Abstract

One hundred and fifty GCN5-like acetyltransferases with amino acid-binding (ACT)-GCN5-related N-acetyltransferase (GNAT) domain organization have been identified in actinobacteria. The ACT domain is fused to the GNAT domain, conferring amino acid-induced allosteric regulation to these protein acetyltransferases (Pat) (amino acid sensing acetyltransferase, (AAPatA)). Members of the AAPatA family share similar secondary structure and are divided into two groups based on the allosteric ligands of the ACT domain: the asparagine (Asn)-activated PatA and the cysteine (Cys)-activated PatA. The former are mainly found in Streptomyces; the latter are distributed in other actinobacteria. We investigated the effect of Asn and Cys on the acetylation activity of Sven_0867 (SvePatA, from Streptomyces venezuelae DSM 40230) and Amir_5672 (AmiPatA, from Actinosynnema mirum strain DSM 43827), respectively, as well as the relationship between the structure and function of these enzymes. These findings indicate that the activity of PatA and acetylation level of proteins may be closely correlated with intracellular concentrations of Asn and Cys in actinobacteria. Amino acid-sensing signal transduction in acetyltransferases may be a mechanism that regulates protein acetylation in response to nutrient availability. Future work examining the relationship between protein acetylation and amino acid metabolism will broaden our understanding of post-translational modifications (PTMs) in feedback regulation.

Introduction

Protein lysine acetylation has emerged as an important metabolic regulatory mechanism in bacteria, since the discovery of reversible acetylation of acetyl–CoA (Ac–CoA) synthetase via enzymatic acetylation, such as acetyltransferase [1] and deacetylase [2]. Protein acetylation also can occur via nonenzymatic reaction, such as chemical acetylation, wherein the intracellular acetyl-phosphate (AcP) plays a critical role [3,4]. The intracellular level of protein acetylation is ultimately defined by the concentration of AcP (or Ac–CoA) and relative activities of acetyltransferases and deacetylases. These two classes of enzymes respond to intracellular nutritional status to control the acetylation of specific proteins that mold the metabolic network [5,6]. Multiple nutrient-mediated signaling pathways regulate the transcription of genes encoding acetyltransferases and deacetylases. In Bacillus subtilis, regulating the expression of the acuA gene, which encodes an acetyltransferase, is under the control of catabolite control protein A (CcpA), a global regulatory protein affected by the quality of the carbon source available to the cell [5]. In Escherichia coli, two acetylation-signaling pathways mediated by cAMP have been identified. The carbon regulating CRP–cAMP complex induces the expression of yfiQ, which encodes the Gcn5-like acetyltransferase YfiQ (also known as PatZ and Pka) [7,8], thus increasing the acetylation level of proteins in response to the intracellular cAMP signal [6]. Furthermore, cAMP–CRP regulates the glucose-induced, AcP-dependent protein acetylation in response to carbon overflow, with specificity of the acetylation determined by the accessibility, reactivity, and 3D microenvironment of the target lysine [9]. In Salmonella enterica, the expression of the genes encoding the acetyltransferase Pat and deacetylase CobB is activated by IolR, a repressor of myo-inositol catabolism involved in the utilization of cyclohexane-1,2,3,4,5,6-hexol, a cyclic polyol abundant in soil, as a carbon source [10].

Allosteric effectors also control the activity of protein acetyltransferases (Pat). In Mycobacterium tuberculosis and Mycobacterium smegmatis, cAMP directly activates the Pat, MtPat (Rv0998) and MsPat (MSMEG_5458) by binding to a cyclic nt-binding domain that is fused to the N-terminus of the catalytic GCN5-related N-acetyltransferase (GNAT) domain [11]. Recently, we found that an ACT domain, fused to the GNAT acetyltransferase of Micromonospora aurantiaca (MaKat), is used for amino acid-induced allosteric regulation of the enzyme [12]. In the present study, we identified a total of 150 putative amino acid sensing acetyltransferases (AAPatA) that have an ACT-GNAT domain organization specific to actinobacteria through bioinformatics analysis. The ACT domain is fused to the GNAT domain, conferring an amino acid-induced allosteric effect to the regulation of protein acetylation. We found that members of the AAPatA family are divided into two groups: the asparagine (Asn)-regulated PatA and cysteine (Cys)-regulated PatA. The former exists mainly in Streptomyces; the latter are distributed in other actinobacteria. Our results present an example of a novel signal transduction mechanism for regulating protein acetylation in actinobacteria via amino acid-sensing acetyltransferases in response to intracellular concentrations of Asn and Cys.

Materials and methods

Strains and reagents

All strains were purchased from the China General Microbiological Culture Collection Center (CGMCC). All the reagents, purchased from Sigma, were of the highest grade available commercially.

Construction of plasmids and overexpression and purification of proteins

All patA and homologous acs genes coding for Ac–CoA synthetase were amplified by PCR using genomic DNA from bacterial strains. After digestion with restriction enzymes, genes coding for PatA and Ac–CoA synthetase (Acs) proteins were ligated into pET-28a, which was predigested with EcoRI and HindIII, generating pET28a-PatA and pET28a-Acs. The overexpression and purification of proteins in the E. coli BL21-DE3 was performed as described previously [12]. Protein concentration was determined using the BCA method.

Acetylation activity and steady-state kinetic assay of PatA acetyltransferases

Acetylation activity of acetyltransferases was measured continuously by a coupled enzymatic assay using a fluorescence plate reader (BioTek Instruments, Winooski, U.S.A.). The production of CoA is measured using pyruvate dehydrogenase mediated reduction of NAD+ to NADH, resulting in an increase in absorbance at 340 nm [13,14]. The optimization of the enzyme-coupled assay and steady-state kinetic analysis of PatA acetyltransferases were performed as described previously [12].

