the S. erythraea proteins involved in the precursor biosynthesis of erythronolide.

Figure 11. Mutagenesis analysis of acetyl−CoA synthetase and glutamine synthetase assays: (A) MS/MS spectra of a tryptic peptide ion SGK (ma) IMR in acetyl−CoA synthetase. (B) MS/MS spectra of a tryptic peptide ion IPITGSNPK (ma) AK in glutamine synthetase. The site of modification is indicated as (ma). In the sequence of the peptide, asterisks represent the neutral loss and the precursor m/z; charge state and mass error are shown for the two spectra. (C) Sequence alignment of acetyl−CoA synthetase: Sace_0337 from S. erythraea, SCO3563 from Streptomyces coelicolor, Rv3667 from Mycobacterium tuberculosis, b4069 from Escherichia coli, BSU29680 from Bacillus subtilis, Dgri_GH23686 from Drosophila grimshawi, and 55902 from Homo sapiens. (D) Purity of wide-type and mutated acetyl−CoA synthetase shown by SDS-PAGE gel. (E) Enzymatic activities of WT, K620 acetyl−CoA synthetase. (F) Sequence alignment of glutamine synthetase: Sace_1623 from S. erythraea, SCO2198 from S. coelicolor, Rv2220 from M. tuberculosis, b3870 from E. coli, MSMEG_4290 from Mycobacterium smegmatis, and BSU6051_17460 from B. subtilis. (G) Purity of wide-type and mutated glutamine synthetase shown by SDS-PAGE gel. (H) Enzymatic activities of WT and K357 glutamine synthetase.

L


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Figure 12. Stoichiometry analysis of lysine malonylome. (A) Scatter plot showing the peptide intensities of the quantifiable lysine malonylated peptides in the two-growth state. The Kmal ratio (WT-stationary phase to WT-exponential phase) and MS signal intensity from stationary phase divided by that from the exponential phase. Blue plot, ratio ≤ 0.5; red plot, 0.5 < ratio ≤ 1; green plot, 1 < ratio ≤ 2; purple plot, 2 < ratio. (B) Schematic illustration of dynamic lysine-malonylation changes identified in the S. erythraea proteins involved in the precursor biosynthesis of erythronolide. Circle in different colors presented the different levels of malonylation. Blue plot, ratio ≤ 0.5; red plot, 0.5 < ratio ≤ 0.67; green plot, 0.67 < ratio ≤ 1.5.

previous study has proven that acetylation on the K620 site of Acs may affect its activity.12 In our study, we identified that the conserved site of Acs is also malonylated. To explore the potential effect of malonylation on the regulation of Acs, we mimicked the malonylated state by converting K to E. Next, we measured the enzymatic activity of Acs and its mutants by using the method we previously used.12 The results showed that activity of AcsK620E has decreased obviously (Figure 11B−C), which is similar to acetylation.12 Furthermore, we speculate that malonylation may also affect the metabolism of amino acids, and we found that glutamine synthetase is malonylated in our results. Glutamine synthetase catalyzes the formation of glutamine from glutamate and the ammonium ion, and the change of its activity may disturb the whole nitrogen metabolism; we used a similar method for generating and purifying Gs, and its mutated enzyme was used for Acs and utilized McCormacks’ method to analyze the activity.51 The site we chose for mutation is K357, which is identified as malonylated in our study, and the site is conversed in large amounts of bacteria (Figure 11D). When the site is converted to E, the activity of GsK357E has just slightly decreased compared with that of GsWT (Figure 11E,F). In conclusion, we suggested that malonylation on both the K620 of Acs and K357 on Gs may be a negative regulatory modification on the two enzyme activities, and the reversible malonylation may have an important influence on the central metabolism and secondary metabolism.

