Archives of Biochemistry and Biophysics 579 (2015) 33–39
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS, from Myxococcus xanthus Manami Oka a, Kaoru Takegawa b, Yoshio Kimura a,⇑ a b
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan Department of Bioscience and Biotechnology, Kyusyu University, Hakozaki, Higashi-ku, Fukuoka, Japan
a r t i c l e
i n f o
Article history: Received 13 March 2015 and in revised form 27 May 2015 Available online 3 June 2015 Keywords: Lysyl-tRNA synthetase Diadenosine tetraphosphate Diadenosine triphosphate Myxococcus xanthus
a b s t r a c t Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap4A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn2+ for the synthesis of Ap4A. Surprisingly, Zn2+ inhibited enzyme activity in the presence of Mn2+. When incubated with ATP, Mn2+, lysine, and inorganic pyrophosphatase, LysS first produced Ap4A and ADP, then converted Ap4A to diadenosine triphosphate (Ap3A), and finally converted Ap3A to ADP, the end product of the reaction. Recombinant LysS retained Ap4A synthase activity without lysine addition. Additionally, when incubated with Ap4A (minus pyrophosphatase), LysS converted Ap4A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Diadenosine tetraphosphate (Ap4A)1 is an important signaling molecule composed of two adenosine moieties joined in a 50 –50 linkage by a chain of four phosphates. It is ubiquitously expressed in both prokaryotic and eukaryotic cells [1,2]. In prokaryotes, the concentration of Ap4A is rapidly increased after cell exposure to various stresses such as heat or oxidative stress [3,4]. Further studies demonstrated that Ap4A metabolism in prokaryotes is also implicated in the regulation of the stress response, pathogenesis, and antibiotic tolerance [5–7]. Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA, specifically pairing amino acids with their cognate anticodons on tRNA. In the absence of cognate tRNA, the majority of aminoacyl-tRNA synthetases synthesize Ap4A in the presence of cognate amino acid and ATP [8]. One highly efficient Ap4A synthase in vitro is the lysyl-tRNA synthetase. Most lysyl-tRNA synthetases, in both eukaryotes and prokaryotes, belong to the class II aminoacyl-tRNA synthetases [9]. Class II aminoacyl-tRNA synthetases are characterized by their unique structure and usually ⇑ Corresponding author. Fax: +81 87 891 3021. E-mail address: [email protected] (Y. Kimura). Abbreviations used: LysS, lysyl-tRNA synthetase; Ap4A, diadenosine tetraphosphate; Ap3A, diadenosine triphosphate; LB, Luria–Bertani broth. 1
http://dx.doi.org/10.1016/j.abb.2015.05.014 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
occur as dimeric or multimeric complexes. Additionally, class II lysyl-tRNA synthetases have two distinct domains: a smaller N-terminal tRNA anticodon binding domain and a larger C-terminal catalytic domain, which contains three conserved motifs [10]. Motif 1 is part of the dimer interface while motifs 2 and 3 assist the binding of ATP and Mg2+ ions. Escherichia coli has two class II lysyl-tRNA synthetases; LysS, which is expressed under all normal growth conditions, and LysU, which is induced by stress such as high temperature, anaerobiosis, low external pH, or the presence of leucine [11–13]. Lysyl-tRNA synthetases produce Ap4A in a two-step reaction. The first step requires Mg2+ and involves activation of the amino acid lysine by ATP to form lysyl-AMP and releases pyrophosphate. In the second step, which requires Zn2+, ATP combines with lysyl-AMP, thereby generating Ap4A. In E. coli LysU, the generated Ap4A is further converted into Ap3A [14]. Myxococcus xanthus has a complex life cycle that includes fruiting body formation; however, the role of Ap4A in the regulation of the M. xanthus life cycle is still unknown. Additionally, the sequence of the M. xanthus genome revealed a lysyl-tRNA synthetase gene (MXAN_4731), designated LysS, whose enzymatic properties are largely unknown. Therefore, we characterized the Ap4A synthase activity of the M. xanthus LysS, and discovered that M. xanthus LysS has different enzymatic characteristics than those of other class II lysyl-tRNA synthetases previously described.
34
B kDa 200 116 97
10 Molecular mass (Da)
A
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
66 44
29
DNA fragment was inserted into a cold shock expression vector, pCold vector (Takara Bio). The plasmid was used for transformation of E. coli. The cells were incubated in Luria-Bertani broth (LB) medium containing ampicillin until the optical density at 600 nm of the culture reached 0.4–0.5 at 37 °C, and then incubated at 15 °C for 20 h. LysS protein with an N-terminal hexahistidine tag was purified by affinity chromatography on a Talon column (Clontech). Also, purified recombinant LysS solution was dialyzed for 72 h against a total of 1.5 liters of 50 mM HEPES (pH 7.5) buffer containing 1 mM EDTA in 3 changes, and then the EDTA was removed by dialysis (5–8 h, three 50 mM HEPES (pH 7.5) buffer changes).
6
5
10
4
10
6
7
8
9
10
11
Elution volume (ml)
Fig. 1. (A) SDS–PAGE analysis of purified recombinant LysS. Protein size is indicated by molecular mass markers in kDa. (B) Molecular mass determination of LysS by gel filtration. (s) molecular markers; (d) LysS.
Methods and materials Expression and purification of LysS lysS gene (MXAN_4731) was amplified by PCR using primers ( 50 -GAAGGTAGGCATATGGCCGAGACCGAAAACAAGAG-30 and 50 -GA CAAGCTTGAATTCCTACTTCGCCAGTGGCTTGAG-30 ). The amplified
A
Enzyme assay The synthesis of Ap4A was assayed in 20 ll of 50 mM HEPES (pH 8.0), 5 mM MnSO4, 5 mM ATP, 2 mM lysine and 5 lM LysS at 37 °C for 30 min. To analyze the time dependence of the synthesis of Ap4A and other nucleotides, the assay was performed in a total volume of 60 ll of 5 mM ATP, 5 mM MnSO4, 2 mM lysine and LysS in the absence and presence of 0.6 units (1.2 lg) inorganic pyrophosphatase from Saccharomyces cerevisiae (Sigma–Aldrich) at 37 °C. Aliquots (5 ll) were removed at successive time intervals and immediately applied onto a resource Q column (GE health). To determine the kinetic parameters Km for ATP, Ap4A synthase activity was measured at ATP concentration ranging 0.1–5 mM, 2 mM lysine and 5 mM MnSO4 in the absence of inorganic pyrophosphatase. Data were analyzed by nonlinear least-squares fitting to the appropriate kinetic equation using Sigma Plot (Systat Software Inc.).
B 100
100 Relative activity (%)
120
Relative activity (%)
120
80 60 40 20 0
60 40 20 0
Mn2+ Ca2+ Co2+ Mg2+ Ni2+ Zn2+ None
C
80
0
5
10 15 MnSO4 (mM)
20
120
Relative activity (%)
100 80 60 40 20 0 0
0.1
0.2
0.3
0.4
0.5
ZnSO4 (mM) Fig. 2. (A) Effects of different metal ions on LysS Ap4A synthase activity. LysS (5 lM) was incubated with 5 mM ATP, 2 mM lysine, and 5 mM of the indicated metal ions in 50 mM HEPES buffer (pH 8.0) at 37 °C for 30 min. Relative activity was calculated by comparison of Ap4A production with each metal to the activity measured in the presence of 5 mM MnSO4, which was set at 100%. Values are presented as mean ± SEM, determined from the average of three independent measurements. (B) Determination of the optimal Mn2+ concentration for Ap4A synthesis catalyzed by LysS. LysS (5 lM) was incubated with 5 mM ATP, 2 mM lysine, and 0, 1, 2.5, 5, 10 or 20 mM MnSO4 in 50 mM HEPES buffer (pH 8.0) at 37 °C for 30 min. (C) Effects of Zn2+ on Ap4A synthase activity. LysS was incubated with 5 mM MnSO4, 5 mM ATP, 2 mM lysine and 0, 0.015, 0.05, 0.15 or 0.5 mM of ZnCl2 at 37 °C for 30 min.