Western blotting assays and peptide fingerprinting using MS

Protein samples were electrophoresed on an SDS/PAGE and then transferred on to a PVDF membrane for 1 h at 100 V. The membrane was blocked in BSA blocking buffer for 2 h at 25°C. We used an acetyl-lysine antibody (Cat# ICP0381, ImmuneChem Pharmaceuticals Inc., Canada) diluted to 1:15000. After incubation at 4°C for 10–12 h, the blot was washed with TBS and Tween 20 (TBST) three-times at ambient temperature. Chemiluminescence was detected using the ECL enhanced system and a luminescent image analyzer (DNR Bio Imaging Systems, Israel). To continuously monitor the acetyl-lysine sites, the acetylated Acs were isolated using the SDS/PAGE and proteins were solubilized according to previously described filter-aided sample preparation (FASP) procedures [15]. Predigested proteins were extracted by solid-phase extraction, separated using Nano-HPLC, and analyzed on an Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Scientific, U.S.A.). Tandem MS (MS/MS) spectra were searched using the MASCOT engine (version 2.2; Matrix Science, United Kingdom).

In vitro assays of Acs

The specific activity of Acs was determined at 30°C in a transparent 384-well microplate reader (Bio Tek Instruments, Winooski, U.S.A.) at 340 nm [16]. The standard reaction mixture contained 100 mM Tris/HCl (pH 7.8), 10 mM malate (pH 7.7), 0.2 mM CoA, 8 mM ATP (pH 7.5), 1 mM NAD+, 10 mM MgCl2, 3 units of malate dehydrogenase, 0.4 units of citrate synthase, and 0.3 μM SveAcs or AmiAcs. The reaction was initiated with 100 mM potassium acetate and the rate was determined continuously for 5 min by measuring NADH production at 340 nm. One unit was defined as the conversion of 1.0 μmol of NAD+ to NADH per min in the presence of saturating levels of acetate.

CD spectroscopy

CD spectra were prepared using a CD spectrophotometer (Applied Photophysics, U.K.) and 1 mm cuvette equilibrated at 25°C. All samples were desalted and dialyzed against buffer P (150 mM KF, 94 mM K2HPO4, and 6 mM KH2PO4, pH 8.0). Samples were diluted to 0.25 μg/μl and incubated with and without 2 mM Asn or 2 mM Cys. The CD spectra were collected between 190 and 260 nm, and three repeat scans were averaged. Parameters of the secondary structure were analyzed using CDNN 2.1 (http://bioinformatik.biochemtech.uni-halle.de/cdnn).

Site-directed mutagenesis and purification of PatA and Acs

Mutations at key sites in PatA and Acs proteins were introduced into pET28a using QuikChange Mutagenesis Kit (TransGen, Beijing, China) according to previously described procedure [12,17]. The mutations were confirmed by DNA sequencing. The overexpression and purification of mutant proteins were performed as described above.

Structural model of the ACT domain of AmiPatA

The structure of the ACT domain of AmiPatA was built by homology modeling from Swiss Model server (https://swissmodel.expasy.org/) using four ACT domains with the greatest similarity to that of AmiPatA (PDB ID: 3W7B, 3O1L, 1PSD, 2J0X) according to the previously described procedure [12]. Discovery Studio 3.5 Client was used as the docking tool to predict amino acid-binding to AAPatA proteins. CHARMM forcefield was used to optimize the model of the ACT domain, and CFF forcefield was used to optimize the structure of Cys. We added Kollman charge to the residues of ACT domain of AmiPatA. LibDock was used to perform molecular docking. The conserved binding site was selected as docking center, and a sphere with a radius of 10 Å were considered as binding region for docking studies.

Sequences and phylogenetic tree analysis of ACT and GNAT domains

Multiple alignments of the ACT and GNAT domains, as well as a phylogenetic analysis of full-length sequences of all ACT-GNAT proteins, were performed using ClustalW 2.1 and MEGA 5.10 software.

Data and statistical analysis

Microsoft Excel was used to quantitate protein concentrations and the activities of PatA, SveAcs, and AmiAcs. GraphPad Prism 5.0 was used for statistical analyses, preparation of graphs, and data analysis. Data are presented as means ± S.E.M. for three independent experiments (n=3); statistical significance was accepted at P≤0.05.

Results

PatA acetyltransferases with ACT-GNAT domain organization are found in actinobacteria

Previous work has shown that MaKat (Micau_1670, from M. aurantiaca) containing an ACT-GNAT domain organization is an amino acid-regulated Pat that can acetylate Ac–CoA synthetase [12]. One hundred and fifty-six proteins containing the ACT-GNAT domain organization were displayed in the InterPro database (v.54.0) and the sequences of these proteins were examined. Six repeated sequences were deleted. A total of 150 unique ACT-GNAT acetyltransferases, which we designated as AAPatAs, are listed in Supplementary Table S1. The AAPatA enzymes are unique to and are distributed in 22 species of actinobacteria. Remarkably, 107 AAPatAs (71.3%) were found in Streptomyces. Some AAPatAs were found in Amycolatopsis (10), Actinoplanes (5), and Micromonospora (5). We performed a phylogenetic tree analysis using the protein sequences of all AAPatAs (Figure 1). Figure 1 shows that the sequence of AAPatA reflects the phylogeny of actinobacteria. In AAPatA sequence phylogeny, organisms are clustered according to orders, such as Streptomycetales (Streptomyces, Kitasatospora), Micromonosporales (Actinoplanes, Micromonospora, Verrucosispora), Pseudonocardiales (Saccharopolyspora, Pseudonocardia, Saccharothrix, Actinosynnema, Kibdelosporangium, Alloactinosynnema, Actinokineospora, Prauserella, Amycolatopsis), and Streptosporangiales (Streptomonospora, Microbispora, Streptosporangium). Exceptions included A4FK80 from Saccharopolyspora erythraea DSM 40517 (Pseudonocardiaceae), which was between Streptomycetales and Micromonosporales, and A0A0N0T6Y4 from Nocardia sp. NRRL S-836 (Corynebacteriales), which was dispersed amongst the Pseudonocardiales. The results revealed the sequence evolution of AAPatA acetyltransferases in actinobacteria, which can be used as a molecular marker for speciation of actinobacteria.

