Acetylation of lysine �-amino groups regulates aminoacyl-tRNA synthetase activity in Escherichia coliReceived for publication, December 1, 2016, and in revised form, April 16, 2017 Published, Papers in Press, April 28, 2017, DOI 10.1074/jbc.M116.770826

Qing Ye‡, Quan-Quan Ji‡, Wei Yan‡, Fang Yang‡, and En-Duo Wang‡§1

From the ‡State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute ofBiochemistry and Cell Biology, Chinese Academy of Science, University of Chinese Academy of Sciences, 320 Yueyang Road,Shanghai 200031, China and the §School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China

Edited by Ronald C. Wek

Previous proteomic analyses have shown that aminoacyl-tRNA synthetases in many organisms can be modified by acetyl-ation of Lys. In this present study, leucyl-tRNA synthetase andarginyl-tRNA synthetase from Escherichia coli (EcLeuRS andEcArgRS) were overexpressed and purified and found to beacetylated on Lys residues by MS. Gln scanning mutagenesisrevealed that Lys619, Lys624, and Lys809 in EcLeuRS and Lys126

and Lys408 in EcArgRS might play important roles in enzymeactivity. Furthermore, we utilized a novel protein expressionsystem to obtain enzymes harboring acetylated Lys at specificsites and investigated their catalytic activity. Acetylation ofthese Lys residues could affect their aminoacylation activity byinfluencing amino acid activation and/or the affinity for tRNA.In vitro assays showed that acetyl-phosphate nonenzymaticallyacetylates EcLeuRS and EcArgRS and suggested that the sirtuinclass deacetylase CobB might regulate acetylation of these twoenzymes. These findings imply a potential regulatory role forLys acetylation in controlling the activity of aminoacyl-tRNAsynthetases and thus protein synthesis.

Aminoacyl-tRNA synthetase (aaRS)2 catalyzes esterificationbetween its cognate amino acid and tRNA to produce amino-acyl-tRNA (aa-tRNA) in the initiation step of translation. Ahigh level of accuracy is essential during aminoacylation toensure quality control during protein synthesis. Disruption oftranslational fidelity can lead to mistranslation, with profoundconsequences for both prokaryotic and eukaryotic cells (1–3).

The 20 aaRSs can be divided into two classes, each with 10members, based on sequence identity and characteristic struc-tural motifs (4). Class I members have two signature peptides

(HIGH and KMSK) located in the active site that form a char-acteristic dinucleotide binding fold (Rossmann fold, �-�-�-�-�). Both leucyl- and arginyl-tRNA synthetases (LeuRS andArgRS) belong to class I aaRSs (5). Like 16 other aaRSs, LeuRScatalyzes aminoacylation of its cognate tRNA in a two-stepreaction: (a) activation of the amino acid with ATP and for-mation of an aminoacyl adenylate and (b) transfer of theaminoacyl moiety from the aminoacyl adenylate to the cog-nate tRNA substrate (5, 6). However, ArgRS, together withglutamyl-tRNA synthetase and glutaminyl-tRNA synthe-tase, requires the presence of the cognate tRNA for aminoacid activation (7, 8).

LeuRS consists of a Rossmann fold domain for aminoacyla-tion, a helix bundle domain for binding the tRNA anticodon, aconnective peptide 1 (CP1) domain for editing mischargedtRNA, a ZN1 module, a leucine-specific domain, and a C-ter-minal domain (CTD) for tRNA binding (9, 10). The aminoacy-lation and editing mechanisms of LeuRS from various specieshave been thoroughly investigated (11–13). ArgRS can bedivided into five domains: an N-terminal additional domain(Add1) for tRNA D-loop recognition, a catalytic Rossmann folddomain, two domains (Ins-1 and Ins-2) inserted in the activesite, and a C-terminal additional domain (Add2) that partici-pates in the binding of the tRNA anticodon (14, 15). It is pecu-liar that the Add1 domain is conserved in ArgRS but not inother class I aaRSs. In most species, ArgRS lacks a canonicalKMSK sequence, and a conserved lysine (Lys) upstream of theHIGH sequence motif in these enzymes stabilizes the transitionstate of the amino acid activation reaction (Arg-AMP forma-tion) to compensate for the loss of the second Lys (K2) in theKMSK motif (16, 17).

Cells are constantly faced with the challenge of changingenvironmental conditions, and post-translational modification(PTM) is one method of dealing with this challenge. PTM canexpand the genetic lexicon by endowing proteins with diversitybeyond that can be achieved with the canonical 20 proteino-genic amino acids. Acetylation of the �-amino group of theN-terminal amino acid (irreversible) or the �-amino group ofinternal Lys residues (reversible) is one type of PTM. In general,acetylation of Lys refers to reversible acetylation, and it canregulate fundamental cellular processes such as transcription,translation, pathways associated with central metabolism, andstress responses (18). Although the essential regulatory role ofLys acetylation in eukaryotes is widely accepted and relatively

This work was supported by Strategic Priority Research Program of the Chi-nese Academy of Sciences Grant XDB19000000 and National Natural Sci-ence Foundation of China Grants 31570792, 31130064, and 91440204. Theauthors declare that they have no conflicts of interest with the contents ofthis article.

1 To whom correspondence should be addressed. Tel.: 86-21-5492-1241; Fax:86-21-5492-1011; E-mail: edwang@sibcb.ac.cn.

2 The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; LeuRS, leu-cyl-tRNA synthetase; ArgRS, arginyl-tRNA synthetase; Acs, acetyl-CoA syn-thetase; WT, wild type; AcK, N�-acetylated lysine; AcP, acetyl-phosphotate;PDB, Protein Data Bank; PTM, post-translational modification; K-Qmutants, proteins that with a substitution of Q for K; KAc mutants, proteinsthat with a substitution of AcK for K; CP1, connective peptide 1; CTD, C-ter-minal domain; Ac-CoA, acetyl coenzyme A; TyrRS, tyrosyl-tRNA synthetase;NAM, nicotinamide; IPTG, isopropyl-1-thio-�-D-galactopyranoside; Ni-NTA,nickel-nitrilotriacetic acid; Ara, arabinose.

crosARTICLE

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well understood, its function in bacteria and archaea remainsmore obscure (19, 20).