35
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
A
B
+ ATP, + lysine, + IPP
+ ATP, + lysine, + IPP
Concentration (mM)
5
Ap3A Lys
Lys
4
ADP Lys ATP lysyl-AMP Ap4A
ADP 3
ATP Lys
2
IPP
1
H2O Lys
Pi
PPi
2Pi
Lys
ATP AMP
Lys
ADP
0 0
Concentration (mM)
C
50
100 150 Reaction time (min)
+ ATP, + lysine,
200
D
IPP
5
5
4
4
3
3
2
2
1
1
+ ATP, + lysine, + Pi
0
0 0
50
100
150
200
0
50
Reaction time (min)
100 150 Reaction time (min)
200
Fig. 3. LysS catalyzed the conversion of ATP to Ap4A, Ap3A or ADP in the presence of lysine. (A and C) LysS (15 lM) was incubated with 5 mM ATP, 2 mM lysine, and 5 mM MnSO4 in the presence (A) or absence (C) of 0.6 U inorganic pyrophosphatase (IPP). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. (B) Diagram of the reaction mechanism described in A. PPi, inorganic pyrophosphate; Pi, inorganic phosphate; IPP, inorganic pyrophosphatase; Lys, lysine. (D) LysS (15 lM) was incubated with 5 mM ATP, 2 mM lysine, and 5 mM MnSO4 in 50 mM potassium phosphate buffer (pH 8.0). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
Analysis of nucleotides by ion exchange HPLC analysis
Gel chromatography LysS was loaded onto a TSKgel SuperSW mAb HR (Tosoh; 7.8 mm 30 cm) equilibrated with 50 mM Tris–HCl buffer, pH 7.5, containing 0.1 M Na2SO4. Protein was eluted at a rate of 0.5 ml/min, and protein absorbance at 280 nm was monitored. The column was calibrated using the following standards: ferritin (440 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa).
120 100 Relative activity (%)
Mononucleotides (AMP, ADP, and ATP) and dinucleotides (Ap3A and Ap4A) in the enzyme assay were detected at 260 nm with a HPLC system using a resource Q column, and the nucleotide concentrations were calculated from the peak areas using standard curves derived from known concentrations of the nucleotides. The mobile phase was composed of solvent A (water) and solvent B (0.7 M ammonium bicarbonate). The gradient elution for solvent A, with a flow rate of 1 ml min1, was 90% (v/v) at 0 min, 75% at 0.5 min, 65% at 10 min, 5% at 13 min, and 0% at 14 min.
80 60 40 20 0
0
2
4
6
8
10
Concentration (mM) Fig. 4. Effects of inorganic pyrophosphate and ADP on LysS Ap4A synthase activity. Reactions containing 5 lM LysS, 5 mM ATP, 2 mM lysine, and 5 mM MnSO4 in 50 mM HEPES buffer, pH 8.0, were incubated with either 0, 0.2, 0.5, 1, 2 or 5 mM inorganic pyrophosphate (d) or 0, 1, 2.5, 5 or 10 mM ADP (s) at 37 °C for 30 min. Relative activity was calculated by comparison of Ap4A production with each concentration to the activity measured in without inorganic pyrophosphate or ADP, which was set at 100%.Values are presented as mean ± SEM, determined from the average of three independent measurements.
Results and discussion Comparison of LysS and other lysyl-tRNA synthetases amino acid sequences The open reading frame of MXAN_4731 (named lysS) encodes a protein of 517 amino acids with a calculated molecular mass of
56 kDa. Similar to other reported class II lysyl-tRNA synthetases, M. xanthus LysS contains two domains and three conserved motifs in the catalytic domain (Supplementary Fig. S1) [15]. The deduced amino acid sequence of LysS was 50% and 40% identical to that of E. coli LysU and human LysRS, respectively.
36
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
A
B Concentration (mM)
Ap4A (nmol/min)
0.5
0
0.5
1.0
1.5
2..0
4 3 2 1 0
5.0
0
50
Lysine (mM)
C Concentration (mM)
lysine, + IPP
5
1.0
0
+ ATP,
+ ATP,
lysine,
100
150
200
Reaction time (min)
IPP
+ ATP,
D
5
5
4
4
3
3
2
2
1
1
lysine, + Pi
0
0 0
50
100
150
200
Reaction time (min)
0
50
100
150
200
Reaction time (min)
Fig. 5. Activity of LysS in the absence of added lysine. (A) LysS (5 lM) was incubated with 5 mM ATP, 5 mM MnSO4, and 0, 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 mM lysine. The rate of Ap4A production was measured and plotted as a function of lysine concentration. (B and C) LysS (15 lM) was incubated with 5 mM ATP and 5 mM MnSO4 in the presence (B) or absence (C) of 0.6 U inorganic pyrophosphatase (IPP). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. (D) LysS (15 lM) was incubated with 5 mM ATP, and 5 mM MnSO4 in 50 mM potassium phosphate buffer (pH 8.0). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
LysS forms a monomer M. xanthus LysS was expressed in E. coli and purified from the soluble fraction using an N-terminal hexahistidine tag (His-tag) and affinity column chromatography. The purified LysS protein was analyzed by SDS–PAGE, which revealed a single band corresponding to a molecular mass of 60 kDa (Fig. 1A). The apparent molecular mass obtained by SDS–PAGE was in agreement with the molecular mass calculated from the predicted amino acid sequence of His-tagged LysS (58 kDa). Class II lysyl-tRNA synthetases are reported to form dimers or tetramers [16]. In fact, human class II lysyl-tRNA synthetases require homodimerization for their aminoacylation function [17]. To determine whether M. xanthus LysS exists in monomeric or multimeric form, we estimated the molecular mass of LysS by gel filtration using the correlation between the elution volume and the logarithm of the molecular mass of known reference proteins (Fig. 1B). Unexpectedly, recombinant M. xanthus LysS eluted as a single peak, and the molecular mass of the native enzyme was estimated to be 68 kDa. This data suggests that recombinant LysS exists as a monomer, despite the presence of the highly conserved motif 1 common to class II enzymes. Zn2+ is not required for Ap4A synthesis by M. xanthus LysS Class II lysyl-tRNA synthetases require Mg2+ and Zn2+ for the first and second step of the reaction mechanism, respectively [18]. To determine the metal cofactor requirement for recombinant
M. xanthus LysS, we measured activity in the presence of a variety of different metals and found that LysS was most active in the presence of Mn2+ (Fig. 2A), and the optimal Mn2+ concentration for full activation of the enzyme ranged from 5 to 20 mM (Fig. 2B). Additionally, we found that the synthase is partially active (72 ± 2% or 24 ± 3%) in the presence of 5 mM Ca2+ or 5 mM Co2+, respectively (Fig. 2A), and has no detectable activity in the presence of Fe2+, Fe3+, and Cu2+ (data not shown). Surprisingly, we found that LysS activity, assayed in the presence of 5 mM Mn2+, was inhibited by the addition of Zn2+ (Fig. 2C). Addition of 15 lM or 150 lM Zn2+ decreased LysS activity by 22 ± 2% and 62 ± 1%, respectively (Fig. 2C). Furthermore, the addition of 150 lM Zn2+ to a dialyzed sample of recombinant protein also inhibited Ap4A synthase activity by 64 ± 3% (data not shown). Also, LysS showed slight Ap4A synthase activity (4 ± 1%) in the presence of 5 mM Mg2+ and 150 lM Zn2+. These data suggest that class II M. xanthus LysS does not require Zn2+ for enzymatic activity. This is similar to many aminoacyl-tRNA synthetases, for example, valine-, histidine- and methionine-specific tRNA synthetases, which are insensitive to the addition of Zn2+ [19]. LysS enzyme kinetics Further characterization of M. xanthus LysS demonstrated that the optimal temperature for Ap4A synthase activity was 50 °C. The relative activity of LysS at 30 °C, 40 °C or 60 °C, as compared to the optimal temperature (50 °C) was 49%, 77% or 45%, respectively (data not shown). LysS activity was most active when pH