Phylogenetic tree analysis of AAPatA sequences

Figure 1
Phylogenetic tree analysis of AAPatA sequences

The phylogenetic tree was made using EMGA 5.10. All ACT-GNAT protein sequences in the InterPro database (v.54) were conducted using ClustalW 2.1 and then construct ed the neighbor tree. The parameters used for the analysis was default value. The specific parameters were as follows: gap opening penalty of pairwise alignment 10, gap extension penalty of pairwise alignment 0.1, gap opening penalty of multiple alignment 10, gap extension penalty of multiple alignment 0.2, gap separation dstance 4, delay divergent cutoff 30%.

Figure 1
Phylogenetic tree analysis of AAPatA sequences

The phylogenetic tree was made using EMGA 5.10. All ACT-GNAT protein sequences in the InterPro database (v.54) were conducted using ClustalW 2.1 and then construct ed the neighbor tree. The parameters used for the analysis was default value. The specific parameters were as follows: gap opening penalty of pairwise alignment 10, gap extension penalty of pairwise alignment 0.1, gap opening penalty of multiple alignment 10, gap extension penalty of multiple alignment 0.2, gap separation dstance 4, delay divergent cutoff 30%.

AAPatA can be divided into two groups: Cys- and Asn-dependent Pat

We overexpressed and purified 24 AAPatA acetyltransferases (indicated by the red dot in Figure 1) from all the ACT-GNAT proteins (Table 1). Like MaKat from M. aurantiaca and other Pat (SlPat, AfPat, 1YGH_A: yeast, 1Z4R_A: human, 1QSN_A: TeT), the most highly conserved motif QXXGX (G/A) for Ac–CoA recognition and binding, and a glutamic acid residue provides as an active site to deprotonate the lysine residue of the acetyl acceptor, were observed in GNAT domains of all 24 AAPatAs (Supplementary Figure S1). The experimental data showed that all AAPatAs can directly acetylate the putative AMP-forming Ac–CoA synthetase (AmiAcs from Actinosynnema mirum strain DSM 43827) in vitro (Supplementary Figure S2). These results demonstrate that AAPatA is a novel type of Pat.

Table 1
Amino acid ligands of 24 AAPatA proteins identified in the present study
No.NamesUniProt accessionSpeciesLigands
SvrPatA A0A0J8BX62 Streptomyces viridochromogenes Asn 
SguPatA A0A089X1X1 Streptomyces glaucescens Asn 
ScbPatA A0A086GIX8 Streptomyces scabiei Asn 
SvtPatA D9XGL8 Streptomyces viridochromogenes DSM 40736 Asn 
SalPatA D6B750 Streptomyces albus J1074 Asn 
ScoPatA Q93IX7 Streptomyces coelicolor ATCC BAA-471 Asn 
SghPatA D5ZV95 Streptomyces ghanaensis ATCC 14672 Asn 
SchPatA A0A0N1JW68 Streptomyces chattanoogensis Asn 
SvgPatA A0A0L8MX44 Streptomyces virginiae Asn 
10 SclPatA E2Q9K6 Streptomyces clavuligerus DSM 738 Asn 
11 SvePatA F2RA54 Streptomyces venezuelae DSM 40230 Asn 
12 SprPatA B5H7N9 Streptomyces pristinaespiralis ATCC 25486 Asn 
13 SfiPatA N0CLA4 Streptomyces fulvissimus DSM 40593 Asn 
14 SvlPatA G2P3F2 Streptomyces violaceusniger Tu 4113 Asn 
15 SmbPatA M3CC83 Streptomyces mobaraensis DSM 40847 Asn 
16 SfrPatA A0A081EMD0 Streptomyces fradiae Asn 
17 KgrPatA A0A0D0PVV0 Kitasatospora griseola Asn 
18 SenPatA A4FK80 Saccharopolyspora erythraea DSM 40517 Cys 
19 AmsPatA I0HG80 Actinoplanes missouriensis DSM 43046 Cys 
20 SroPatA D2AVA5 Streptosporangium roseum DSM 43021 Cys 
21 PdxPatA F4CS81 Pseudonocardia dioxanivorans DSM 44775 Cys 
22 AmiPatA C6WC99 Actinosynnema mirum DSM 43827 Cys 
23 AdePatA M2YM44 Amycolatopsis decaplanina DSM 44594 Cys 
24 AazPatA M2QMD0 Amycolatopsis azurea DSM 43854 Cys 
No.NamesUniProt accessionSpeciesLigands
SvrPatA A0A0J8BX62 Streptomyces viridochromogenes Asn 
SguPatA A0A089X1X1 Streptomyces glaucescens Asn 
ScbPatA A0A086GIX8 Streptomyces scabiei Asn 
SvtPatA D9XGL8 Streptomyces viridochromogenes DSM 40736 Asn 
SalPatA D6B750 Streptomyces albus J1074 Asn 
ScoPatA Q93IX7 Streptomyces coelicolor ATCC BAA-471 Asn 
SghPatA D5ZV95 Streptomyces ghanaensis ATCC 14672 Asn 
SchPatA A0A0N1JW68 Streptomyces chattanoogensis Asn 
SvgPatA A0A0L8MX44 Streptomyces virginiae Asn 
10 SclPatA E2Q9K6 Streptomyces clavuligerus DSM 738 Asn 
11 SvePatA F2RA54 Streptomyces venezuelae DSM 40230 Asn 
12 SprPatA B5H7N9 Streptomyces pristinaespiralis ATCC 25486 Asn 
13 SfiPatA N0CLA4 Streptomyces fulvissimus DSM 40593 Asn 
14 SvlPatA G2P3F2 Streptomyces violaceusniger Tu 4113 Asn 
15 SmbPatA M3CC83 Streptomyces mobaraensis DSM 40847 Asn 
16 SfrPatA A0A081EMD0 Streptomyces fradiae Asn 
17 KgrPatA A0A0D0PVV0 Kitasatospora griseola Asn 
18 SenPatA A4FK80 Saccharopolyspora erythraea DSM 40517 Cys 
19 AmsPatA I0HG80 Actinoplanes missouriensis DSM 43046 Cys 
20 SroPatA D2AVA5 Streptosporangium roseum DSM 43021 Cys 
21 PdxPatA F4CS81 Pseudonocardia dioxanivorans DSM 44775 Cys 
22 AmiPatA C6WC99 Actinosynnema mirum DSM 43827 Cys 
23 AdePatA M2YM44 Amycolatopsis decaplanina DSM 44594 Cys 
24 AazPatA M2QMD0 Amycolatopsis azurea DSM 43854 Cys 