In Escherichia coli, although several putative protein acety-lases are present in the genome, Gcn5-like YfiQ, which is highlysimilar to the acetyltransferase Pat in Salmonella enterica, is theonly confirmed acetyltransferase to date (21–23), and CobB isthe predominate deacetylase, which belongs the NAD�-depen-dent sirtuin family (22–24). Recently, the serine hydrolaseYcgC was identified as a Zn2�- and NAD�-independentdeacetylase that regulates a distinct set of substrates from CobB(25). One well studied target of protein acetylation is acetyl-CoA synthetase (Acs), which activates acetate to the high-en-ergy intermediate acetyl coenzyme A (Ac-CoA; acetate � ATP �CoA 3 AMP � PPi � Ac-CoA). Reversible acetylation of acatalytic core Lys residue conserved in Acs enzymes from bac-teria to human (Lys609 in S. enterica Acs) could regulate enzymeactivity, because it blocks ATP-dependent adenylation of ace-tate, preventing the formation of acetyl-AMP and the subse-quent production of Ac-CoA (18, 21). In S. enterica, Acs Lys609

is regulated by a protein acetylation/deacetylation system thatincludes Pat and CobB (21), and this system also coordinatescarbon source utilization and metabolic flux by controlling theacetylation of metabolic enzymes (26). Interestingly, acetyla-tion was recently found to be mediated nonenzymatically inmitochondria of both prokaryotes and eukaryotes (20, 23,27–31). In E. coli, the majority of acetylation occurs indepen-dently of YfiQ, and the glycolysis intermediate acetyl-phos-phate (AcP) is associated with a global shift in protein acetyla-tion, whereas CobB regulates a subset of these chemicalacetylation events (20, 23, 28).

Some aaRSs are modified by phosphorylation, which influ-ences multidrug tolerance in E. coli and the reactive oxygenspecies defense mechanism in mammalian cells (32, 33).Despite growing knowledge, studies focusing on other forms ofPTM of aaRSs are few in number. A large-scale proteomic sur-vey demonstrated that some aaRSs from E. coli, S. enterica,Bacillus subtilis, Drosophila melanogaster, Mus musculus, Rat-tus norvegicus, and Homo sapiens are acetylated, and bioinfor-matics and network analysis of acetylation sites found aa-tRNAbiosynthesis pathway enriched in some species (23, 26, 31,34 – 42). Furthermore, some of the identified acetylated Lys res-

idues are conserved, and it would be intriguing to decipher theexact role of acetylation of aaRSs.

Herein, we discovered that EcLeuRS and EcArgRS are mod-ified by acetylation of Lys residues in vivo. By utilizing an engi-neered Methanosarcina barkeri pyrrolysyl-tRNA synthetase(MbPylRS)/MbtRNACUA pair (N�-acetyllysyl-tRNA synthe-tase/tRNACUA pair, pAcKRS) system (43), we obtained en-zymes harboring acetylation at specific sites and investigatedthe biochemical properties of EcLeuRS-KAcs and EcArgRS-KAcs. We also examined the molecular mechanism controllingregulation of the acetylation of EcLeuRS and EcArgRS and iden-tified CobB and AcP as possible regulatory factors. Acetylationappears to be a mechanism for adjusting the activity of aaRSsand thereby controlling protein synthesis.

Results

MS revealed acetylation at 11 Lys residues in EcLeuRS

Previous studies demonstrated that LeuRS from E. coli, Sac-charomyces cerevisiae, R. norvegicus, H. sapiens, and other spe-cies can be acetylated (38, 40, 41, 44). To identify the acetylationsites in EcLeuRS, we overexpressed ecleuS that encodesEcLeuRS with a N-terminal His6 tag in E. coli BL21 and purifiedthe recombinant protein by Ni2�-NTA affinity chromatogra-phy. 11 Lys residues were detected to be acetylated in threeindependent MS analyses. They span the entire protein andinclude Lys619 and Lys624 in the KMSK signature sequence(Figs. 1 and 2, A and B).

To understand the effect of acetylation on the aminoacyla-tion activity of EcLeuRS, we separately mutated all 11 Lys resi-dues to Gln, because this residue lacks a positive charge on theside chain and is a good mimic of acetylated Lys. We assayed theaminoacylation activity of the K-Q mutants and found thatmutations at Lys619 and Lys624 in the amino acid activationactive site and Lys809 in the CTD displayed decreased amino-acylation activity compared with WT EcLeuRS, whereas theaminoacylation activity of mutants at all other sites wasunchanged (Fig. 2C). Lys402 is the only residue in the CP1domain among these 11 residues, and the co-crystal structureof EcLeuRS with tRNALeu and Leu (PDB number 4ARC) indi-cated that Lys402 lies on the surface of the CP1 pocket and

Figure 1. Identification of acetylation at Lys619 of EcLeuRS by MS. MS/MS spectrum of a tryptic peptide from EcLeuRS (DAAGHELVYTGMSKAcMSK) showsacetylation of Lys (KAc), confirmed as Lys619 by sequence alignment with the known sequence of EcLeuRS. Most major fragmentation ions matched predictedb or y ions.

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points away from the domain core, suggesting it is not likely tobe essential for the editing function. Indeed, Ile-tRNALeu

deacylation assays showed that the post-transfer editing activ-ity of the EcLeuRS-K402Q mutant remained unchanged com-pared with native EcLeuRS (data not shown).

Characterization of KAc mutants reveals that Lys acetylationreduces EcLeuRS enzyme activity

To further explore the influence of acetylation on EcLeuRS,we used a previously described system to incorporateN�-acetyl-L-Lys (AcK) at specific sites to generate EcLeuRS-KAcs in situ (Fig. 3) (43). We transformed E. coli BL21 cells withtwo plasmids: pAcKRS encoding the N�-acetyllysyl-tRNA syn-thetase/tRNACUA pair that activates AcK and recognizes theUAG codon and another encoding EcLeuRS in which the Lystriplet codon was substituted with TAG.

Sequence alignment showed that Lys619 and Lys809 ofEcLeuRS are conserved in LeuRSs from various species,whereas Lys624 is basically conserved in prokaryotic LeuRSs(Fig. 4A). Following overexpression as described above,EcLeuRS-K619Ac, EcLeuRS-K624Ac, and EcLeuRS-K809Ac

were purified and confirmed to be 90% homogeneous by SDS-PAGE (data not shown). Western blotting confirmed the incor-poration of AcK into EcLeuRS (Fig. 4B), and comparison of CDspectra of EcLeuRS-WT and EcLeuRSAcs confirmed thatEcLeuRSAcs were properly folded (data not shown).