37
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
ranged between 8 and 9 and 80% activity remained in a 0.2 M HEPES buffer (pH 7.0). The Km value of M. xanthus LysS for ATP in the presence of 2 mM lysine was 1.3 ± 0.1 mM. The Km of LysS for ATP was approximately 5-fold lower than those of E. coli LysU [14]. Additionally, the kcat of M. xanthus LysS in the absence of inorganic pyrophosphatase was 0.088 ± 0.013 S1. LysS exhibits Ap4A, Ap3A and ADP synthase activities In the presence of lysine, ATP, Mg2+, Zn2+, and inorganic pyrophosphatase, E. coli LysU has dual Ap4A and Ap3A synthase activity [14]. LysU first produces Ap4A from ATP, and then, in a second reaction, converts Ap4A into Ap3A using ADP. In addition, Chen and Xu reported that E. coli LysU finally catalyzes Ap3A into ADP [20]. In the process, inorganic phosphate is generated from pyrophosphate by inorganic pyrophosphatase, and ADP is generated from inorganic phosphate and lysyl-AMP by LysU. We found that when M. xanthus LysS was incubated with ATP, Mn2+, inorganic pyrophosphatase and lysine for 200 min, LysS produced Ap4A and ADP after 25 min of incubation, followed by conversion of Ap4A to Ap3A. Ap3A was then converted to ADP, the end product of the reaction, by 150 min (Fig. 3A). This result suggests that the reaction of lysyl-AMP with ATP to give Ap4A, or with ADP to give Ap3A, is a reversible reaction while the reaction of lysyl-AMP with phosphate to give ADP is an irreversible reaction (Fig. 3B). Additionally, AMP was gradually generated through the 200-min reaction, suggesting that LysS may hydrolyze lysyl-AMP to yield AMP and lysine. In contrast, to M. xanthus LysS, which
A
+ Ap4A, + lysine,
constantly produced ADP, E. coli LysU did not produce ADP during the conversion of Ap4A to Ap3A [14]. In the absence of inorganic pyrophosphatase, LysS produced Ap4A, and the Ap4A was in equilibrium with ATP after 50 min of incubation (Fig. 3C), suggesting that ATP may be formed from the released inorganic pyrophosphate and lysyl-AMP. Accumulation of a small amount of ADP was also observed, indicating that LysS may hydrolyze Ap4A into two molecules of ADP. This inference is in agreement with the results obtained by Goerlich et al. who suggested that E. coli phenylalanyl-tRNA synthetase also degrades Ap4A into 2 mol of ADP [8]. When LysS was incubated with ATP in the presence of 50 mM potassium phosphate, LysS generated Ap4A and ADP; however, Ap3A was hardly detected in the reaction (Fig. 3D), suggesting that phosphate may be more readily combined with lysyl-AMP than ADP by LysS, and/or Ap3A may be generated from lysyl-AMP and ADP after 200 min of incubation. It has been reported that inorganic pyrophosphate inhibits Ap4A synthesis by aminoacyl-tRNA synthetases through competitive inhibition of the ATP reaction with aminoacyl-adenylate [8,21]. To confirm whether inorganic pyrophosphate inhibits the Ap4A synthase activity of LysS, LysS was incubated with inorganic pyrophosphate in the presence of 5 mM ATP. Addition of increasing concentrations of inorganic pyrophosphate (0 to 5 mM) led to the inhibition of Ap4A synthesis (Fig. 4). We next examined whether ADP inhibits the catalysis of Ap4A formation. Similarly, the addition of ADP inhibited the Ap4A synthase activity of LysS (Fig. 4). Ap4A production decreased by 22 ± 2% and 52 ± 2% at 1 mM and 5 mM ADP, respectively. In the presence of ADP, Ap3A was not synthesized by LysS during 30 min of incubation. These results suggest
B
IPP
+ Ap4A, + lysine,
IPP
Concentration (mM)
2 Lys PPi
ATP Lys
Ap4A
1
H2O Lys
Lys PPi
H2O
lysyl-AMP ADP
2ADP
Lys
AMP
ADP Lys
Ap3A 0 0
50
100
150
200
Reaction time (min)
C
D
+ Ap4A, + lysine, + IPP
Concentration (mM)
3
+ Ap4A,
lysine,
IPP
2
2 1 1
0
0 0
50
100
Reaction time (min)
150
200
0
50
100
150
200
Reaction time (min)
Fig. 6. Conversion of Ap4A by LysS in the absence of ADP. (A and C) LysS (24 lM) was incubated with 2 mM Ap4A, 2 mM lysine and 5 mM MnSO4 in the absence (A) or presence (C) of 0.3 U inorganic pyrophosphatase. (B) Diagram of the reaction mechanism described in A. PPi, inorganic pyrophosphate; Lys, lysine. (D) LysS (24 lM) was incubated with 2 mM Ap4A and 5 mM MnSO4. Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
38
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
that ADP may act as a competitive inhibitor of LysS in the first step reaction (lysyl-AMP formation from lysine and ATP). Additionally, because the reversal of the second step did not occur during a 30-min incubation, it can be inferred that Ap3A may be generated from ADP and lysyl-AMP arising from Ap4A via this reversal. In E. coli LysU, the reversal of the second step allows Ap3A formation from ADP and lysyl-AMP [14]. On the other hand, Ap4A synthase activity of LysS was not inhibited by addition of AMP (data not shown). The activity of most class II aminoacyl-tRNA synthetases is found to absolutely require the presence of added cognate amino acids, except for a human glycyl-tRNA synthetase [8,22]. When LysS was incubated with a range of lysine concentrations (0– 5 mM) in the presence of 5 mM MnSO4 and 5 mM ATP, we observed production of Ap4A even in the absence of added lysine (Fig. 5A). However, the rate of Ap4A production by LysS in the absence of added lysine was about 1.7-fold lower than in the presence of 0.05 mM to 5 mM lysine. Goerlich et al. reported that E. coli LysRS also exhibited considerable synthesis in the absence of lysine, because 1.2 lM dialyzed LysRS contained approximately 0.2 lM endogeneous lysine [8]. In this study, the Ap4A synthase activity of dialyzed LysS was approximately 3-fold lower in the absence of added lysine than in the presence of 2 mM lysine (data not shown). During the expression of recombinant M. xanthus LysS in E. coli, intracellular lysine in E. coli cells may bind to the active site of recombinant LysS. Dialysis may not be able to dissociate lysine or lysyl-AMP from LysS by that may enable to catalyze in vitro formation of Ap4A by LysS in the absence of added lysine.