The ACT domain binds to more than one amino acid, by inducing conformational changes, it can generate variation in the activity of the catalytic domain. To identify the amino acid ligands of ACT domains in AAPatA acetyltransferases, we investigated the activities of all AAPatAs acetylating putative Ac–CoA synthetases with and without the addition of the 20 L-amino acids. Acetylation was measured continuously using a coupled enzymatic assay to monitor increase in absorbance at 340 nm (ΔA340). These increases resulted from the formation of NADH by pyruvate dehydrogenase because of AAPatA-dependent CoA production [1214]. The ACT domain allosterically regulates acetylation activity of AAPatA in response to addition of amino acids, as shown in Supplementary Figure S2. Adding Cys or Asn increased the values of ΔA340, while no change in the values of ΔA340 were observed after the addition of other amino acids. These increases in the values of ΔA340 suggest that the acetylation activity of AAPatAs is enhanced by the conformational changes resulting from Cys or Asn binding the ACT domain. The amino acid ligands of AAPatAs are listed in Table 1. We found that members of the AAPatA family are divided into two groups based on the allosteric ligands of the ACT domain: the Asn-regulated PatAs and Cys-regulated PatAs. The former are mainly found in Streptomyces; the latter are distributed in other actinobacteria. Furthermore, we performed a phylogenetic analysis of the sequences that make up the GNAT and ACT domains in the AAPatA proteins. As shown in Figure 2, the two groups were clustered according to the binding ligands, Asn (indicated by the red lines) or Cys (indicated by the blue lines) in both ACT and GNAT domains.

Phylogenetic analysis of the GNAT and ACT domain sequences in AAPatA proteins

Figure 2
Phylogenetic analysis of the GNAT and ACT domain sequences in AAPatA proteins

The GNAT and ACT domains of the 24 AAPatA proteins were analyzed using a phylogenetic tree analysis. The two groups were clustered according to the amino acids Asn (indicated by the red lines) and Cys (indicated by the blue lines) in ACT and GNAT domains. The phylogenetic tree was made using EMGA 5.10. All the GNAT or ACT domain sequences were conducted using ClustalW 2.1 and then constructed the neighbor tree. The parameters used for the analysis was default value. The specific parameters were as follows: gap opening penalty of pairwise alignment 10, gap extension penalty of pairwise alignment 0.1, gap opening penalty of multiple alignment 10, gap extension penalty of multiple alignment 0.2, gap separation dstance 4, delay divergent cutoff 30%.

Figure 2
Phylogenetic analysis of the GNAT and ACT domain sequences in AAPatA proteins

The GNAT and ACT domains of the 24 AAPatA proteins were analyzed using a phylogenetic tree analysis. The two groups were clustered according to the amino acids Asn (indicated by the red lines) and Cys (indicated by the blue lines) in ACT and GNAT domains. The phylogenetic tree was made using EMGA 5.10. All the GNAT or ACT domain sequences were conducted using ClustalW 2.1 and then constructed the neighbor tree. The parameters used for the analysis was default value. The specific parameters were as follows: gap opening penalty of pairwise alignment 10, gap extension penalty of pairwise alignment 0.1, gap opening penalty of multiple alignment 10, gap extension penalty of multiple alignment 0.2, gap separation dstance 4, delay divergent cutoff 30%.

Multiple alignments of ACT domains in the 24 PatA proteins revealed that all ACT domains contain the conserved βαββαβ-fold and the highly conserved glycine residue between the first β1 strand and the first α1 helix; Asn-binding ACT domains have higher sequence conservation than the Cys-binding ACT domains (Figure 3). The β1 and α1 are the conserved motifs for amino acid recognition and binding [18]. As indicated by the red frame in Figure 3, conserved residues, such as DXPGXL, at the interface of β1 and α1, may play an important role in the binding of specific amino acids. Bioinformatics analysis and coupled enzymatic assay showed that AAPatAs, the protein lysine acetyltransferases (KATs) that acetylate the Ac–CoA synthetase, are divided into two groups: Cys_AAPatA and Asn_PatA. Next, we characterized AmiPatA (Cys_AAPatA) from A. mirum DSM 43827 and SvePatA (Asn_PatA) from S. venezuelae DSM 40230.

Multiple alignment of ACT domains

Figure 3
Multiple alignment of ACT domains

(A) An alignment of amino acid sequences that make up the ACT domains of 17 proteins regulated by Asn. (B) Multiple alignment of amino acid sequences that make up the seven ACT domains of AAPatA regulated by Cys. The red frame shows important region between the first β1 strand and the first α1 helix containing a highly conserved glycine residue.

Figure 3
Multiple alignment of ACT domains

(A) An alignment of amino acid sequences that make up the ACT domains of 17 proteins regulated by Asn. (B) Multiple alignment of amino acid sequences that make up the seven ACT domains of AAPatA regulated by Cys. The red frame shows important region between the first β1 strand and the first α1 helix containing a highly conserved glycine residue.