CoA to propanoyl−CoA are also malonylated, including enoyl−CoA hydratase−isomerase (echA), which catalyzes the biosynthesis of acryloyl−CoA from 3-hydroxy-propionyl−CoA and methylmalonate−semialdehyde dehydrogenase (mmsA2 and mmsA3), which converts the methylmalonate semialdehyde to propanoyl−CoA. The result suggests that lysine malonylation may affect the relative activities of metabolic enzymes in the precursor-supplied pathways and modulate metabolic flux for the biosynthesis of erythromycin. When comparing with acetylome, we found that malonylated enzymes are also enriched in that pathway. There are no enzymes malonylated for producing erythromycin in direct process, such as EryBII (sace_0727), which is acetylated. However, considering the lower level in cells for malonylation than acetylation, we infer that more malonylated substrates will be identified with the development of mass spectrometry technologies. According to this result, other acylomes that are linked with the erythromycin biosynthesis pathway, such as succinylome, methylmalonylome and propionylome, are further developed in S. erythraea. Effect of Malonylation on Acetyl−CoA Synthetase and Glutamine Synthetase

The acetate-scavenging, AMP-forming acetyl−CoA synthetase (Acs) belongs to the acyl−adenylate-forming superfamily, which are ubiquitous enzymes whose activity is central to the metabolism of prokaryotic and eukaryotic cells. It plays a vital role in central metabolism and erythromycin biosynthesis pathway (Figure 10), which is important for maintaining adequate levels of Acs−CoA and supplies precursors for both propionyl−CoA and methylmalonyl−CoA. A proposed acetylation motif (PXXXXGK) was conserved from prokaryotes to eukaryotes (Figure 11A) in acetyl−CoA synthetase. Our

Quantitative Analysis of Malonylome in the Two-Growth State in S. erythraea

Our previous study has demonstrated that erythromycin biosynthesis is directly correlated with the time course.52 A typical erythromycin production curve indicates that the M


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Journal of Proteome Research specific secondary metabolite for S. erythraea increases when the stationary phase begins. So we infer that malonylation may change on a number of or just a few malonylated sites of some important enzymes during the different growth states. Using a stable isotope dimethyl labeling-based quantitative proteomics approach, we analyzed the change in Kmal site levels between the latter exponential phase and the middle stationary phase of that bacteria on the basis of the levels of Kmal peptides and those of protein expression. After we collected cells from the two-growth state, we prepared the protein lysates and digested them from the two groups in parallel. Stable isotope dimethyl labeling was then performed according to the published protocol. Briefly speaking, the same amount of tryptic digests (3 mg) were labeled with light (CH2O; NaBH3CN; +28 Da) and heavy (CD2O; NaBH3CN; +32 Da) dimethyl labeling reagents, respectively. After the confirmation of the labeling efficiency, the two groups of peptides were mixed and then separated by the Sep-Pak Vac 6 cm3 (1 g) tC18 cartridges (Waters) with a gradient elution (9%, 15%, 20%, 25%, 30%, and 80% acetonitrile). A total of three fractions were acquired after combination, which were used for antimalonyllysine pan antibody enrichment and then analyzed by Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). The results indicates that the quantification of unique 158 malonylated sites on 100 proteins by MaxQuant analysis (Tables S-3 and S-4). After the normalization to protein abundance, the distribution of malonylated site relative abundances were presented. The median ratio of the quantifiable Kmal sites was 0.97 (Figure 12A). These result suggests that there is no obvious difference among the two-growth states. However, when we analyze the feeder pathways of erythromycin precursor biosynthesis, we found something interesting. Propionyl−CoA and methylmalony−CoA are the direct precursors for the production of 6deoxyerythronolide B. In the S. erythraea, the methylmalonyl− CoA mutase pathway, which catalyzes the reversible isomerization of succinyl−CoA from the Krebs cycle, the fatty acid catabolic pathway, which catalyzes the production of propionyl−CoA through long-chain acyl−CoA degradation, and the methylmalonate semialdehyde dehydrogenase, which converts methylmalonate semialdehyde to propionyl−CoA, are connected to the methylmalonyl−semialdehyde pools and propionyl−CoA pools. Comparing with the other two pathways, the malonylation level of the enzyme through the Valine degradation changed obviously. In the six sites in the methylmalonate semialdehyde dehydrogenase (mmsA2), the level of malonylation is higher in the latter exponential phase than that in the middle stationary phase (Figure 12B). After consideration of the possible negative regulation of malonylation on the enzymes, such as Acs and Gs, we infer that malonylation may act as an important switch for tuning the concentration of propionyl−CoA in cells, which closes that pathway when the bacteria grows and opens it as the bacteria begins to produce the secondary metabolite, erythromycin. Further studies are need to prove the effect of malonylation on that important enzyme.