A
LysS incubated with ATP, Mn2+, and inorganic pyrophosphatase (without lysine addition) first generated Ap4A after 100 min which was followed by a slow decrease in Ap4A (Fig. 5B). Additionally, ADP was gradually generated throughout the 200-min reaction and only small amounts of Ap3A were produced in these conditions. This reaction progressed more slowly than in the presence of lysine. In the absence of added lysine and inorganic pyrophosphatase, Ap4A was in equilibrium with ATP after 100 min of incubation (Fig. 5C). LysS also generated Ap4A and ADP in the presence of 50 mM potassium phosphate (Fig. 5D) in a reaction that proceeded more slowly than in the presence of inorganic pyrophosphatase (Fig. 5B). Ap4A degradation by LysS E. coli LysU does not degrade Ap4A when it is incubated with Ap4A, lysine, Mg2+, Zn2+, and inorganic phosphatase [14]. In contrast, E. coli phenylalanyl-tRNA synthetase degraded Ap4A into almost equal amounts of ATP and AMP in the absence of inorganic phosphatase [8]. M. xanthus LysS converted Ap4A to yield ATP and AMP when Mn2+ and lysine were present; however, formation of ATP was higher than that of AMP (Fig. 6A). When 0.5 mM inorganic pyrophosphate was included in the reaction mixture, formation of ATP was also accelerated (data not shown). These results suggest that formation of ATP from lysyl-AMP and pyrophosphate (pyrophosphorolysis) proceeds faster than the hydrolysis of lysyl-AMP to lysine and AMP in the presence of pyrophosphate (Fig. 6B) [8]. In addition, LysS hydrolyzed Ap4A symmetrically
B
+ Ap4A, + ADP, + lysine, IPP
+ Ap4A, + ADP, + lysine,
IPP
Concentration (mM)
2
ATP Lys
Ap4A
1
Lys
Lys PPi
Lys PPi
lysyl-AMP ATP ADP ADP Lys
Lys
Ap3A 0 0
Concentration (mM)
C
50
100 150 Reaction time (min)
200
D
+ Ap4A, + ADP, + lysine, + IPP
4
2
2
1
0
+ Ap4A, + ADP,
lysine,
IPP
0 0
50
100
150
Reaction time (min)
200
0
50
100 150 Reaction time (min)
200
Fig. 7. Conversion of Ap4A by LysS in the presence of ADP. (A and C) 8 lM LysS was incubated with 2 mM Ap4A, 2 mM ADP, 2 mM lysine and 5 mM MnSO4 in the absence (A) or presence (C) of 0.3 U inorganic pyrophosphatase. (B) Diagram of the reaction mechanism described in A. PPi, inorganic pyrophosphate; Lys, lysine. (D) LysS (8 lM) was incubated with 2 mM Ap4A, 2 mM ADP and 5 mM MnSO4. Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
yielding ADP, because Ap3A under these conditions was produced throughout the duration of the experiment. In the presence of inorganic pyrophosphatase, added Ap4A was rapidly converted to ADP and Ap3A without apparent accumulation of ATP, because the released pyrophosphate from ATP is hydrolyzed by pyrophosphatase (Fig. 6C). On the other hand, LysS hydrolyzed Ap4A to yield ADP as a major cleavage product in the absence of added lysine and inorganic pyrophosphatase (Fig. 6D). LysS incubated with 2 mM Ap4A, 2 mM ADP, and 2 mM lysine generated Ap3A and ATP (Fig. 7A). In this condition, accumulation of ATP, but not AMP, was observed. This result suggests that LysS does not hydrolyze lysyl-AMP to lysine and AMP in the presence of high concentration of ADP, because the lysyl-AMP is rapidly combined with ADP to form Ap3A (Fig. 7B). In the presence of inorganic pyrophosphatase, LysS converted Ap4A rapidly into Ap3A and ADP with low level of ATP. Ap3A was then converted to ADP, the end product of the reaction, by 200 min (Fig. 7C). This is similar to E. coli LysU, which also generates Ap3A in the presence of a mixture of ADP and Ap4A; however, the same accumulation in ADP was not observed [14]. In the absence of added lysine, Ap4A hydrolysis with ADP progressed more slowly than in the presence of lysine (Fig. 7D). In conclusion, we demonstrated that the class II M. xanthus LysS has Ap4A, Ap3A and ADP synthase activity without the requirement for Zn2+ and recombinant LysS showed considerable Ap4A synthase activity in the absence of added lysine. In addition, LysS converted Ap4A mainly into AMP and ATP, or ADP in the presence or absence of lysine, respectively. These results suggested that LysS has different enzymatic properties from the class II lysyl-tRNA synthetases reported previously. Acknowledgments This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25440087).
39
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2015.05.014. References [1] L.L. Kisselev, J. Justesen, A.D. Wolfson, L.Y. Frolova, FEBS Lett. 427 (1998) 157– 163. [2] P.C. Zamecnik, M.L. Stephenson, C.M. Janeway, K. Randerath, Biochem. Biophys. Res. Commun. 24 (1966) 91–97. [3] P.C. Lee, B.R. Bochner, B.N. Ames, Proc. Natl. Acad. Sci. U.S.A. 80 (1983) 7496– 7500. [4] P.C. Lee, B.R. Bochner, B.N. Ames, J. Biol. Chem. 258 (1983) 6827–6834. [5] B.R. Bochner, P.C. Lee, S.W. Wilson, C.W. Cutler, B.N. Ames, Cell 37 (1984) 225– 232. [6] T.M. Ismail, C.A. Hart, A.G. McLennan, J. Biol. Chem. 278 (2003) 32602–32607. [7] S. Hansen, K. Lewis, M. Vulic, Antimicrob. Agents Chemother. 52 (2008) 2718– 2726. [8] O. Goerlich, R. Foeckler, E. Holler, Eur. J. Biochem. 126 (1982) 135–139. [9] S. Blanquet, P. Plateau, S. Onesti, Class II Lysyl-tRNA Synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), The Aminoacyl-tRNA Synthetases, Eurekah, Georgetown, TX, 2005, pp. 227–240. [10] G. Desogus, F. Todone, P. Brick, S. Onesti, Biochemistry 39 (2000) 8418–8425. [11] F. Lévéque, P. Plateau, P. Dessen, S. Blanquet, Nucleic Acids Res. 18 (1990) 305– 312. [12] F. Lévéque, M. Gazeau, M. Fromant, S. Blanquet, P. Plateau, J. Bacteriol. 173 (1991) 7903–7910. [13] A. Brevet, J. Chen, F. Lévéque, S. Blanquet, P. Plateau, J. Biol. Chem. 270 (1995) 14439–14444. [14] M. Wright, N. Boonyalai, J.A. Tanner, A.D. Hindley, A.D. Miller, FEBS J. 273 (2006) 3534–3544. [15] G. Eriani, M. Delarue, O. Poch, J. Gangloff, D. Moras, Nature 347 (1990) 203– 206. [16] M. Ibba, D. Söll, Annu. Rev. Biochem. 69 (2000) 617–650. [17] M. Guo, M. Ignatov, K. Musier-Forsyth, P. Schimmel, X.-L. Yang, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 2331–2336. [18] X. Chen, N. Boonyalai, C. Lau, C. Thipayang, Y. Xu, M. Wright, M.D. Miller, FEBS J. 280 (2013) 102–114. [19] S. Blanquet, P. Plateau, A. Brevet, Mol. Cell. Biochem. 52 (1983) 3–11. [20] X. Chen, X. Yuhong, Acta Biophys. Sin. 29 (2013) 35–45. [21] L. Dibbelt, U. Pachmann, H.G. Zachau, Nucleic Acid Res. 8 (1980) 4021–4039. [22] R.-T. Guo, Y.E. Chong, M. Guo, X.-L. Yang, J. Biol. Chem. 284 (2009) 28968– 28976.