Cys enhances protein lysine acetylation by AmiPatA derived from A. mirum

To examine whether Cys regulates the enzymatic activity of A. mirum-derived AmiPatA, we used a spectrophotometric assay to assess the acetylation of AmiAcs by AmiPatA in the presence and absence of Cys. As shown in Figure 4A, the acetylation of Acs was enhanced in the presence of Cys, indicating an increase in the activity of the acetyltransferase AmiAcs. The initial rate of acetylation of AmiAcs by AmiPatA (indicated by the formation of NADH) was estimated to be 3.8 μM/min, which increased to 6.8 μM/min (an increase by 179%) in the presence of Cys. The acetylation level of AmiAcs was assessed by Western blotting using an anti-acetyl-lysine antibody (Anti-AcK) in the presence or absence of Cys. Cys increased the acetylation level of AmiAcs (Figure 4B); furthermore, increases in the acetylation level of AmiAcs were dependent on the concentration of Cys (Figure 4C). When the concentration of Cys was increased from 0.004 to 0.5 mM, the acetylation levels also increased. The concentrations of Cys ligand that saturated the acetylation of the protein were in the submillimolar range. The concentration is within the endogenous ranges reported for Cys, which are estimated to be 0.1–0.2 mM in E. coli cells [19], indicating that intracellular Cys is able to exert an effect on activity of AmiPatA enzyme in vivo. To investigate the effect of acetylation on enzyme activity, AmiAcs was incubated with AmiPatA in the presence or absence of Ac–CoA for 3 h. The activity of AmiAcs was reduced by half in the presence of Ac–CoA and AmiPatA, indicating that lysine acetylation effectively decreased the activity of AmiAcs (Figure 4D). The acetylated AmiAcs protein was subjected to trypsin digestion, and the generated peptides were analyzed by MS. The acetylated lysine site (SGK (620) IMR) was identified in the conserved PXXXXGK motif of the AMP-forming acyl–CoA synthetases; this motif is recognized by the previously reported bacterial acetyltransferases [17] (Supplementary Figure S3). We further determined the kinetic parameters of the AmiPatA catalyzed acetylation reaction of AmiAcs in the absence and presence of Cys using a coupled enzymatic assay previously described [12]. The data are shown in Table 2. The Km and kcat values of AmiPatA for AmiAcs are 45.5 μM and 0.03 s−1, respectively. Cys was able to decrease the Km value of AmiPatA for AmiAcs by three-fold, and increase kcat value by 1.5-fold.

Cys-mediated allosteric regulation of AmiPatA activity for protein acetylation

Figure 4
Cys-mediated allosteric regulation of AmiPatA activity for protein acetylation

(A) Acetyltransferase activity of AmiPatA was measured using a coupled enzymatic assay with and without Cys. (B) Acetylation of AmiAcs by AmiPatA. AmiAcs (2 μM) was incubated alone or in the presence of AmiPatA (0.3 μM), Ac–CoA (60 μM), or Cys (2 mM) in a volume of 100 μl at 37°C for 1.5 h, followed by analysis using SDS/PAGE. Levels of acetylation were detected by Western blot assay using an anti-AcK antibody. (C) Acetylation levels of AmiAcs were measured using Western blot assay. Varying concentrations of Cys, ranging from 0 μM to 2.5 mM, were added to the solution of AmiAcs (2 μM), AmiPatA (0.3 μM), and Ac–CoA (60 μM), followed by incubation for 1.5 h at 37°C. (D) Time-dependent inactivation of AmiAcs by acetylation. The activity of AmiAcs was measured at different time intervals during incubation with AmiPatA. ● 5 μM AmiAcs; ▪ 5 μM AmiAcs and 0.2 μM AmiPatA; ▲ 5 μM AmiAcs, 0.2 μM AmiPatA, and 60 μM Ac–CoA ▼5 μM AmiAcs, 60 μM Ac–CoA, 0.2 μM AmiPatA and 2 mM Cys. Each data point indicate an average of three independent assays; mean ± S.E.M. (P<0.05; n=3 experiments).

Figure 4
Cys-mediated allosteric regulation of AmiPatA activity for protein acetylation

(A) Acetyltransferase activity of AmiPatA was measured using a coupled enzymatic assay with and without Cys. (B) Acetylation of AmiAcs by AmiPatA. AmiAcs (2 μM) was incubated alone or in the presence of AmiPatA (0.3 μM), Ac–CoA (60 μM), or Cys (2 mM) in a volume of 100 μl at 37°C for 1.5 h, followed by analysis using SDS/PAGE. Levels of acetylation were detected by Western blot assay using an anti-AcK antibody. (C) Acetylation levels of AmiAcs were measured using Western blot assay. Varying concentrations of Cys, ranging from 0 μM to 2.5 mM, were added to the solution of AmiAcs (2 μM), AmiPatA (0.3 μM), and Ac–CoA (60 μM), followed by incubation for 1.5 h at 37°C. (D) Time-dependent inactivation of AmiAcs by acetylation. The activity of AmiAcs was measured at different time intervals during incubation with AmiPatA. ● 5 μM AmiAcs; ▪ 5 μM AmiAcs and 0.2 μM AmiPatA; ▲ 5 μM AmiAcs, 0.2 μM AmiPatA, and 60 μM Ac–CoA ▼5 μM AmiAcs, 60 μM Ac–CoA, 0.2 μM AmiPatA and 2 mM Cys. Each data point indicate an average of three independent assays; mean ± S.E.M. (P<0.05; n=3 experiments).

Table 2
Kinetic analysis of AmiPatA on AmiAcs
EnzymeSubstrateKm/μMkcat/s−1kcat/Km (M−1s−1)
AmiPatA Ac–CoA 5.5 ± 1.1 0.19 ± 0.008 (3.6 ± 0.8) × 104 
 AmiAcs 45.5 ± 7.3 0.30 ± 0.014 (6.8 ± 1.4) × 103 
AmiPatA (Cys) Ac–CoA 4.2 ± 0.6 0.33 ± 0.010 (8.2 ± 0.7) × 104 
 AmiAcs 15.9 ± 1.8 0.54 ± 0.014 (3.4 ± 0.5) × 104 
EnzymeSubstrateKm/μMkcat/s−1kcat/Km (M−1s−1)
AmiPatA Ac–CoA 5.5 ± 1.1 0.19 ± 0.008 (3.6 ± 0.8) × 104 
 AmiAcs 45.5 ± 7.3 0.30 ± 0.014 (6.8 ± 1.4) × 103 
AmiPatA (Cys) Ac–CoA 4.2 ± 0.6 0.33 ± 0.010 (8.2 ± 0.7) × 104 
 AmiAcs 15.9 ± 1.8 0.54 ± 0.014 (3.4 ± 0.5) × 104 