now, no malonylome data were available in any high G+C Gram-positive actinomycetes or even in bacteria. Knowledge on lysine malonylation and its functions is sparse. In our study, we reported the first investigation of lysine malonylation from the perspective of a proteome-wide analysis of the actinomycete S. erythraea. Compared with the previous study about the acetylome of S. erythraea, we improved the process to completely explore the possible malonylated proteins through separation of the peptides by Sep-Pak Vac 6 cm3 (1 g) tC18 cartridges after digestion. A total of 192 malonylated sites on 132 proteins are the largest malonylome data set in bacteria to the best our knowledge. According to GO analysis and KEGG analysis, malonylated proteins are enriched in the ribosome and the central metabolic pathway, which is similar to acetylation. In the motif analysis, three similar motifs were identified, which were defined as K(X6)Kmal, Kmal(X6)K, and K(X5)Kmal, and we acquired four clusters in the protein−protein interaction network with the higher degrees by using MCODE plugin tool in Cytoscape according to the STRING database. After that, we performed serine, threonine, tyrosine, and histidine phosphorylation through TiO2 enrichment and LC− ESI−MS/MS analysis and identified 238 sites on 158 proteins. We wanted to explore the potential cross-talk between malonylation, acetylation, and phosphorylation. We found 52 proteins from S. erythraea were shared between lysine acetylation and serine, threonine, tyrosine, and histidine phosphorylation; 25 proteins were shared between lysine malonylation and serine, threonine, tyrosine, and histidine phosphorylation; and 87 proteins and 51 sites from S. erythraea were shared between lysine acetylation and lysine malonylation. Subsequently, we replaced lysine with glutamic acid to mimic the effect of malonylation on the two sites of acetyl−CoA synthetase and glutamine synthetase, and the results showed that malonylation may have a potential negative regulation on the two important enzyme in central metabolism and secondary metabolism. Furthermore, we quantified the difference in Kmal substrate levels between the bacteria in the exponential phase and the stationary phase on the basis of the levels of Kmal peptides and those of protein expression. The whole level of the malonylation almost presented the consistency, while a key enzyme mmsA2, which controls the production of the propionyl−CoA pools from valine degradation, bears a higher malonylated abundance in the exponential phase than that in the stationary phase. Although we infer that malonylation may inhibit the activity of the important enzyme, more experiments are need to be addressed in the further study. In the article, we proved that the function of malonylation may be similar to acetylation through bioinformatics and biochemical analysis; there is something diverse between the two PTMs.53 Malonylation belongs to acidic modifications, which changes the charge on lysine from +1 to −1 under physiological conditions. That peculiarity of malonylation is more similar to phosphorylation than acetylation, which changes the charge on lysine from +1 to 0. Thus, we can infer that malonylation may mimic the function as the phosphorylation on lysine, and the difference of function between the two acylations is an interesting direction to explore. Our further task is to explore other acylomes that may affect the production of erythromycin, such as methylmalonylome and propionylome, and to investigate the real function roles of

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CONCLUSIONS Lysine malonylation is a dynamic and reversible PTM in prokaryotes that may regulate gene expression, protein synthesis, and enzyme activity. Actinomycetes are filamentous bacteria that are the major producers of therapeutic antibiotics, and malonyl−CoA is the one of the precursor metabolites for therapeutic antibiotics and anticancer drugs. However, until N



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ACKNOWLEDGMENTS This work was supported by grants from the China NSF (21276079 and 21335003) and SRFDP (20120074110009) of the Chinese Ministry of Education, the National Key Technologies R & D Programs (2014AA02150), and Fundamental Research Funds for the Central Universities.

acylation on the identified vital metabolism enzymes, such as mmsA2, which are more closely involved in erythromycin biosynthesis pathway than is acetyl−CoA synthetase. In addition, there is no evidence about the effective acyltransferases and deacylases for malonylation and other nonacetyllysine acylations in microrganisms, although CobB has a limited ability to act as an eraser for removing the succinyl groups of substrates in the recent research.54