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS, from Myxococcus xanthus Manami Oka a, Kaoru Takegawa b, Yoshio Kimura a,⇑ a b
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan Department of Bioscience and Biotechnology, Kyusyu University, Hakozaki, Higashi-ku, Fukuoka, Japan
a r t i c l e
i n f o
Article history: Received 13 March 2015 and in revised form 27 May 2015 Available online 3 June 2015 Keywords: Lysyl-tRNA synthetase Diadenosine tetraphosphate Diadenosine triphosphate Myxococcus xanthus
a b s t r a c t Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap4A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn2+ for the synthesis of Ap4A. Surprisingly, Zn2+ inhibited enzyme activity in the presence of Mn2+. When incubated with ATP, Mn2+, lysine, and inorganic pyrophosphatase, LysS first produced Ap4A and ADP, then converted Ap4A to diadenosine triphosphate (Ap3A), and finally converted Ap3A to ADP, the end product of the reaction. Recombinant LysS retained Ap4A synthase activity without lysine addition. Additionally, when incubated with Ap4A (minus pyrophosphatase), LysS converted Ap4A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Diadenosine tetraphosphate (Ap4A)1 is an important signaling molecule composed of two adenosine moieties joined in a 50 –50 linkage by a chain of four phosphates. It is ubiquitously expressed in both prokaryotic and eukaryotic cells [1,2]. In prokaryotes, the concentration of Ap4A is rapidly increased after cell exposure to various stresses such as heat or oxidative stress [3,4]. Further studies demonstrated that Ap4A metabolism in prokaryotes is also implicated in the regulation of the stress response, pathogenesis, and antibiotic tolerance [5–7]. Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA, specifically pairing amino acids with their cognate anticodons on tRNA. In the absence of cognate tRNA, the majority of aminoacyl-tRNA synthetases synthesize Ap4A in the presence of cognate amino acid and ATP [8]. One highly efficient Ap4A synthase in vitro is the lysyl-tRNA synthetase. Most lysyl-tRNA synthetases, in both eukaryotes and prokaryotes, belong to the class II aminoacyl-tRNA synthetases [9]. Class II aminoacyl-tRNA synthetases are characterized by their unique structure and usually ⇑ Corresponding author. Fax: +81 87 891 3021. E-mail address: [email protected] (Y. Kimura). Abbreviations used: LysS, lysyl-tRNA synthetase; Ap4A, diadenosine tetraphosphate; Ap3A, diadenosine triphosphate; LB, Luria–Bertani broth. 1
http://dx.doi.org/10.1016/j.abb.2015.05.014 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
occur as dimeric or multimeric complexes. Additionally, class II lysyl-tRNA synthetases have two distinct domains: a smaller N-terminal tRNA anticodon binding domain and a larger C-terminal catalytic domain, which contains three conserved motifs [10]. Motif 1 is part of the dimer interface while motifs 2 and 3 assist the binding of ATP and Mg2+ ions. Escherichia coli has two class II lysyl-tRNA synthetases; LysS, which is expressed under all normal growth conditions, and LysU, which is induced by stress such as high temperature, anaerobiosis, low external pH, or the presence of leucine [11–13]. Lysyl-tRNA synthetases produce Ap4A in a two-step reaction. The first step requires Mg2+ and involves activation of the amino acid lysine by ATP to form lysyl-AMP and releases pyrophosphate. In the second step, which requires Zn2+, ATP combines with lysyl-AMP, thereby generating Ap4A. In E. coli LysU, the generated Ap4A is further converted into Ap3A [14]. Myxococcus xanthus has a complex life cycle that includes fruiting body formation; however, the role of Ap4A in the regulation of the M. xanthus life cycle is still unknown. Additionally, the sequence of the M. xanthus genome revealed a lysyl-tRNA synthetase gene (MXAN_4731), designated LysS, whose enzymatic properties are largely unknown. Therefore, we characterized the Ap4A synthase activity of the M. xanthus LysS, and discovered that M. xanthus LysS has different enzymatic characteristics than those of other class II lysyl-tRNA synthetases previously described.
34
B kDa 200 116 97
10 Molecular mass (Da)
A
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
66 44
29
DNA fragment was inserted into a cold shock expression vector, pCold vector (Takara Bio). The plasmid was used for transformation of E. coli. The cells were incubated in Luria-Bertani broth (LB) medium containing ampicillin until the optical density at 600 nm of the culture reached 0.4–0.5 at 37 °C, and then incubated at 15 °C for 20 h. LysS protein with an N-terminal hexahistidine tag was purified by affinity chromatography on a Talon column (Clontech). Also, purified recombinant LysS solution was dialyzed for 72 h against a total of 1.5 liters of 50 mM HEPES (pH 7.5) buffer containing 1 mM EDTA in 3 changes, and then the EDTA was removed by dialysis (5–8 h, three 50 mM HEPES (pH 7.5) buffer changes).
6
5
10
4
10
6
7
8
9
10
11
Elution volume (ml)
Fig. 1. (A) SDS–PAGE analysis of purified recombinant LysS. Protein size is indicated by molecular mass markers in kDa. (B) Molecular mass determination of LysS by gel filtration. (s) molecular markers; (d) LysS.
Methods and materials Expression and purification of LysS lysS gene (MXAN_4731) was amplified by PCR using primers ( 50 -GAAGGTAGGCATATGGCCGAGACCGAAAACAAGAG-30 and 50 -GA CAAGCTTGAATTCCTACTTCGCCAGTGGCTTGAG-30 ). The amplified
A
Enzyme assay The synthesis of Ap4A was assayed in 20 ll of 50 mM HEPES (pH 8.0), 5 mM MnSO4, 5 mM ATP, 2 mM lysine and 5 lM LysS at 37 °C for 30 min. To analyze the time dependence of the synthesis of Ap4A and other nucleotides, the assay was performed in a total volume of 60 ll of 5 mM ATP, 5 mM MnSO4, 2 mM lysine and LysS in the absence and presence of 0.6 units (1.2 lg) inorganic pyrophosphatase from Saccharomyces cerevisiae (Sigma–Aldrich) at 37 °C. Aliquots (5 ll) were removed at successive time intervals and immediately applied onto a resource Q column (GE health). To determine the kinetic parameters Km for ATP, Ap4A synthase activity was measured at ATP concentration ranging 0.1–5 mM, 2 mM lysine and 5 mM MnSO4 in the absence of inorganic pyrophosphatase. Data were analyzed by nonlinear least-squares fitting to the appropriate kinetic equation using Sigma Plot (Systat Software Inc.).