Lys619 and Lys624 are located in or downstream of the con-served KMSK loop, which, together with the HIGH motif, isessential for the amino acid activation activity. EcLeuRS-K619Ac completely lost its Leu activation and leucylation activ-ities (Fig. 4, C and D, and Tables 1 and 2). The amino acidactivation and aminoacylation activities of EcLeuRS-K624Ac

were also determined. Even though EcLeuRS-K624Ac severelylost its activation activity (Fig. 4C and Table 1), the catalyticefficiency (kcat/Km) in aminoacylation was not that severelydamaged (Fig. 4D and Table 2). The total effect might bebecause tRNA charging is the rate-limiting step. Kd valuesbetween EcLeuRSs and tRNALeu calculated by fluorescencequenching showed that the binding affinity of EcLeuRS-K619Ac

and-K624Ac with tRNALeu was not altered compared withthat of EcLeuRS-WT (Table 3). These results suggest acety-lation of these two Lys residues (especially Lys619) might leadto a conformational change in the synthetic active sitepocket, decreasing the Leu activation and aminoacylationactivities (Fig. 4E).

The flexibly linked CTD in LeuRS makes contacts withtertiary structural base pairs and the long variable arm oftRNALeu (10, 45). The ternary complex structure of EcLeuRS,tRNALeu

UAA, and Leu in the editing conformation (PDB num-ber 4ARC) revealed that Lys809 is located on the edge of one�-sheet in the CTD. The Leu activation activity of EcLeuRS-

Figure 2. Identification of Lys residues acetylated in EcLeuRS. A, the overall ternary structure of EcLeuRS and its cognate tRNALeu together with Leu in theediting conformation (PDB number 4ARC). B, schematic diagram of EcLeuRS. RF, Rossmann fold. C, aminoacylation assays screening potential crucial Lysresidues. Left panel, mutation of Lys619, Lys624, and Lys809 to Gln damaged EcLeuRS canonical activities. Right panel, mutation of other Lys residues had a slightlynegative effect on EcLeuRS canonical activities. The results are averages plus standard deviations from three independent experiments.

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K809Ac was not changed compared with that of WT EcLeuRS;consistent with that, Lys809 is distant from the activation activesite core region (Fig. 4C and Table 1). The crystal structure ofEcLeuRS (PDB number 4ARC) showed that the side chain ofLys809 lies at a distance of 3.16 Å away from the phosphategroup of the U47I ribose backbone of tRNALeu (Fig. 4F).EcLeuRS-K809Ac had a similar kcat toward EctRNALeu as didEcLeuRS-WT (5.0 s�1 for EcLeuRS-K809Ac, 5.7 s�1 for WT);nevertheless, the affinity for the cognate EctRNALeu (Km, 6.7�M for EcLeuRS-K809Ac and 1.2 �M for WT) was decreased,and the catalytic efficiency (kcat/Km) of EcLeuRS-K809Ac (0.7s�1 �M�1) was only 15% that of EcLeuRS-WT (4.8 s�1 �M�1;Table 2). In addition, the Kd of EcLeuRS-K809Ac with tRNALeu

was 1.4-fold that of the native enzyme, implying a decrease inbinding affinity between LeuRS and tRNALeu (Table 3). Theinteraction between enzyme and tRNALeu was partially dis-rupted by the acetylation of Lys809.

These results indicate that acetylation of Lys619, Lys624, andLys809 could potentially inhibit either amino acid activation ortRNA-charging activities of EcLeuRS. Among these residues,Lys619 and Lys809 in EcLeuRS are the residues whose acetylationlead to a sharp reduction of catalytic efficiency. In addition, theaminoacylation activity of EcLeuRSAcs was comparable withthe corresponding K-Q mutants (Figs. 2C and 4D), suggestingthat Gln is a suitable mimic of acetylated Lys.

EcLeuRS is acetylated by AcP rather than YfiQ, and CobB candeacetylate EcLeuRSAc

Determining the enzyme responsible for acetylatingEcLeuRS is of particular interest. At present, the Gcn5-likeacetyltransferase YfiQ is the only known enzyme that acetylatesthe �-NH2 group of Lys in E. coli (23). However, purified YfiQwas unable to transfer the acetyl group of Ac-CoA to EcLeuRSin vitro (Fig. 5A). The metabolism of AcP, a high-energy inter-mediate between acetate and Ac-CoA, has been shown to alterglobal acetylation levels in vivo, and AcP acetylates proteinsnonenzymatically at multiple Lys residues in vitro (23). Giventhat AcP is a critical regulator of acetylation in bacteria, weincubated purified EcLeuRS with AcP and detected an increasein EcLeuRS acetylation (no acetylation signal is detected on WTenzyme before AcP treatment), implying a potential role forAcP in the acetylation of LeuRS (Fig. 5A). We refer to this AcP-derived form of EcLeuRS as EcLeuRSAc.

CobB, belonging to the sirtuin class, is the main deacetylasein E. coli (22–24). To determine whether CobB is involved indeacetylation of EcLeuRSAc, deacetylation assays were per-formed with EcLeuRSAc as the substrate for CobB. The resultsshowed that CobB could deacetylate EcLeuRSAc, and the pres-ence of the CobB inhibitor NAM, or the absence of its cofactor,NAD�, rendered CobB inactive (Fig. 5B). We also tested theactivity of CobB on purified EcLeuRS-K619Ac and EcLeuRS-K809Ac, and Western blotting showed that CobB effectivelydecreased their acetylation in a time-dependent manner (Fig.5C). Additionally, aminoacylation assays showed that aftertreatment with CobB, EcLeuRS-K619Ac and EcLeuRS-K809Ac

recovered aminoacylation activity to some extent (Fig. 5D). Theabove results suggest CobB deacetylates EcLeuRS in vitro.

Acetylation of EcArgRS influences its catalytic rate

To investigate whether EcArgRS is regulated by acetylation,similar experiments to those described above were performedon EcArgRS. MS (repeated three times) detected acetylationat five Lys residues in EcArgRS including Lys126, which isupstream of the HIGH motif in the activation site (Figs. 6 and 7)(16, 17). As described above for EcLeuRS, we performed Gln-scanning mutagenesis on these five Lys residues of EcArgRSand found that K-Q mutants of Lys126 and Lys408 displayed adecrease in aminoacylation activity (Fig. 7C). Sequence align-ment showed that Lys126 and Lys408 are highly conservedamong prokaryotic and eukaryotic ArgRSs (Fig. 8A). Asdescribed above for EcLeuRS, we utilized the pAcKRS system togenerate EcArgRS-KAcs, and CD spectra confirmed that theirsecondary structures were not altered by the point mutations(data not shown).

