Identification and characterization of two types of amino acid-regulated acetyltransferases in actinobacteria

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.

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.

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 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].

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).

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 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).

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.

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.

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.

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.

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.

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.

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 [12–14]. 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.

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.

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 K_m and k_cat values of AmiPatA for AmiAcs are 45.5 μM and 0.03 s⁻¹, respectively. Cys was able to decrease the K_m value of AmiPatA for AmiAcs by three-fold, and increase k_cat value by 1.5-fold.

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].

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.

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.

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.

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 N^ε-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.

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.

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].

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

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