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REFERENCES

(1) Huang, H.; Sabari, B. R.; Garcia, B. A.; Allis, C. D.; Zhao, Y. SnapShot: histone modifications. Cell 2014, 159, 458−458. (2) Huang, H.; Lin, S.; Garcia, B. A.; Zhao, Y. Quantitative proteomic analysis of histone modifications. Chem. Rev. 2015115, 2376− 2418.10.1021/cr500491u (3) Wellen, K. E.; Thompson, C. B. A two-way street: reciprocal regulation of metabolism and signaling Protein lysine acylation and cysteine succination by intermediates of energy metabolism. Nat. Rev. Mol. Cell Biol. 2012, 13, 270−276. (4) L'Hernault, S. W.; Rosenbaum, J. L. Chlamydomonas alphatubulin is posttranslationally modified by acetylation on the epsilonamino group of a lysine. Biochemistry 1985, 24, 473−478. (5) Starai, V. J.; Celic, I.; Cole, R. N.; Boeke, J. D.; EscalanteSemerena, J. C. Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 2002, 298, 2390−2392. (6) Starai, V. J.; Escalante-Semerena, J. C. Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica. J. Mol. Biol. 2004, 340, 1005−1012. (7) Gardner, J. G.; Escalante-Semerena, J. C. Biochemical and mutational analyses of AcuA, the acetyltransferase enzyme that controls the activity of the acetyl coenzyme a synthetase (AcsA) in Bacillus subtilis. J. Bacteriol. 2008, 190, 5132−5136. (8) Castaño-Cerezo, S.; Bernal, V.; Blanco-Catalá, J.; Iborra, J. L.; Cánovas, M. cAMP-CRP co-ordinates the expression of the protein acetylation pathway with central metabolism in Escherichia coli. Mol. Microbiol. 2011, 82, 1110−1128. (9) Nambi, S.; Basu, N.; Visweswariah, S. S. cAMP-regulated protein lysine acetylases in Mycobacteria. J. Biol. Chem. 2010, 285, 24313− 24323. (10) Crosby, H. A.; Pelletier, D. A.; Hurst, G. B.; EscalanteSemerena, J. C. System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases. J. Biol. Chem. 2012, 287, 15590−15601. (11) Xu, J.; You, D.; Leng, P.; Ye, B. Allosteric regulation of a protein acetyltransferase in Micromonospora aurantiaca by the amino acids cysteine and arginine. J. Biol. Chem. 2014, 289, 27034−27045. (12) You, D.; Yao, L.; Huang, D.; Escalante-Semerena, J. C.; Ye, B. Acetyl-CoA synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with single GNAT domain in Saccharopolyspora erythraea. J. Bacteriol. 2014, 196, 3169−3178. (13) Shi, L.; Tu, B. P. Protein acetylation as a means to regulate protein function in tune with metabolic state. Biochem. Soc. Trans. 2014, 42, 1037−1042. (14) Olsen, J. V.; Mann, M. Status of large-scale analysis of posttranslational modifications by mass spectrometry. Mol. Cell. Proteomics 2013, 12, 3444−3452. (15) Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.; Walther, T. C.; Olsen, J. V.; Mann, M. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325, 834−840. (16) Schwer, B.; Eckersdorff, M.; Li, Y.; Silva, J. C.; Fermin, D.; Kurtev, M. V.; Giallourakis, C.; Comb, M. J.; Alt, F. W.; Lombard, D. B. Calorie restriction alters mitochondrial protein acetylation. Aging Cell 2009, 8, 604−606. (17) van Oudenhove, L.; Devreese, B. A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics. Appl. Microbiol. Biotechnol. 2013, 97, 4749−4762. (18) Porteus, B.; Kocharunchitt, C.; Nilsson, R. E.; Ross, T.; Bowman, J. P. Utility of gel-free, label-free shotgun proteomics