EcArgRS-K126Ac lost Arg activation and arginylation activi-ties and its affinity with tRNAArg did not change compared withWT enzyme (Fig. 8, B and C; see Table 6). Lys126 lies in theupstream of the HIGH (HVGH in EcArgRS) sequence withinthe catalytic pocket of EcArgRS, where it compensates for thelack of a canonical KMSK (especially the second Lys, K2) inArgRS in most species and thus makes an important contribu-tion to the aminoacylation reaction (Fig. 8D) (16, 17). Acetyla-tion of Lys126 in EcArgRS led to the complete loss of arginyla-

Figure 3. Flow diagram of the overexpression of site-directed AcK-incor-porated proteins in E. coli BL21 (DE3). Taking EcLeuRS as an example,pAcKRS and pET22b(�)-ecleuS were co-transformed in E. coli BL21 (DE3), andengineered pAcKRS were induced by the addition of Ara. Subsequently, IPTGwas added to induce the production of EcLeuRS in the presence of NAM, aninhibitor of CobB. With the assistance of pAcKRS, EcLeuRS was translated infull-length form with incorporation of AcK or in truncated form (terminatingat the Lys codon mutation site). All other experimental details were asdescribed previously (43).

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tion, consistent with previous experiments on TtArgRS-K116Gthat also showed a complete loss of activation and aminoacyla-tion activities (17).

We next focused on the Lys408 residue in the Add2 domainthat is implicated in the binding of the tRNAArg anticodonregion (15, 46). EcArgRS-K408Ac displayed a low Arg activationactivity (Fig. 8B and Table 4) and aminoacylation activity (Fig.

8C and Table 5), and kinetic parameters revealed a weakeraffinity for EctRNAArg (Km � 9.0 �M) compared with WTenzyme (Km � 2.7 �M). The kcat (6.9 s�1) was also decreased to24% that of WT (28.4 s�1), and the overall effect was a decreasein the catalytic efficiency (kcat/Km, 0.8 s�1 �M�1) to only 8% that

Figure 4. Effect of acetylation of Lys residues on the Leu activation and aminoacylation activities of EcLeuRS. A, sequence alignment of LeuRSs fromvarious species in regions homologous to Lys619, Lys624, and Lys809 in EcLeuRS. Ec, E. coli; Aa, Aquifex aeolicus; Bs, B. subtilis; Sco, Streptomyces coelicolor; Hs,H. sapiens; Ph, Pyrococcus horikoshii; mt, mitochondrial; ct, cytoplasmic. B, Western blotting confirming the incorporation of AcK in EcLeuRS-K619Ac, EcLeuRS-K624Ac, and EcLeuRS-K809Ac. C, Leu activation of EcLeuRS-K619Ac, EcLeuRS-K624Ac, and EcLeuRS-K809Ac. D, aminoacylation of EcLeuRS-K619Ac, EcLeuRS-K624Ac, and EcLeuRS-K809Ac resembling that of the K-Q mutants. E, closer view of the orientation of Lys619 and Lys624 relative to the conserved HIGH and KMSKmotifs (HMGH and KMSK in EcLeuRS, depicted in dark blue; PDB code 4ARC). F, closer view of the interaction between EcLeuRS Lys809 and EctRNALeu U47I (PDBcode 4ARC). The results are the averages and standard deviations from three independent experiments, and all Western blots were repeated.

Table 1Observed rate constants (kobs) of EcLeuRSs in Leu activationAll parameters are average values from three independent determinations withstandard deviations. nm, nonmeasurable (too low to be measured).

Enzymes kobs Relative values

s�1 %EcLeuRS-WT 55.0 � 3.6 100EcLeuRS-K619Ac nmEcLeuRS-K624Ac 4.4 � 0.6 8EcLeuRS-K809Ac 48.3 � 5.1 88

Table 2Kinetic parameters of EcLeuRS and derived site-specific acetylatedvariants for EctRNALeu in aminoacylationAll parameters are average values from three independent determinations withstandard deviations. nm, nonmeasurable (too low to be measured).

Enzymes Km kcat kcat/Km

Relative catalyticefficiency

�M s�1 s�1 �M�1 %EcLeuRS-WT 1.2 � 0.3 5.7 � 0.3 4.8 100EcLeuRS-K619Ac nm nmEcLeuRS-K624Ac 1.3 � 0.2 2.8 � 0.3 2.2 46EcLeuRS-K809Ac 6.7 � 0.7 5.0 � 0.4 0.7 15

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of the native enzyme (10.5 s�1 �M�1) (Table 5). The Kd value ofEcArgRS-K408Ac with tRNAArg (0.40 �M) was also increased by�1.7-fold compared with EcArgRS-WT (0.24 �M; Table 6).Lys408 is located in the hairpin following helix �13 of EcArgRS,corresponding to Lys455 in PhArgRS. In the crystal structure ofPhArgRS (PDB number 2ZUE), Lys455 lies within an �-helixadjacent to the phosphate group of A38 in the anticodon loop oftRNAArg (Fig. 8E). In yeast and E. coli, C35 is one of the majoridentity elements of tRNAArg (47, 48). Acetylation of Lys408

may distort the neighboring region that interacts with thetRNAArg anticodon and consequently decrease the affinity ofEcArgRS for tRNAArg. Given that EcArgRS requires tRNAArg asthe activator during Arg activation, the decrease in the EcAr-gRS-K408Ac activation activity could be partly due to a loss inaffinity with tRNAArg. Overall, the in vitro data show that theacetylation of EcArgRS (Lys126 and Lys408) could severelydecrease its aminoacylation activity.

EcArgRS acetylation appears to be regulated by AcP and CobB

We also attempted to identify enzymes or other moleculesinvolved in acetylation of EcArgRS. In vitro assays showed thatAcP acetylated purified EcArgRS (Fig. 9A), but YfiQ did not(data not shown) (no acetylation signal is detected on WTenzyme before AcP treatment). Furthermore, in vitro CobBdeacetylated EcArgRSAc, the product of EcArgRS followingtreatment with AcP (Fig. 9B). Purified EcArgRS-K126Ac andEcArgRS-K408Ac variants were also deacetylated by CobB, andas described above, addition of NAM or the absence of NAD�

caused CobB to lose its deacetylation activity (Fig. 9C). AcP andCobB therefore appear to control acetylation and deacetylationof EcArgRS, consistent with the results discovered above forEcLeuRS.

Discussion

AaRSs are found to be acetylated

Some aaRSs are post-translationally modified. In E. coli,tRNAGlu-bound glutamyl-tRNA synthetase can be phosphory-lated at Ser239 in the KMSK motif by the eukaryote-like serine-threonine kinase HipA. This PTM results in a loss of aminoacy-lation activity, which increases uncharged tRNAGlu loading atthe A site of the ribosome, triggering (p)ppGpp formation andfacilitating multidrug tolerance (32).