B 100
100 Relative activity (%)
120
Relative activity (%)
120
80 60 40 20 0
60 40 20 0
Mn2+ Ca2+ Co2+ Mg2+ Ni2+ Zn2+ None
C
80
0
5
10 15 MnSO4 (mM)
20
120
Relative activity (%)
100 80 60 40 20 0 0
0.1
0.2
0.3
0.4
0.5
ZnSO4 (mM) Fig. 2. (A) Effects of different metal ions on LysS Ap4A synthase activity. LysS (5 lM) was incubated with 5 mM ATP, 2 mM lysine, and 5 mM of the indicated metal ions in 50 mM HEPES buffer (pH 8.0) at 37 °C for 30 min. Relative activity was calculated by comparison of Ap4A production with each metal to the activity measured in the presence of 5 mM MnSO4, which was set at 100%. Values are presented as mean ± SEM, determined from the average of three independent measurements. (B) Determination of the optimal Mn2+ concentration for Ap4A synthesis catalyzed by LysS. LysS (5 lM) was incubated with 5 mM ATP, 2 mM lysine, and 0, 1, 2.5, 5, 10 or 20 mM MnSO4 in 50 mM HEPES buffer (pH 8.0) at 37 °C for 30 min. (C) Effects of Zn2+ on Ap4A synthase activity. LysS was incubated with 5 mM MnSO4, 5 mM ATP, 2 mM lysine and 0, 0.015, 0.05, 0.15 or 0.5 mM of ZnCl2 at 37 °C for 30 min.
35
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
A
B
+ ATP, + lysine, + IPP
+ ATP, + lysine, + IPP
Concentration (mM)
5
Ap3A Lys
Lys
4
ADP Lys ATP lysyl-AMP Ap4A
ADP 3
ATP Lys
2
IPP
1
H2O Lys
Pi
PPi
2Pi
Lys
ATP AMP
Lys
ADP
0 0
Concentration (mM)
C
50
100 150 Reaction time (min)
+ ATP, + lysine,
200
D
IPP
5
5
4
4
3
3
2
2
1
1
+ ATP, + lysine, + Pi
0
0 0
50
100
150
200
0
50
Reaction time (min)
100 150 Reaction time (min)
200
Fig. 3. LysS catalyzed the conversion of ATP to Ap4A, Ap3A or ADP in the presence of lysine. (A and C) LysS (15 lM) was incubated with 5 mM ATP, 2 mM lysine, and 5 mM MnSO4 in the presence (A) or absence (C) of 0.6 U inorganic pyrophosphatase (IPP). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. (B) Diagram of the reaction mechanism described in A. PPi, inorganic pyrophosphate; Pi, inorganic phosphate; IPP, inorganic pyrophosphatase; Lys, lysine. (D) LysS (15 lM) was incubated with 5 mM ATP, 2 mM lysine, and 5 mM MnSO4 in 50 mM potassium phosphate buffer (pH 8.0). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
Analysis of nucleotides by ion exchange HPLC analysis
Gel chromatography LysS was loaded onto a TSKgel SuperSW mAb HR (Tosoh; 7.8 mm 30 cm) equilibrated with 50 mM Tris–HCl buffer, pH 7.5, containing 0.1 M Na2SO4. Protein was eluted at a rate of 0.5 ml/min, and protein absorbance at 280 nm was monitored. The column was calibrated using the following standards: ferritin (440 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa).
120 100 Relative activity (%)
Mononucleotides (AMP, ADP, and ATP) and dinucleotides (Ap3A and Ap4A) in the enzyme assay were detected at 260 nm with a HPLC system using a resource Q column, and the nucleotide concentrations were calculated from the peak areas using standard curves derived from known concentrations of the nucleotides. The mobile phase was composed of solvent A (water) and solvent B (0.7 M ammonium bicarbonate). The gradient elution for solvent A, with a flow rate of 1 ml min1, was 90% (v/v) at 0 min, 75% at 0.5 min, 65% at 10 min, 5% at 13 min, and 0% at 14 min.
80 60 40 20 0
0
2
4
6
8
10
Concentration (mM) Fig. 4. Effects of inorganic pyrophosphate and ADP on LysS Ap4A synthase activity. Reactions containing 5 lM LysS, 5 mM ATP, 2 mM lysine, and 5 mM MnSO4 in 50 mM HEPES buffer, pH 8.0, were incubated with either 0, 0.2, 0.5, 1, 2 or 5 mM inorganic pyrophosphate (d) or 0, 1, 2.5, 5 or 10 mM ADP (s) at 37 °C for 30 min. Relative activity was calculated by comparison of Ap4A production with each concentration to the activity measured in without inorganic pyrophosphate or ADP, which was set at 100%.Values are presented as mean ± SEM, determined from the average of three independent measurements.
Results and discussion Comparison of LysS and other lysyl-tRNA synthetases amino acid sequences The open reading frame of MXAN_4731 (named lysS) encodes a protein of 517 amino acids with a calculated molecular mass of
56 kDa. Similar to other reported class II lysyl-tRNA synthetases, M. xanthus LysS contains two domains and three conserved motifs in the catalytic domain (Supplementary Fig. S1) [15]. The deduced amino acid sequence of LysS was 50% and 40% identical to that of E. coli LysU and human LysRS, respectively.