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00131. Figure S-1: INTERPRO domain analysis for lysine malonylated proteins in S. erythraea. Figure S-2: Parameters of network of malonylated protein in Figures 6 and 7. Analysis for networks of malonylated proteins using Cytoscape (2.8.2) software and STRING database. Figure S-3: Parameters of MCODE tool for clusters of malonylated protein in Figure 7A−D. Figure S-4: The details of four clusters with high scores by using cytoscope. Figure S-5: Overview of the phosphorylated peptides enrichment results: (A) overview of the phosphorylated peptides enrichment used in this study; (B) distribution of singly, doubly, and triply phosphorylated peptides; and (C) pie chart representation of identified phosphorylation site distribution. (PDF) Figure S-6: All 192 malonylated spectras and all 220 phosphorylated spectras together with all 158 malonylated spectras in the quantitative malonylome analysis identified in this study. (PDF) Table S-1: List of all 192 identified Kmal peptides identified in the present study. Table S-2: List of all 220 identified phosphorylated peptides identified by MaxQuant software. Table S-3:.List of protein quantification results in the stable isotope dimethyl labeling S. erythraea samples. Table S-4: List of sites quantifiable for Kmal in the two-growth state in S. erythraea. Table S-5: Gene ontology (GO) analysis of lysine-malonylated proteins. Table S-6: KEGG pathway enrichment analysis of lysinemalonylated proteins. Table S-7: Enrichment analysis for INTERPRO domains of lysine-malonylated proteins. Table S-8: Motif analysis for lysine-malonylated proteins. Table S-9: Protein interaction networks of malonylated proteins using the STRING database by MCODE. Table S-10: List of primers used in the study. (XLSX)

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*Tel/fax: 008621-64252094; e-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. O


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Journal of Proteome Research approaches to investigate microorganisms. Appl. Microbiol. Biotechnol. 2011, 90, 407−416. (19) Yu, B. J.; Kim, J. A.; Moon, J. H.; Ryu, S. E.; Pan, J. G. The diversity of lysine-acetylated proteins in Escherichia coli. J. Mol. Biol. 2008, 18, 1529−1536. (20) Zhang, J.; Sprung, R.; Pei, J. M.; Tan, X.; Kim, S.; Zhu, H.; Liu, C.; Grishin, N. V.; Zhao, Y. Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli. Mol. Cell. Proteomics 2008, 8, 215−225. (21) Zhang, K.; Zheng, S.; Yang, J. S.; Chen, Y.; Cheng, Z. Comprehensive profiling of protein lysine acetylation in Escherichia coli. J. proteome Res. 2013, 12, 844−851. (22) Wang, Q.; Zhang, Y.; Yang, C.; Xiong, H.; Lin, Y.; Yao, J.; Li, H.; Xie, L.; Zhao, W.; Yao, Y.; Ning, Z.; Zeng, R.; Xiong, Y.; Guan, K.; Zhao, S.; Zhao, G. Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science 2010, 327, 1004− 1007. (23) Kim, D.; Yu, B.; Lee, Y. J.; Choi, S. G.; Kang, S.; Pan, J. G.; Kim, J. A. The acetylproteome of Gram-positive model bacterium Bacillus subtilis. Proteomics 2013, 13, 1726−1736. (24) Okanishi, H.; Kim, K.; Masui, R.; Kuramitsu, S. Acetylome with Structural Mapping Reveals the Significance of Lysine Acetylation in Thermus thermophilus. J. Proteomics Res. 2013, 12, 3952−3968. (25) Zhang, Y.; Wu, Z.; Wan, X.; Liu, P.; Zhang, J.; Ye, Y.; Zhao, Y.; Tan, M. Comprehensive profiling of lysine acetylome in Staphylococcus aureus. Sci. China: Chem. 2014, 57, 732−738. (26) Pan, J.; Ye, Z.; Cheng, Z.; Peng, X.; Wen, L.; Zhao, F. Systematic analysis of the lysine acetylome in Vibrio parahemolyticus. J. proteome Res. 2014, 13, 3294−3302. (27) Cai, L.; Tu, B. P. On acetyl-CoA as a gauge of cellular metabolic state. Cold Spring Harbor Symp. Quant. Biol. 2011, 76, 195−202. (28) Huang, D.; Li, Z.; You, D.; Zhou, Y.; Ye, B. Lysine acetylproteome analysis suggests its roles on primary and secondary metabolism in Saccharopolyspora erythraea. Appl. Microbiol. Biotechnol. 2015, 99, 1399−1413. (29) Tan, M.; Luo, H.; Lee, S.; Jin, F.; Yang, J. S.; Montellier, E.; Buchou, T.; Cheng, Z.; Rousseaux, S.; Rajagopal, N.; Lu, Z.; Ye, Z.; Zhu, Q.; Wysocka, J.; Ye, Y.; Khochbin, S.; Ren, B.; Zhao, Y. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 2011, 146, 1016−1028. (30) Rosen, R.; Becher, D.; Büttner, K.; Biran, D.; Hecker, M.; Ron, E. Z. Probing the active site of homoserine trans-succinylase. FEBS Lett. 2004, 577, 386−392. (31) Zhang, Z.; Tan, M.; Xie, Z.; Dai, L.; Chen, Y.; Zhao, Y. Identification of lysine succinylation as a new post-translational modification. Nat. Chem. Biol. 2011, 7, 58−63. (32) Tan, M.; Peng, C.; Anderson, K.; Chhoy, P.; Xie, Z.; Dai, L.; Park, J.; Chen, Y.; Huang, H.; Zhang, Y.; Ro, J.; Wagner, G. R.; Green, M. F.; Madsen, A. S.; Schmiesing, J.; Peterson, B. S.; Xu, G.; Ilkayeva, O. R.; Muehlbauer, M. J.; Braulke, T.; Mühlhausen, C.; Backos, D. S.; Olsen, C. A.; McGuire, P. J.; Pletcher, S. D.; Lombard, D. B.; Hirschey, M. D.; Zhao, Y. Lysine glutarylation is a protein posttranslational modification regulated by SIRT5. Cell Metab. 2014, 19, 605−617. (33) Jiang, T.; Zhou, X.; Taghizadeh, K.; Dong, M.; Dedon, P. C. Nformylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 60−65. (34) Peng, C.; Lu, Z.; Xie, Z.; Cheng, Z.; Chen, Y.; Tan, M.; Luo, H.; Zhang, Y.; He, W.; Yang, K.; Zwaans, B. M.; Tishkoff, D.; Ho, L.; Lombard, D.; He, T. C.; Dai, J.; Verdin, E.; Ye, Y.; Zhao, Y. The first identification of lysine malonylation substrates and its regulatory enzyme. Mol. Cell. Proteomics 2011, 10, 012658. (35) Colak, G.; Pougovkina, O.; Dai, L.; Tan, M.; Te Brinke, H.; Huang, H.; Cheng, Z.; Park, J.; Wan, X.; Liu, X.; Yue, W. W.; Wanders, R. J.; Locasale, J. W.; Lombard, D. B.; de Boer, V. C.; Zhao, Y. Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation. Mol. Cell. Proteomics 2015, 14, 3056−3071.