The ACT domain is an amino acid-binding domain involved in amino acid metabolism; 3-phosphoglycerate dehydrogenase (3PGDH) in E. coli was the first enzyme found to contain the ACT domain [20] and folds with a ferredoxin-like topology. In the ACT domain, the most conserved part is the region at the interface between the first strand (β1) and the first helix (α1); the residues binding specific amino acids via hydrogen bonding are found at this conserved region [21]. To investigate key residues used to bind Cys in AmiPatA, the structure of the ACT domain in AmiPatA was modeled using four ACT domains with the greatest similarity (query cover: 85%, identity: 25%) to that of AmiPatA (PDB ID: 3W7B, 3O1L, 1PSD, 2J0X). The docking result showed that the residues involved in hydrogen bonding with Cys were Asp16, Ser17, Gly19, Ala20, and Leu21; in particular, Asp16 and Ser17 were located in the loop between the first strand (β1) and the first helix (α1) of the AmiPatA ACT domain (Figure 5A). The characteristic Gly19, followed by a hydrophobic residue, is the most conserved residue in the helix, necessary for maintaining the conformation of the strand–helix interface. Far-UV CD spectroscopy was conducted to confirm the conformational change in AmiPatA after the addition of Cys (Figure 5B). CD spectra, recorded from 190 to 260 nm, showed an increase in ellipticity at 208 and 222 nm, indicating a decrease in the α-helical content of AmiPatA after the addition of Cys and suggesting that the binding of Cys altered the secondary structure of the protein. The characteristics of the secondary structure, calculated using CDNN version 2.1, indicated the presence of approximately 45.1% α-helices and 11.1% β-sheets in AmiPatA, and 35.8% α-helices and 15.8% β-sheets in AmiPatA after the addition of Cys. This phenomenon was observed in some proteins regulated allosterically by effectors [22,23].

Specific binding residues of Cys in the ACT domain of AmiPatA

Figure 5
Specific binding residues of Cys in the ACT domain of AmiPatA

(A) A structural model of the ACT domain of AmiPatA shows that the possible residues of AmiPatA interact with Cys via hydrogen bonding between the first strand (β1) and the first helix (α1). (B) Secondary structure (dotted line) and conformational change of AmiPatA, after the addition of Cys (full line) and subtraction of buffer baseline, are shown from 190 to 260 nm. (C, D) The conformational changes in AmiPatA mutants (D16A and S17A) were evaluated using CD in the presence and absence of Cys. (E, F) Initial rate of the formation of NADH and Western blotting analysis are shown in the AmiPatA mutants D16A and S17A. The gray values were quantitated by densitometry using ImageJ software.

Figure 5
Specific binding residues of Cys in the ACT domain of AmiPatA

(A) A structural model of the ACT domain of AmiPatA shows that the possible residues of AmiPatA interact with Cys via hydrogen bonding between the first strand (β1) and the first helix (α1). (B) Secondary structure (dotted line) and conformational change of AmiPatA, after the addition of Cys (full line) and subtraction of buffer baseline, are shown from 190 to 260 nm. (C, D) The conformational changes in AmiPatA mutants (D16A and S17A) were evaluated using CD in the presence and absence of Cys. (E, F) Initial rate of the formation of NADH and Western blotting analysis are shown in the AmiPatA mutants D16A and S17A. The gray values were quantitated by densitometry using ImageJ software.

To further identify the key residues involved in the binding of Cys, we mutated the acidic amino acid Asp16 and the hydrophilic amino acid Ser17 to a neutral and hydrophobic amino acid alanine. The CD spectra of D16A and S17A mutant proteins showed that Cys does not induce changes in the secondary structures of AmiPatA mutants (Figure 5C,D). We also used an enzyme-coupled assay and Western blotting analysis to investigate the acetylation activity of the two mutants with and without the addition of Cys. As shown in Figure 5E,F, no increases in acetylation were observed in D16A and S17A mutants after the addition of Cys; this agrees with the CD assays that indicated no conformational changes induced by the addition of Cys. These results suggest that Asp16 and Ser17 at the interface between β1 and α1 may somehow affect the Cys-binding of AmiPatA. Taken together, these results indicate that AmiPatA has protein-acetylating acetyltransferase activity and that this activity is regulated allosterically by Cys binding.

Asn enhances protein lysine acetylation by SvePatA from S. venezuelae

Finally, we characterized SvePatA from S. venezuelae as a model representative of Asn-binding AAPatA acetyltransferases. To investigate the effect of Asn on the activity of SvePatA, we determined the initial rate of SveAcs acetylation by SvePatA with and without the addition of Asn. For this, we used a coupled enzymatic assay, where the amount of CoA released after SveAcs acetylation was measured by the formation of NADH from NAD+ via pyruvate dehydrogenase. As shown in Figure 6A, SvePatA acetylated SveAcs in the absence of Asn (2.23 nmol of NADH formed per min/ml), and the rate of acetylation was increased by 176% in the presence of Asn (3.92 nmol of NADH formed per min/ml). Western blotting was conducted to detect the level of SveAcs acetylation with an anti-Ack antibody in the presence and absence of Asn. Acetylation of SveAcs was clearly observed after incubation with SvePatA and Ac–CoA; acetylation was significantly increased in the presence of Asn (Figure 6B). Moreover, the increase in the acetyltransferase activity of SvePatA was dependent on the concentration of Asn (Figure 6C). Previous work showed that Asn can reach cytoplasmic concentration of 0.51 mM in E. coli cells [24], which is comparable with the concentration of Asn needed to regulate allosterically activity of SvePatA enzyme. To examine the effect of acetylation on enzyme activity, SveAcs was incubated with SvePatA in the presence of Ac-CoA for 2 h. The activity of SveAcs was reduced by approximately 60%, indicating that lysine acetylation effectively decreased the activity of SveAcs (Figure 6D). These results demonstrated that AAPatA proteins possessing the ACT-GNAT domain in non-Streptomycetaceae actinobacteria can function as acetyltransferases regulated allosterically by Asn.