36
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
A
B Concentration (mM)
Ap4A (nmol/min)
0.5
0
0.5
1.0
1.5
2..0
4 3 2 1 0
5.0
0
50
Lysine (mM)
C Concentration (mM)
lysine, + IPP
5
1.0
0
+ ATP,
+ ATP,
lysine,
100
150
200
Reaction time (min)
IPP
+ ATP,
D
5
5
4
4
3
3
2
2
1
1
lysine, + Pi
0
0 0
50
100
150
200
Reaction time (min)
0
50
100
150
200
Reaction time (min)
Fig. 5. Activity of LysS in the absence of added lysine. (A) LysS (5 lM) was incubated with 5 mM ATP, 5 mM MnSO4, and 0, 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 mM lysine. The rate of Ap4A production was measured and plotted as a function of lysine concentration. (B and C) LysS (15 lM) was incubated with 5 mM ATP and 5 mM MnSO4 in the presence (B) or absence (C) of 0.6 U inorganic pyrophosphatase (IPP). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. (D) LysS (15 lM) was incubated with 5 mM ATP, and 5 mM MnSO4 in 50 mM potassium phosphate buffer (pH 8.0). Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
LysS forms a monomer M. xanthus LysS was expressed in E. coli and purified from the soluble fraction using an N-terminal hexahistidine tag (His-tag) and affinity column chromatography. The purified LysS protein was analyzed by SDS–PAGE, which revealed a single band corresponding to a molecular mass of 60 kDa (Fig. 1A). The apparent molecular mass obtained by SDS–PAGE was in agreement with the molecular mass calculated from the predicted amino acid sequence of His-tagged LysS (58 kDa). Class II lysyl-tRNA synthetases are reported to form dimers or tetramers [16]. In fact, human class II lysyl-tRNA synthetases require homodimerization for their aminoacylation function [17]. To determine whether M. xanthus LysS exists in monomeric or multimeric form, we estimated the molecular mass of LysS by gel filtration using the correlation between the elution volume and the logarithm of the molecular mass of known reference proteins (Fig. 1B). Unexpectedly, recombinant M. xanthus LysS eluted as a single peak, and the molecular mass of the native enzyme was estimated to be 68 kDa. This data suggests that recombinant LysS exists as a monomer, despite the presence of the highly conserved motif 1 common to class II enzymes. Zn2+ is not required for Ap4A synthesis by M. xanthus LysS Class II lysyl-tRNA synthetases require Mg2+ and Zn2+ for the first and second step of the reaction mechanism, respectively [18]. To determine the metal cofactor requirement for recombinant
M. xanthus LysS, we measured activity in the presence of a variety of different metals and found that LysS was most active in the presence of Mn2+ (Fig. 2A), and the optimal Mn2+ concentration for full activation of the enzyme ranged from 5 to 20 mM (Fig. 2B). Additionally, we found that the synthase is partially active (72 ± 2% or 24 ± 3%) in the presence of 5 mM Ca2+ or 5 mM Co2+, respectively (Fig. 2A), and has no detectable activity in the presence of Fe2+, Fe3+, and Cu2+ (data not shown). Surprisingly, we found that LysS activity, assayed in the presence of 5 mM Mn2+, was inhibited by the addition of Zn2+ (Fig. 2C). Addition of 15 lM or 150 lM Zn2+ decreased LysS activity by 22 ± 2% and 62 ± 1%, respectively (Fig. 2C). Furthermore, the addition of 150 lM Zn2+ to a dialyzed sample of recombinant protein also inhibited Ap4A synthase activity by 64 ± 3% (data not shown). Also, LysS showed slight Ap4A synthase activity (4 ± 1%) in the presence of 5 mM Mg2+ and 150 lM Zn2+. These data suggest that class II M. xanthus LysS does not require Zn2+ for enzymatic activity. This is similar to many aminoacyl-tRNA synthetases, for example, valine-, histidine- and methionine-specific tRNA synthetases, which are insensitive to the addition of Zn2+ [19]. LysS enzyme kinetics Further characterization of M. xanthus LysS demonstrated that the optimal temperature for Ap4A synthase activity was 50 °C. The relative activity of LysS at 30 °C, 40 °C or 60 °C, as compared to the optimal temperature (50 °C) was 49%, 77% or 45%, respectively (data not shown). LysS activity was most active when pH
37
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
ranged between 8 and 9 and 80% activity remained in a 0.2 M HEPES buffer (pH 7.0). The Km value of M. xanthus LysS for ATP in the presence of 2 mM lysine was 1.3 ± 0.1 mM. The Km of LysS for ATP was approximately 5-fold lower than those of E. coli LysU [14]. Additionally, the kcat of M. xanthus LysS in the absence of inorganic pyrophosphatase was 0.088 ± 0.013 S1. LysS exhibits Ap4A, Ap3A and ADP synthase activities In the presence of lysine, ATP, Mg2+, Zn2+, and inorganic pyrophosphatase, E. coli LysU has dual Ap4A and Ap3A synthase activity [14]. LysU first produces Ap4A from ATP, and then, in a second reaction, converts Ap4A into Ap3A using ADP. In addition, Chen and Xu reported that E. coli LysU finally catalyzes Ap3A into ADP [20]. In the process, inorganic phosphate is generated from pyrophosphate by inorganic pyrophosphatase, and ADP is generated from inorganic phosphate and lysyl-AMP by LysU. We found that when M. xanthus LysS was incubated with ATP, Mn2+, inorganic pyrophosphatase and lysine for 200 min, LysS produced Ap4A and ADP after 25 min of incubation, followed by conversion of Ap4A to Ap3A. Ap3A was then converted to ADP, the end product of the reaction, by 150 min (Fig. 3A). This result suggests that the reaction of lysyl-AMP with ATP to give Ap4A, or with ADP to give Ap3A, is a reversible reaction while the reaction of lysyl-AMP with phosphate to give ADP is an irreversible reaction (Fig. 3B). Additionally, AMP was gradually generated through the 200-min reaction, suggesting that LysS may hydrolyze lysyl-AMP to yield AMP and lysine. In contrast, to M. xanthus LysS, which
A
+ Ap4A, + lysine,
constantly produced ADP, E. coli LysU did not produce ADP during the conversion of Ap4A to Ap3A [14]. In the absence of inorganic pyrophosphatase, LysS produced Ap4A, and the Ap4A was in equilibrium with ATP after 50 min of incubation (Fig. 3C), suggesting that ATP may be formed from the released inorganic pyrophosphate and lysyl-AMP. Accumulation of a small amount of ADP was also observed, indicating that LysS may hydrolyze Ap4A into two molecules of ADP. This inference is in agreement with the results obtained by Goerlich et al. who suggested that E. coli phenylalanyl-tRNA synthetase also degrades Ap4A into 2 mol of ADP [8]. When LysS was incubated with ATP in the presence of 50 mM potassium phosphate, LysS generated Ap4A and ADP; however, Ap3A was hardly detected in the reaction (Fig. 3D), suggesting that phosphate may be more readily combined with lysyl-AMP than ADP by LysS, and/or Ap3A may be generated from lysyl-AMP and ADP after 200 min of incubation. It has been reported that inorganic pyrophosphate inhibits Ap4A synthesis by aminoacyl-tRNA synthetases through competitive inhibition of the ATP reaction with aminoacyl-adenylate [8,21]. To confirm whether inorganic pyrophosphate inhibits the Ap4A synthase activity of LysS, LysS was incubated with inorganic pyrophosphate in the presence of 5 mM ATP. Addition of increasing concentrations of inorganic pyrophosphate (0 to 5 mM) led to the inhibition of Ap4A synthesis (Fig. 4). We next examined whether ADP inhibits the catalysis of Ap4A formation. Similarly, the addition of ADP inhibited the Ap4A synthase activity of LysS (Fig. 4). Ap4A production decreased by 22 ± 2% and 52 ± 2% at 1 mM and 5 mM ADP, respectively. In the presence of ADP, Ap3A was not synthesized by LysS during 30 min of incubation. These results suggest
B
IPP
+ Ap4A, + lysine,
IPP
Concentration (mM)
2 Lys PPi
ATP Lys
Ap4A
1
H2O Lys
Lys PPi
H2O
lysyl-AMP ADP
2ADP
Lys
AMP
ADP Lys
Ap3A 0 0
50
100
150
200
Reaction time (min)
C
D
+ Ap4A, + lysine, + IPP
Concentration (mM)
3
+ Ap4A,
lysine,
IPP
2
2 1 1
0
0 0
50
100
Reaction time (min)
150
200
0
50
100
150
200
Reaction time (min)
Fig. 6. Conversion of Ap4A by LysS in the absence of ADP. (A and C) LysS (24 lM) was incubated with 2 mM Ap4A, 2 mM lysine and 5 mM MnSO4 in the absence (A) or presence (C) of 0.3 U inorganic pyrophosphatase. (B) Diagram of the reaction mechanism described in A. PPi, inorganic pyrophosphate; Lys, lysine. (D) LysS (24 lM) was incubated with 2 mM Ap4A and 5 mM MnSO4. Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
38
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
that ADP may act as a competitive inhibitor of LysS in the first step reaction (lysyl-AMP formation from lysine and ATP). Additionally, because the reversal of the second step did not occur during a 30-min incubation, it can be inferred that Ap3A may be generated from ADP and lysyl-AMP arising from Ap4A via this reversal. In E. coli LysU, the reversal of the second step allows Ap3A formation from ADP and lysyl-AMP [14]. On the other hand, Ap4A synthase activity of LysS was not inhibited by addition of AMP (data not shown). The activity of most class II aminoacyl-tRNA synthetases is found to absolutely require the presence of added cognate amino acids, except for a human glycyl-tRNA synthetase [8,22]. When LysS was incubated with a range of lysine concentrations (0– 5 mM) in the presence of 5 mM MnSO4 and 5 mM ATP, we observed production of Ap4A even in the absence of added lysine (Fig. 5A). However, the rate of Ap4A production by LysS in the absence of added lysine was about 1.7-fold lower than in the presence of 0.05 mM to 5 mM lysine. Goerlich et al. reported that E. coli LysRS also exhibited considerable synthesis in the absence of lysine, because 1.2 lM dialyzed LysRS contained approximately 0.2 lM endogeneous lysine [8]. In this study, the Ap4A synthase activity of dialyzed LysS was approximately 3-fold lower in the absence of added lysine than in the presence of 2 mM lysine (data not shown). During the expression of recombinant M. xanthus LysS in E. coli, intracellular lysine in E. coli cells may bind to the active site of recombinant LysS. Dialysis may not be able to dissociate lysine or lysyl-AMP from LysS by that may enable to catalyze in vitro formation of Ap4A by LysS in the absence of added lysine.