Acetylation regulates proteins involved in transcription,amino acid, nucleotide and protein biosynthesis, protein fold-

Figure 5. AcP and CobB regulate acetylation of EcLeuRS. A, AcP but not YfiQ acetylates EcLeuRS in vitro. B, CobB removal of the acetyl moiety of EcLeuRSAc.C, CobB deacetylation of EcLeuRS-K619Ac and EcLeuRS-K809Ac. In the presence of NAM or the absence of NAD�, CobB is inactivated. D, incubation with CobBrecovers the aminoacylation activity of EcLeuRS-K619Ac (left panel) and EcLeuRS-K809Ac (right panel). All experiments were conducted at least twice. Whenquantifying of the relative amount of AcK signal/His signal, the sample without NAD� and NAM was defined as 100%.

Table 3Kd values between tRNALeu and EcLeuRSs determined by fluorescencequenchingAll parameters are average values from three independent determinations withstandard deviations.

Enzymes Kd Relative values

�M -foldEcLeuRS-WT 0.19 � 0.01 1.0EcLeuRS-K619Ac 0.21 � 0.03 1.1EcLeuRS-K624Ac 0.18 � 0.01 0.9EcLeuRS-K809Ac 0.27 � 0.02 1.4

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ing, and detoxification responses in various species. Wefocused on aaRSs and found that many are acetylated on Lysresidues located both on the surface and within the catalyticcore or the tRNA binding domain in others’ papers. In the pres-ent work, we confirmed the acetylation of two class Ia aaRSs inE. coli by three independent MS experiments. The residuesfound to be acetylated on these two aaRSs are not exactly thesame as what were found in the studies of Weinert et al. (23)and Kuhn et al. (28). This might result from the use of differentE. coli strains under various growth stages. Differences of nutri-ents in the media used might also influence the acetylation.In addition, we purified overexpressed proteins, rather thanendogenous aaRSs. However, acetylation of some crucial resi-dues that had been identified by the above authors (like Lys619

and Lys624 in EcLeuRS and Lys126 and Lys408 in EcArgRS) wereconsistently identified in our studies. We screened several Lysresidues at which acetylation may negatively regulate theaminoacylation activity of enzyme by using a Gln scanningmutagenesis approach. Furthermore, we used the pAcKRS sys-

tem to express and purify EcLeuRS and EcArgRS acetylated atspecific sites (43) and characterized the effect of acetylation onthe catalytic properties.

The catalytic activity of aaRSs is regulated by acetylation

Acetylation of Lys619 and Lys809 greatly impacted the amino-acylation activity of EcLeuRS, and both residues are highly con-served among LeuRS in various species (Fig. 4A). Lys622 is thesecond Lys in the KMSK motif (K2), which is the key residuethat directly interacts with Leu-AMP and believed to stabilizethe negatively charged transition state of the first reaction stepin class I aaRSs. Mutation of this residue led to a severe loss ofenzymatic activity (49 –51). Acetylation of the adjacent resi-dues, Lys619 and Lys624 caused significant inhibition of the firststep of aminoacylation. In Bacillus stearothermophilus tyrosyl-tRNA synthetase (TyrRS), another class I aaRS, 230KFGK233

corresponds to the signature sequence KMSK. Previous datashowed that the BsTyrRS-K230N mutant lost its activationactivity, indicating that the first Lys in the KMSK is crucial to

Figure 6. Identification of acetylation at Lys126 of EcArgRS by MS. MS/MS spectrum of a tryptic peptide from EcArgRS (QTIVVDYSAPNVAKAcEMHVGHLR)showing acetylation of Lys (KAc), confirmed as Lys126 by sequence alignment of the peptide with the known sequence of EcArgRS. Most major fragmentationions matched predicted b or y ions.

Figure 7. Lys residues acetylated in EcArgRS. A, crystal structure of PhArgRS complexed with PhtRNAArg and ANP (PDB code 2ZUE). B, schematic diagram ofEcArgRS. RF, Rossmann fold. C, aminoacylation assay screening of potentially crucial Lys residues. Left panel, mutation of Lys126 and Lys408 damages theenzymatic activities of EcArgRS. Right panel, mutation of other Lys residues has a slight negative effect on the activities EcArgRS. The results are the averagesand standard deviations from three independent experiments.

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the activation of tyrosine (49). Lys619 is the first Lys in the sig-nature sequence of EcLeuRS, and Lys624 is adjacent to the sig-nature sequence. Acetylation of the two Lys residues shouldinhibit the active site of amino acid activation and thus influ-ence the activation and aminoacylation activities of EcLeuRS.

Hsu et al. (52) utilized Ala scanning mutagenesis to identifyspecific sites in the CTD that may be important for RNA-pro-tein interactions and found that mutation of Lys809 had a neg-ligible effect on aminoacylation catalytic efficiency. By contrast,

Figure 8. Effect of acetylation of Lys on the Leu activation and aminoacylation activities of EcArgRS. A, sequence alignment of ArgRSs from variousspecies in regions homologous to Lys126 and Lys408 of EcArgRS. The abbreviations are the same as those in Fig. 4, except for two species (Se, S. enterica; Sc,S. cerevisiae). B, Arg activation of EcArgRS-KAcs. C, aminoacylation of EcArgRS-KAcs resembling that of the K-Q mutants. D, orientation of PhArgRS Lys132

(homologous to Lys126 in EcArgRS) relative to the conserved HIGH motif (HMGH in PhArgRS, depicted in dark blue). E, closer view of the interaction betweenPhArgRS Lys455 (corresponding to Lys408 in EcArgRS) and PhtRNAArg A38. The results are the averages and standard deviations from three independentexperiments.

Table 4Observed rate constants (kobs) of EcArgRSs in the presence ofEctRNAArg in Arg activationAll parameters are average values from three independent determinations withstandard deviations. nm, nonmeasurable (too low to be measured).

Enzymes kobs Relative values

s�1 %EcArgRS-WT 43.5 � 2.7 100EcArgRS-K126Ac nmEcArgRS-K408Ac 10.7 � 0.6 25

Table 5Kinetic parameters of EcArgRS and derived site-specific acetylatedvariants for EctRNAArg in aminoacylationAll parameters are average values from three independent determinations withstandard deviations. nm, nonmeasurable (too low to be measured).

Enzymes Km kcat kcat/Km

Relative catalyticefficiency

�M s�1 s�1 �M�1 %EcArgRS-WT 2.7 � 0.1 28.4 � 3.4 10.5 100EcArgRS-K126Ac nm nmEcArgRS-K408Ac 9.0 � 0.6 6.9 � 0.5 0.8 8

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our results suggest acetylation of Lys809 has a marked negativeeffect on the affinity for EctRNALeu.