Asn-mediated allosteric regulation of SvePatA activity for protein acetylation

Figure 6
Asn-mediated allosteric regulation of SvePatA activity for protein acetylation

(A) Acetyltransferase activity of SvePatA was measured using a coupled enzymatic assay with and without the addition of Asn. (B) Acetylation of SveAcs by SvePatA. SveAcs (2 μM) was incubated alone or in the presence of SvePatA (0.3 μM), Ac–CoA (60 μM), and Asn (2 mM) in a volume of 100 μl at 37°C for 1.5 h, followed by an analysis using SDS/PAGE. Levels of SveAcs acetylation were detected by Western blotting using a specific anti-AcK antibody. (C) Levels of SveAcs acetylation were measured using a Western blot assay. Varying concentrations of Asn, in the range from 0 μM to 5 mM, were added to a solution containing SveAcs (2 μM), SvePatA (0.3 μM), and Ac-CoA (60 μM), followed by incubation for 1.5 h at 37°C. (D) Time-dependent inactivation of SveAcs by acetylation. The activity of SveAcs was measured at different time intervals during incubation with SvePatA. ● 5 μM SveAcs; ▪ 5 μM SveAcs and 0.2 μM SvePatA; ▲ 5 μM SveAcs, 0.2 μM SvePatA, and 60 μM Ac-CoA ▼5 μM SveAcs, 60 μM Ac-CoA, 0.2 μM SvePatA, and 2 mM Asn. Each data point indicate the average from three independent assays. Data are represented as mean ± S.E.M., n=3 experiments; the significance level was set at P<0.05.

Figure 6
Asn-mediated allosteric regulation of SvePatA activity for protein acetylation

(A) Acetyltransferase activity of SvePatA was measured using a coupled enzymatic assay with and without the addition of Asn. (B) Acetylation of SveAcs by SvePatA. SveAcs (2 μM) was incubated alone or in the presence of SvePatA (0.3 μM), Ac–CoA (60 μM), and Asn (2 mM) in a volume of 100 μl at 37°C for 1.5 h, followed by an analysis using SDS/PAGE. Levels of SveAcs acetylation were detected by Western blotting using a specific anti-AcK antibody. (C) Levels of SveAcs acetylation were measured using a Western blot assay. Varying concentrations of Asn, in the range from 0 μM to 5 mM, were added to a solution containing SveAcs (2 μM), SvePatA (0.3 μM), and Ac-CoA (60 μM), followed by incubation for 1.5 h at 37°C. (D) Time-dependent inactivation of SveAcs by acetylation. The activity of SveAcs was measured at different time intervals during incubation with SvePatA. ● 5 μM SveAcs; ▪ 5 μM SveAcs and 0.2 μM SvePatA; ▲ 5 μM SveAcs, 0.2 μM SvePatA, and 60 μM Ac-CoA ▼5 μM SveAcs, 60 μM Ac-CoA, 0.2 μM SvePatA, and 2 mM Asn. Each data point indicate the average from three independent assays. Data are represented as mean ± S.E.M., n=3 experiments; the significance level was set at P<0.05.

As shown in Figure 7A, CD spectra of SvePatA displayed an increase in ellipticity at 208 and 222 nm after the addition of Asn, indicating that the binding of Asn altered the secondary structure of the protein. The docking result of Asn to SvePatA showed that the residues involved in hydrogen bonding with Asn were Asp109, Lys112, Pro113, Glu114, and Cys139 (Figure 7B). To further identify the key residues involved in the binding of Asn, we mutated the acidic amino acid Lys112 and amino acid Pro113 to alanine. Allosteric effect of Asn on SvePatA activity was still observed in two mutants K112A and P113A (Figure 7C,D), indicating that Lys112 and Pro113 were not involved in the Asn binding of SvePatA.

The binding of Asn with SvePatA

Figure 7
The binding of Asn with SvePatA

(A) Secondary structure (dotted line) and conformational change of SvePatA, after the addition of 2 mM Asn (full line) and subtraction of buffer baseline, are shown from 190 to 260 nm. (B) The docking result of Asn to SvePatA shows that the possible residues of SvePatA interact with Asn via hydrogen bonds. (C, D) The acetyltransferase activities of SvePatA mutants (K112A and P113A) in the presence of Asn.

Figure 7
The binding of Asn with SvePatA

(A) Secondary structure (dotted line) and conformational change of SvePatA, after the addition of 2 mM Asn (full line) and subtraction of buffer baseline, are shown from 190 to 260 nm. (B) The docking result of Asn to SvePatA shows that the possible residues of SvePatA interact with Asn via hydrogen bonds. (C, D) The acetyltransferase activities of SvePatA mutants (K112A and P113A) in the presence of Asn.

Discussion

N-lysine acetylation is a dynamic, reversible, and regulatory post-translational modification (PTM) used to modulate enzyme activity in prokaryotes. Accumulating evidence suggests that metabolic networks are co-ordinated via reversible acetylation of enzymes that regulate cellular metabolism to maintain homeostasis in the rapidly changing microenvironment. The present study examined the archetypical acetyltransferases AAPatAs possessing GNAT and ACT domains, and shows a novel signaling pathway for regulating the acetylation of cellular proteins. These findings indicate that in actinobacteria, acetyltransferase activity and protein acetylation may be tightly correlated with intracellular amino acid metabolism.

The conventional mechanism for reversible Nε-acetylation of proteins is enzyme-catalyzed acetylation and deacetylation, which relies on KATs and deacetylases (HDACs). Reversible enzyme-catalyzed acetylation is regulated at different levels, such as transcriptionally and allosterically, to adjust the acetylation of specific proteins responding to intracellular nutritional signals. In B. subtilis, the acuA gene encoding an acetyltransferase is controlled by the carbon catabolite control protein, CcpA [5]. In E. coli, the cAMP receptor protein CRP induces the expression of yfiQ, which encodes the acetyltransferase YfiQ, thus increasing the acetylation level of proteins in response to the intracellular cAMP signal [6]. In S. enterica, lolR, the repressor of myo-inositol catabolism, activates the transcription of pat and cobB [10].