(36) Kim, S. C.; Sprung, R.; Chen, Y.; Xu, Y.; Ball, H.; Pei, J.; Cheng, T.; Kho, Y.; Xiao, H.; Xiao, L.; Grishin, N. V.; White, M.; Yang, X. J.; Zhao, Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol. Cell 2006, 23, 607−618. (37) Chen, Y.; Kwon, S. W.; Kim, S. C.; Zhao, Y. An integrated approach for manual verification of peptides identified by searching protein sequence databases with tandem mass spectra. J. Proteome Res. 2005, 4, 998−1005. (38) Huang, D. W.; Sherman, B. T.; Lempicki, R. A. Systematic and integrative analysis of large gene lists using david bioinformatics resources. Nat. Protoc. 2008, 4, 44−57. (39) Apweiler, R.; Attwood, T. K.; Bairoch, A.; Bateman, A.; Birney, E.; Biswas, M.; Bucher, P.; Cerutti, L.; Corpet, F.; Croning, M. D.; Durbin, R.; Falquet, L.; Fleischmann, W.; Gouzy, J.; Hermjakob, H.; Hulo, N.; Jonassen, I.; Kahn, D.; Kanapin, A.; Karavidopoulou, Y.; Lopez, R.; Marx, B.; Mulder, N. J.; Oinn, T. M.; Pagni, M.; Servant, F.; Sigrist, C. J.; Zdobnov, E. M. The InterPro database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Res. 2001, 29, 37−40. (40) Franceschini, A.; Szklarczyk, D.; Frankild, S.; Kuhn, M.; Simonovic, M.; Roth, A.; Lin, J.; Minguez, P.; Bork, P.; Jensen, L. J.; von Mering, C. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013, 41, D808−D815. (41) Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.; Heck, A. J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 2009, 4, 484−494. (42) Xie, Z.; Dai, J.; Dai, L.; Tan, M.; Cheng, Z.; Wu, Y.; Boeke, J. D.; Zhao, Y. Lysine succinylation and lysine malonylation in histones. Mol. Cell. Proteomics 2012, 11, 100−107. (43) Nishida, Y.; Rardin, M. J.; Carrico, C.; He, W.; Sahu, A. K.; Gut, P.; Najjar, R.; Fitch, M.; Hellerstein, M.; Gibson, B. W.; Verdin, E. SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target. Mol. Cell 2015, 59, 321−332. (44) Shao, J.; Xu, D.; Hu, L.; Kwan, Y. W.; Wang, Y.; Kong, X.; Ngai, S. M. Systematic analysis of human lysine acetylation proteins and accurate prediction of human lysine acetylation through bi-relative adapted binomial score Bayes feature representation. Mol. BioSyst. 2012, 8, 2964−2973. (45) Xie, L.; Liu, W.; Li, Q.; Chen, S.; Xu, M.; Huang, Q.; Zeng, J.; Zhou, M.; Xie, J. First Succinyl-Proteome Profiling of Extensively Drug-Resistant Mycobacterium tuberculosis Revealed Involvement of Succinylation in Cellular Physiology. J. Proteome Res. 2015, 14, 107− 119. (46) Choudhary, C.; Weinert, B. T.; Nishida, Y.; Verdin, E.; Mann, M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat. Rev. Mol. Cell Biol. 2014, 15, 536−550. (47) van Noort, V.; Seebacher, J.; Bader, S.; Mohammed, S.; Vonkova, I.; Betts, M. J.; Kuhner, S.; Kumar, R.; Maier, T.; O’Flaherty, M.; Rybin, V.; Schmeisky, A.; Yus, E.; Stulke, J.; Serrano, L.; Russell, R. B.; Heck, A. J.; Bork, P.; Gavin, A. C. Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium. Mol. Syst. Biol. 2012, 8, 571. (48) Licona-Cassani, C.; Lim, S.; Marcellin, E.; Nielsen, L. K. Temporal Dynamics of the Saccharopolyspora erythraea Phosphoproteome. Mol. Cell. Proteomics 2014, 13, 1219−1230. (49) Yang, M.; Wang, Y.; Chen, Y.; Cheng, Z.; Gu, J.; Deng, J.; Bi, L.; Chen, C.; Mo, R.; Wang, X.; Ge, F. Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis. Mol. Cell. Proteomics 2015, 14, 796−811. (50) Weinert, B. T.; Iesmantavicius, V.; Wagner, S. A.; Schölz, C.; Gummesson, B.; Beli, P.; Nyström, T.; Choudhary, C. Acetylphosphate is a critical determinant of lysine acetylation in E. coli. Mol. Cell 2013, 51, 265−272. (51) McCormack, D. K.; Farnden, K. J.; Boland, M. J. Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules. Arch. Biochem. Biophys. 1982, 218, 561−571. P


Article

Journal of Proteome Research (52) Li, Y. Y.; Chang, X.; Yu, W. B.; Li, H.; Ye, Z. Q.; Yu, H.; Liu, B. H.; Zhang, Y.; Zhang, S. L.; Ye, B. C.; Li, Y. X. Systems perspectives on erythromycin biosynthesis by comparative genomic and transcriptomic analyses of S. erythraea E3 and NRRL23338 strains. BMC Genomics 2013, 14, 523. (53) Hirschey, M. D.; Zhao, Y. Metabolic Regulation by Lysine Malonylation, Succinylation, and Glutarylation. Mol. Cell. Proteomics 2015, 14, 2308−2315. (54) Colak, G.; Xie, Z.; Zhu, A. Y.; Dai, L.; Lu, Z.; Zhang, Y.; Wan, X.; Chen, Y.; Cha, Y. H.; Lin, H.; Zhao, Y.; Tan, M. Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli. Mol. Cell. Proteomics 2013, 12, 3509−3520.

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