Previously, relatively less attention has been paid to EcArgRS.Lys126, the residue upstream the signature sequence HIGH(HVGH), makes up for the absence of K2 in the KMSK motif inEcArgRS (17), and acetylation of key residues in the activationpocket leads to the complete loss of activation activity and con-sequent aminoacylation activity. Lys408 (PhArgRS Lys455) is inthe vicinity of the tRNAArg anticodon region that harbors theidentity element C35 (PDB number 2ZUE), and acetylation ofLys408 inhibited amino acid activation and transfer of the argi-nyl group from Arg-AMP to the 3�end of tRNAArg. Theseresults suggest for the first time that acetylation regulates theamino acid activation and aminoacylation activities of EcArgRSand EcLeuRS.

Acetylation of EcArgRS and EcLeuRS was first reported inprevious MS studies, including acetylation of Lys residues sur-rounding or within the conserved HIGH and KMSK motifs (23,40, 42). We noticed that the conserved Lys residue upstream of

the HIGH motif was also acetylated in various ArgRSs (Lys156 inthe yeast cytosol, Lys139 in R. norvegicus mitochondria, andLys205 in the H. sapiens, R. norvegicus, and M. musculus cytosol;Fig. 10A) (35, 38, 39, 54). These results indicate that Lys acety-lation is a conserved mechanism for regulating the catalyticactivity of aaRSs in particular conditions.

Recently, TyrRS has also been shown to be highly acetylatedin response to oxidative stress. This aaRS is primarily acetylatedon Lys244 near the nuclear localization signal, and the acetyla-

Figure 9. AcP and CobB regulate acetylation of EcArgRS. A, AcP acetylates EcArgRS in vitro. B, CobB removes the acetyl moiety of EcArgRSAc. C, CobBdeacetylates EcArgRS-K126Ac and other acetylated variants. CobB deacetylation activity is lost in the presence of NAM or the absence of NAD�. All experimentswere performed at least twice. When quantifying of the relative amount of AcK signal/His signal, the group without NAD� and NAM was defined as 100%.

Figure 10. Sequence alignment of the HIGH region and the acetatemetabolism pathway. A, sequence alignment of the HIGH region withArgRSs from various species. Lys residues preceding the HIGH motif (indi-cated by an arrow, Lys126 in EcArgRS) were found to be acetylated by MS.Abbreviations are the same as Figs. 4 and 8, except for two species (Rn, R. nor-vegicus; Mm, M. musculus). B, acetate metabolism pathway in E. coli (23). Pta,AckA, and Acs are crucial enzymes involved in the interconversion of Ac-CoAand acetate.

Table 6Kd values between tRNAArg and EcArgRSs determined by fluorescencequenchingAll parameters are average values from three independent determinations withstandard deviations.

Enzymes Kd Relative value

�M -foldEcArgRS-WT 0.24 � 0.03 1.0EcArgRS-K126Ac 0.26 � 0.02 1.1EcArgRS-K408Ac 0.40 � 0.03 1.7

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tion inhibits the aminoacylation activity of TyrRS. Acetylation,which is regulated by PCAF and sirtuin 1, also promotes TyrRStranslocation from cytoplasm to the nucleus and protectsagainst DNA damage caused by oxidative stress in mammaliancells and zebrafish. This study provided us with other perspec-tives about the biological role of aaRS acetylation (55).

AcP and CobB appear to regulate acetylation of EcArgRS andEcLeuRS

Our results showed that AcP and CobB may regulate acety-lation of EcLeuRS and EcArgRS in vitro. We also tried toexplore the acetylation state of these two endogenous aaRSs,but haven’t yet found a physiological state that leads to an obvi-ous increase in the acetylation of aaRSs. In a previous study (23),the global acetylation state of E. coli was found to be elevated ingrowth-arrested (GE) cells compared with those in the expo-nential phase (EP). This accumulation required AcP, and mostacetylation was independent of YfiQ. Additionally, CobB cansuppress chemical acetylation by AcP in growing and GE cells(23), and proteins functioning in translation, transcription andcentral metabolism are acetylated, with a considerable numberof the acetylated sites regulated by AcP (28).

The supplementary data in the study of Weinert et al. (23)showed that acetylation of Lys126 in EcArgRS can differ in E. colicells cultured in M9 minimal medium on different geneticbackground and under different metabolic states. Acetylationof EcArgRS Lys126 in stationary phase BL21 cells was �10-foldhigher compared with EP cells, indicating elevation of acetyla-tion during this stage. Furthermore, acetylation of this site in EP�ackA BW25113 cells was increased �10-fold compared withEP control cells, suggesting that elevation of AcP levels (�ackA)by genetic manipulation could stimulate acetylation at specificsites (Fig. 10B). Acetylation of EcArgRS Lys126 in EP �ptaBW25113 cells was 62.5% that of the level measured in EP WTcells, indicating that a decrease in AcP in �pta cells reduces theacetylation of Lys126 (Fig. 10B). In addition, deletion of the yfiQgene had no direct effect on the acetylation of Lys126 in EP�yfiQ MG1655 cells compared with EP control MG1655 cells,consistent with our in vitro assay results that similarly indicatedthat YfiQ is not involved in the acetylation of the equivalentresidue in EcArgRS. Further studies should focus on investigat-ing the ability of CobB to deacetylate EcLeuRSAc and EcAr-

gRSAc in vivo, as well as the physiological significance of acety-lation. In addition, questions about whether there are someundiscovered acetyltransferases that can take AcP as a sub-strate and catalyze acetylation should be addressed.

Acylation of aaRSs might regulate the metabolic state of cells

In E. coli and yeast, acetylation is much less abundant thanphosphorylation, and succinylation of aaRSs (Lys619 and Lys624

on EcLeuRS) has recently been reported (56). Nonenzymaticprotein acylation has been linked to negative regulation of pro-tein function because carbon stress and deacylases are evolvedto reverse this form of PTM in both prokaryotes and eukaryotes(29). Our results, together with those of previous studies, sug-gest that acetylation and other forms of acylation may inhibitthe activity of aaRSs in response to environmental stresses.Under normal conditions, aaRSs may endure basal-level acety-lation. When there is a stimulus that requires cells to reducetheir growth rates, cells could utilize an economical way to slowdown protein synthesis. Environmental stresses may causeAcP-mediated acetylation of aaRSs, which inhibits their amino-acylation activities, and the deacetylase CobB may reverse thisPTM to recover aa-tRNA biosynthesis when conditionsimprove (Fig. 11). It is very interesting to understand exactlyhow acetylation specificity is achieved. To our understanding,first, even though AcP can acetylate peptides nonenzymaticallyat high concentrations, with some salt and Mg2�, selectivityand specificity can increase (28). Second, it is possible that theremight be an/some undiscovered acetyltransferase(s) that canutilize AcP as a substrate and catalyze acetylation (23); lastly,deacetylase could preferentially remove acetylation on somespecific sites. Moreover, cross-talk between different types ofacylation may be important and should be investigated.Whether acetylation could also affect other functions of aaRSsbeyond translation, as demonstrated by TryRS, is a fascinatingto question to be answered (55).