There are three classes of KATs that catalyze the donation of an acetyl group from acetyl-coenzyme A (Ac-CoA) to an ε-amino group of a deprotonated lysine. These KATs include the GNAT family, the MYST family, and the p300/CBP family [25]. The genomes of actinobacteria encode approximately 40–80 GNAT acetyltransferases, 1–2 NAD+-dependent sirtuin deacetylases, or 1 NAD+-independent protein deacetylase. The genome of S. coelicolor encodes 77 putative GNAT acetyltransferases (Pfam00583), 2 sirtuin-type deacetylases (SCO0452, SCO6464), and an AcuC-like NAD+-independent deacetylase (SCO3330). The genome of S. avermitilis encodes 43 putative GNAT acetyltransferases, 1 sirtuin-type deacetylase (SAV_537), and 1 AcuC-like deacetylase (SAV_4729). The genome of S. lividans encodes 72 putative GNAT acetyltransferases, 2 sirtuin-type protein deacetylases (EFD65580, EFD71509), and 1 NAD+-independent deacetylase (EFD68590). The genome of Saccharopolyspora erythraea encodes 42 putative GNAT acetyltransferases, 1 NAD+-dependent deacetylase (SACE_3798), and 1 NAD+-independent deacetylase (SACE_1779). Thus, the number of GNAT acetyltransferases in a bacterial species may reflect the metabolic complexity of the species. Thus far, all the known bacterial Pat identified contain the GNAT domain having low sequence homology, and sharing a conserved motif A, sequence (R/Q)XXGX(G/A) for Ac–CoA recognition and binding, and a glutamate residue providing as an active site. It is worth noting that there has been considerable debate about the function of the conserved glutamate as a catalytic base [26,27]. In prokaryotes, some Pat have two different domain organizations: the multidomain (Class I) and single-domain (Class II). Several single-domain GNAT proteins have been shown to have acetyltransferase activity. These Class II enzymes, which are approximately 200 residues long, include BsAcuA from B. subtilis [28], RpKatA from Rhodopseudomonas palustris [29], and SacAcuA from Saccharopolyspora erythraea [17].

Class Iα Pat, such as the first discovered SePat in S. enterica [1], the homologs (EcPatZ from E. coli and RpPat from R. palustris) and variants (SlPatA from Streptomyces lividans) of SePat [6,29,30], are 800-1000 residues long and contain a GNAT catalytic and NAD+-forming CoA ligase domains; the function of the latter is undetermined, but it has homology with ADP-forming acyl–CoA synthetases. The catalytic mechanism and domains function of SePat and EcPatZ have been investigated [31,32]. Biochemical and thermodynamic analyses of SePat revealed that SePat has two distinct sites for Ac–CoA binding, and shows the positive co-operativity (with a Hill coefficient of 2.2 ± 0.2) where binding of Ac–CoA to one site increases the affinity of a second binding site [31]. Recent work found that the kinetic data of EcPatZ for Ac–CoA display atypical sigmoidal activity, suggesting that EcPatZ also reveals the positive co-operativity with a Hill coefficient of 7.91 ± 0.22 [32]. Positive co-operativity observed in class Iα multidomain acetyltransferases may be a result of subunit interactions and oligomerization in the presence of Ac–CoA. More recently, an interesting work demonstrated that cAMP directly binds to SeAcs, and inhibits its activity through competitive binding to the ATP/AMP pocket, whose binding with cAMP promotes SeAcs acetylation, and further resulting in inhibition of SeAcs activity [33].

Class Iβ Pat are allosteric enzymes with two domains in which an effector-binding regulatory domain is fused to the GNAT catalytic domain. The cAMP-sensing acetyltransferases (MtPatA in Mycobacterium tuberculosis and MsPatA in M. smegmatis) [34,35], NADP-sensing acetyltransferase (MxKat in Myxococcus xanthus) [36], and amino acid-sensing acetyltransferase (MaKat in Micromonospora aurantiaca) [12] are examples of Class Iβ Pat. These enzymes have regulatory domains that bind to an effector (e.g. cAMP, NADP, or amino acids) to allosterically regulate the activities of Pat; this presents a novel mechanism for directly connecting the levels of protein acetylation with intracellular concentration of metabolites. Class Iβ enzymes are not widely distributed in microorganisms. cAMP-sensing acetyltransferases are present only in mycobacteria, while the NADP-sensing acetyltransferase is present only in M. xanthus. In the present study, we found that the amino acid-sensing AAPatA acetyltransferases are divided into two groups based on allosteric effectors: the Asn-regulated PatA and Cys-regulated PatA. The former are found only in Streptomycetaceae; the latter are distributed in other actinobacteria (Pseudonocardiaceae, Micromonosporaceae, Nocardiopsaceae, and Streptosporangiaceae). Class Iβ Pat with different regulatory domains use diverse metabolic signals to regulate lysine acetylation in response to varying physiological conditions and environmental changes. To investigate the function of Class Iβ enzymes, it is necessary to identify their physiological substrates. A universal stress protein USP (MSMEG_4207), Acs, and eight additional acyl-CoA synthetases may be the possible acetylated protein targets of MsPatA, and their acetylation may be dependent upon the levels of intracellular cAMP [11,12,34]. No acetylated protein targets of other Class Iβ enzymes have been identified. It is possible that AAPatA may acetylate enzymes involved in Asn and Cys metabolism and may regulate these pathways in response to intracellular Asn or Cys concentrations. More work is needed to identify the physiological substrates of Class Iβ enzymes and to elucidate the effect of acetylation on the regulation of metabolic enzyme activity.

Author contribution

B.-C.Y., Y.-X.L. and X.-X.L. conceived and designed the experiments. Y.-X.L. and X.-X.L. performed the experiments and analysed the experimental data. B.-C.Y. and W.-B.L. wrote the paper.

Funding

This work is supported by the China NSF [grant numbers 21335003, 21575089, 21276079]; the National Key Technologies R&D Programs [grant number 2014AA021502], the Fundamental Research Funds for the Central Universities and the Natural Science Foundation of Shanghai [grant number 14ZR1409600].

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • AAPatA

    amino acid sensing acetyltransferase

  •  
  • Ac–CoA

    acetyl–CoA

  •  
  • AcP

    acetyl-phosphate

  •  
  • Acs

    acetyl–CoA synthetase

  •  
  • anti-Ack

    anti-acetyl-lysine antibody

  •  
  • CcpA

    catabolite control protein A

  •  
  • GNAT

    Gcn5-related N-acetyltransferase

  •  
  • KAT

    lysine acetyltransferase

  •  
  • Pat

    protein acetyltransferase

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Supplementary data