Concluding remarks

Herein, we confirmed acetylation of EcLeuRS and EcArgRSin vivo and identified the Lys residues involved. To investigatethe significance of this form of PTM, we engineered K-Qmutants to identify residues that may affect the aminoacylationactivity and employed a novel site-directed AcK incorporation

Figure 11. Proposed acetylation mechanism for aaRSs. In this model, AcP nonenzymatically acetylates aaRSs, which negatively regulates their aminoacy-lation activities, and this PTM is removed by CobB to recover aaRS function and maintain cellular homeostasis (29).

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system to prepare EcLeuRS and EcArgRS acetylated at specificsites. Characterization of the amino acid activation and tRNA-charging activities of these EcLeuRS-KAc and EcArgRS-KAc

variants confirmed that acetylation of several Lys residues neg-atively regulates their catalytic activities. Subsequent in vitroassays suggest that AcP might be the source of the nonenzy-matic acetylation of EcLeuRS and EcArgRS, and CobB is likelyto be responsible for deacetylation. This work extends ourunderstanding of acetylation of aaRSs. Whether this type ofPTM is prevalent and physiologically important remains to beelucidated.

Experimental procedures

Materials

L-Leu, L-Arg, L-Ile, AcP (potassium lithium salt lithiumpotassium acetyl phosphate), Ac-CoA, AcK (N�-acetyl-L-Lys),NaBu, NAD�, nicotinamide (NAM), MgCl2, NaCl, KCl, KF,ATP, Tris-HCl, HEPES, Na4PPi, inorganic pyrophosphate,DTT, activated charcoal, and His6-tagged monoclonal antibodywere purchased from Sigma-Aldrich. Polyclonal antibody rec-ognizing AcK was bought from Cell Signaling Technology(Danvers, MA). Amicon ultra-15 centrifugal filters and nitro-cellulose membranes (0.22 �m) were obtained from MerckMillipore. A Bradford protein assay kit was bought from Bio-Rad. Isopropyl-1-thio-�-D-galactopyranoside (IPTG) and pep-tone were purchased from Amresco (Solon, OH). L-[3H]Leu,L-[3H]Ile, L-[3H]Arg, [32P]Na4PPi, and [�-32P]ATP wereobtained from Perkin-Elmer. Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography agarose was purchased fromQiagen. Superdex 75 resin and PVDF membranes were pur-chased from GE Healthcare. dNTP mixtures, arabinose (Ara),Tween 20, Triton X-100, BSA, Na4PPi, KH2PO4, and K2HPO4were purchased from Sangon (Shanghai, China). Oligonucleo-tide primers were synthesized by Invitrogen. A DNA fragmentrapid purification kit and a plasmid extraction kit wereobtained from Yuanpinghao Biotech (Tianjin, China). Proteinstandard markers, T4 ligase, restriction endonucleases andZeba spin desalting columns were obtained from Thermo Sci-entific (Waltham, MA). The KOD-plus mutagenesis kit andKOD-plus Neo enzyme were purchased from TOYOBO(Osaka, Japan), and DNA sequencing was performed by Bio-sune (Shanghai, China). The pAcKRS system was gift from Prof.Jiang-yun Wang. Competent E. coli Top10 and BL21 (DE3)cells were prepared in our laboratory.

Gene cloning, mutagenesis, protein expression, andpurification

Plasmid pET30a(�)-ecleuS encoding EcLeuRS with a N-ter-minal His6 tag was constructed previously in our lab (57). Muta-tion of ecleuS was performed by PCR as reported (57). EcLeuRSand its K-Q mutants were purified by Ni-NTA affinity chroma-tography as reported (58). To obtain site-directed EcLeuRS-KAc, we constructed pET22b(�)-ecleuS encoding EcLeuRSwith a C-terminal His6 tag, in which target Lys codons wereseparately mutated to TAG. We co-transformed E. coli BL21(DE3) cells with pAcKRS and pET22b(�)-ecleuS with a C-ter-minal His6 tag. As described previously (43), EcLeuRS-KAcswere overexpressed in E. coli BL21 (DE3) following the addition

of Ara and IPTG, in the presence of ampicillin, chlorampheni-col, 10 mM NAM, and AcK (Fig. 3). EcLeuRS-KAc was purifiedby Ni-NTA affinity chromatography, followed by gel-filtrationchromatography with a Superdex 75. The purity of the prepa-rations was assessed by SDS-PAGE, and protein concentrationwas determined using the Bradford protein assay kit. EcLeuRSenzymes were stored in buffer containing 20 mM potassiumphosphate (pH 6.8) and 1 mM DTT (58).

The gene encoding EcArgRS was amplified from plasmidpUC18-ecargS and inserted into pET28a(�) (59). WT EcArgRSand K-Q mutants were purified by Ni-NTA affinity chromatog-raphy as described above for EcLeuRS. Because EcArgRS with aC-terminal His6 tag displayed no activity (data not shown),EcArgRS-KAc enzymes were also expressed using thepET28a(�) vector. EcArgRS-KAc enzymes were purified bytwo-step chromatography as described above for EcLeuRS andstored in buffer containing 20 mM potassium phosphate (pH7.5) and 1 mM DTT (59). Cloning of genes encoding YfiQ,CobB, and YcgC and purification of the recombinant enzymeswas performed as reported previously (25, 26).

CD spectroscopy

The secondary structures of EcLeuRS, EcArgRS, and theirvariants were determined by CD spectroscopy as described pre-viously (9).

Acquisition of tRNAs

E. coli tRNALeuCAG (EctRNALeu) and E. coli tRNAArg

ICG(EctRNAArg) were isolated from the corresponding overexpres-sion strains as previously described (60, 61). The charging levelof EctRNALeu and EctRNAArg was measured with 1 �M corre-sponding EcLeuRS and EcArgRS for more than 20 min. BothtRNAs harbored �1400 pmol/A260 units of accepting activity.

Mass spectrometry

E. coli BL21 (DE3) cells harboring the corresponding plas-mids containing ecleuS or ecargS were inoculated from over-night culture in a ratio of 1:100 to 2 YT medium. When theA600 reached 0.6 – 0.8, ecleuS and ecargS genes were induced byIPTG in the presence of 10 mM NAM in 22 °C for 6 h. The cellspellets were lysed by sonication in the presence of PMSF. Aftercentrifugation, the supernatants were applied to the Ni-NTAcolumn. Then His6-taggged EcLeuRS and EcArgRS were puri-fied by the affinity chromatography. After SDS-PAGE, the tar-get bands were separated, excised, and sent to ShanghaiApplied Protein Technology (Shanghai, China). Protein bandswere in-gel digested, subjected to nanoLC to separate theresultant peptides, and identified by mass spectrometry(Thermo Finnigan, Silicon Valley, CA). Data processing andanalysis of raw files were conducted using Proteomics Tools3.1.6 and Mascot 2.2.

Amino acid activation, aminoacylation, misaminoacylation,and deacetylation assays

Assays of EcLeuRS were performed as previously described(62). The amino acid activation of EcLeuRS and its mutants wasassayed by monitoring ATP-PPi exchange reactions at 37 °C inreaction mixture containing 100 mM HEPES (pH 7.8), 10 mM

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MgCl2, 10 mM KF, 4 mM ATP, 2 mM [32P] NaPPi, 5 mM Leu, 0.1mg/ml BSA, and 10 nM enzyme. The aminoacylation activity ofEcLeuRS was monitored at 37 °C in reaction mixtures contain-ing 100 mM Tris-HCl (pH 7.8), 30 mM KCl, 12 mM MgCl2, 2 mM

DTT, 4 mM ATP, 10 �M tRNALeu, 40 �M [3H] Leu, 0.1 mg/mlBSA, and 1 nM enzymes. In aminoacylation reaction, kineticconstants for EcLeuRS and its mutants were determined in thepresence of tRNALeu at concentrations between 0.5 and 30 �M.Misacylation assays were performed in a similar system to thatof aminoacylation, except 40 �M [3H] Ile (30 Ci/mmol) and 1�M WT-EcLeuRS were used instead of 40 �M [3H] Leu and 1 nM

enzyme. Deacylation assays with [3H] Ile-EctRNALeu were car-ried out in buffer containing 100 mM Tris-HCl (pH 7.5), 30 mM

KCl, 12 mM MgCl2, 2 mM DTT, and 1 �M [3H] Ile-EctRNALeu.20 nM enzymes were used to initiate reactions at 37 °C.

ATP-PPi exchange assays of EcArgRSs were performed at37 °C in reaction mixtures containing 130 mM Tris-HCl (pH7.2), 6 mM MgCl2, 2 mM ATP, 2 mM [32P] NaPPi, 2 mM Arg, 0.1mg/ml BSA, and tRNAArg at saturating concentrations. 5 nM

EcArgRSs was used to initiate ATP-PPi exchange reactions.Aminoacylation assays of EcArgRS and its variants were per-formed in reaction mixtures containing 50 mM Tris-HCl (pH7.8), 80 mM KCl, 8 mM MgCl2, 0.5 mM DTT, 4 mM ATP, 10 �M

tRNAArg, 100 �M [3H] Arg, 0.1 mg/ml BSA, and 1 nM enzymesat 37 °C (59). In aminoacylation reaction, kinetic constants forEcArgRS and its mutants were determined in the presence oftRNAArg between 0.5 and 80 �M (63).

Determination of the tRNALeu dissociation constant (Kd) byfluorescence quenching assays

0.1 �M proteins in 400 �l of equilibrium titration buffer con-taining 100 mM Tris-HCl (pH 8.2), 12 mM MgCl2, and 0.5 mM

DTT were excited at 280 nm in a quartz cuvette, and the appro-priate emission wavelength was monitored at room tempera-ture as described previously (64). Maximum emission ofEcLeuRS and EcArgRS was observed at 340 nm, and this wasused to measure the fluorescence intensity of enzymes titratedwith their cognate tRNAs. The final concentration of tRNAsranged from 0.06 to 4.21 �M, and the total volume of titrationscontaining tRNA was less than 20 �l (1/20 of the original vol-ume). We calculated the Kd values by plotting the fluorescenceintensity change (a.u.) against final tRNA concentration (�M)according to the “one site-specific binding” option in GraphPadPrism software. BSA was used as a control.

In vitro acetylation/deacetylation assays

YfiQ-mediated acetylation assays were performed at 37 °C inreaction mixtures containing 50 mM Tris-HCl (pH 8.0), 1 mM

DTT, 10% glycerol, 0.2 mM Ac-CoA, and 5 mM NaBu (26).Acetylation of EcLeuRS and EcArgRS by AcP was carried outwith freshly prepared 10 mM AcP for 1 h in reaction buffercontaining 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 40 mM KCl,1 mM DTT, and enzyme at 37 °C.

In vitro deacetylation reactions were carried out as describedpreviously with purified EcLeuRS or EcArgRS and purifiedCobB in reaction mixtures containing 40 mM HEPES (pH 7.0), 6mM MgCl2, 1 mM NAD�, 1 mM DTT, and 10% glycerol at 37 °C(26). To investigate the effect of deacetylation by CobB on theaminoacylation activities of EcLeuRS-KAc, enzymes were incu-

bated with or without CobB in deacetylation buffer for 1 h at37 °C and subsequently diluted to 5 nM to initiate the amino-acylation reaction. For deacetylation of EcLeuRSAcs and EcAr-gRSAcs (corresponding enzymes preincubated with AcP), pro-teins were pretreated (desalted) using spin desalting columns.

Western blotting

For the detection of AcK, PVDF membranes were blocked inbuffer containing 50 mM Tris-HCl (pH 7.5), 1% peptone, and10% (v/v) Tween 20 and subsequently incubated with dilutedprimary antibody in 50 mM Tris-HCl and 0.1% peptone (53). Allother Western blotting assays were conducted using conven-tional methods.

Author contributions—Q. Y. and E.-D. W. designed the study, ana-lyzed the data, and wrote the paper. Q.-Q. J. assisted with the obtain-ing of clones and the preparation of samples for mass spectrometry.W. Y. participated in determination of the secondary structure ofEcLeuRS and EcArgRS by CD analysis. F. Y. helped in the prepara-tion of proteins. QY performed all the other experiments. All of theauthors reviewed the results and approved the final version of themanuscript.

Acknowledgments—We are grateful to Professor Jiang-Yun Wang(Institute of Biophysics, Chinese Academy of Sciences) for providingpAcKRS plasmid. We also thank Prof. Guo-Ping Zhao, Dr. Wei Zhao,Dr. Xu-Feng Cen, and Dr. Xiao-Biao Han (Institute of Plant Physiol-ogy and Ecology, Chinese Academy of Sciences) and Gisela Geoghegan(School of Medicine, University of Utah) for valuable advice.

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Qing Ye, Quan-Quan Ji, Wei Yan, Fang Yang and En-Duo WangEscherichia coliin

Acetylation of lysine ?-amino groups regulates aminoacyl-tRNA synthetase activity

doi: 10.1074/jbc.M116.770826 originally published online April 28, 20172017, 292:10709-10722.J. Biol. Chem. 

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