A
LysS incubated with ATP, Mn2+, and inorganic pyrophosphatase (without lysine addition) first generated Ap4A after 100 min which was followed by a slow decrease in Ap4A (Fig. 5B). Additionally, ADP was gradually generated throughout the 200-min reaction and only small amounts of Ap3A were produced in these conditions. This reaction progressed more slowly than in the presence of lysine. In the absence of added lysine and inorganic pyrophosphatase, Ap4A was in equilibrium with ATP after 100 min of incubation (Fig. 5C). LysS also generated Ap4A and ADP in the presence of 50 mM potassium phosphate (Fig. 5D) in a reaction that proceeded more slowly than in the presence of inorganic pyrophosphatase (Fig. 5B). Ap4A degradation by LysS E. coli LysU does not degrade Ap4A when it is incubated with Ap4A, lysine, Mg2+, Zn2+, and inorganic phosphatase [14]. In contrast, E. coli phenylalanyl-tRNA synthetase degraded Ap4A into almost equal amounts of ATP and AMP in the absence of inorganic phosphatase [8]. M. xanthus LysS converted Ap4A to yield ATP and AMP when Mn2+ and lysine were present; however, formation of ATP was higher than that of AMP (Fig. 6A). When 0.5 mM inorganic pyrophosphate was included in the reaction mixture, formation of ATP was also accelerated (data not shown). These results suggest that formation of ATP from lysyl-AMP and pyrophosphate (pyrophosphorolysis) proceeds faster than the hydrolysis of lysyl-AMP to lysine and AMP in the presence of pyrophosphate (Fig. 6B) [8]. In addition, LysS hydrolyzed Ap4A symmetrically
B
+ Ap4A, + ADP, + lysine, IPP
+ Ap4A, + ADP, + lysine,
IPP
Concentration (mM)
2
ATP Lys
Ap4A
1
Lys
Lys PPi
Lys PPi
lysyl-AMP ATP ADP ADP Lys
Lys
Ap3A 0 0
Concentration (mM)
C
50
100 150 Reaction time (min)
200
D
+ Ap4A, + ADP, + lysine, + IPP
4
2
2
1
0
+ Ap4A, + ADP,
lysine,
IPP
0 0
50
100
150
Reaction time (min)
200
0
50
100 150 Reaction time (min)
200
Fig. 7. Conversion of Ap4A by LysS in the presence of ADP. (A and C) 8 lM LysS was incubated with 2 mM Ap4A, 2 mM ADP, 2 mM lysine and 5 mM MnSO4 in the absence (A) or presence (C) of 0.3 U inorganic pyrophosphatase. (B) Diagram of the reaction mechanism described in A. PPi, inorganic pyrophosphate; Lys, lysine. (D) LysS (8 lM) was incubated with 2 mM Ap4A, 2 mM ADP and 5 mM MnSO4. Concentrations of ATP, Ap4A, Ap3A, and ADP were plotted at the indicated time points during the reaction. ((s) ATP, (d) Ap4A, (N) ADP, (j) Ap3A, (.) AMP).
M. Oka et al. / Archives of Biochemistry and Biophysics 579 (2015) 33–39
yielding ADP, because Ap3A under these conditions was produced throughout the duration of the experiment. In the presence of inorganic pyrophosphatase, added Ap4A was rapidly converted to ADP and Ap3A without apparent accumulation of ATP, because the released pyrophosphate from ATP is hydrolyzed by pyrophosphatase (Fig. 6C). On the other hand, LysS hydrolyzed Ap4A to yield ADP as a major cleavage product in the absence of added lysine and inorganic pyrophosphatase (Fig. 6D). LysS incubated with 2 mM Ap4A, 2 mM ADP, and 2 mM lysine generated Ap3A and ATP (Fig. 7A). In this condition, accumulation of ATP, but not AMP, was observed. This result suggests that LysS does not hydrolyze lysyl-AMP to lysine and AMP in the presence of high concentration of ADP, because the lysyl-AMP is rapidly combined with ADP to form Ap3A (Fig. 7B). In the presence of inorganic pyrophosphatase, LysS converted Ap4A rapidly into Ap3A and ADP with low level of ATP. Ap3A was then converted to ADP, the end product of the reaction, by 200 min (Fig. 7C). This is similar to E. coli LysU, which also generates Ap3A in the presence of a mixture of ADP and Ap4A; however, the same accumulation in ADP was not observed [14]. In the absence of added lysine, Ap4A hydrolysis with ADP progressed more slowly than in the presence of lysine (Fig. 7D). In conclusion, we demonstrated that the class II M. xanthus LysS has Ap4A, Ap3A and ADP synthase activity without the requirement for Zn2+ and recombinant LysS showed considerable Ap4A synthase activity in the absence of added lysine. In addition, LysS converted Ap4A mainly into AMP and ATP, or ADP in the presence or absence of lysine, respectively. These results suggested that LysS has different enzymatic properties from the class II lysyl-tRNA synthetases reported previously. Acknowledgments This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25440087).
39
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2015.05.014. References [1] L.L. Kisselev, J. Justesen, A.D. Wolfson, L.Y. Frolova, FEBS Lett. 427 (1998) 157– 163. [2] P.C. Zamecnik, M.L. Stephenson, C.M. Janeway, K. Randerath, Biochem. Biophys. Res. Commun. 24 (1966) 91–97. [3] P.C. Lee, B.R. Bochner, B.N. Ames, Proc. Natl. Acad. Sci. U.S.A. 80 (1983) 7496– 7500. [4] P.C. Lee, B.R. Bochner, B.N. Ames, J. Biol. Chem. 258 (1983) 6827–6834. [5] B.R. Bochner, P.C. Lee, S.W. Wilson, C.W. Cutler, B.N. Ames, Cell 37 (1984) 225– 232. [6] T.M. Ismail, C.A. Hart, A.G. McLennan, J. Biol. Chem. 278 (2003) 32602–32607. [7] S. Hansen, K. Lewis, M. Vulic, Antimicrob. Agents Chemother. 52 (2008) 2718– 2726. [8] O. Goerlich, R. Foeckler, E. Holler, Eur. J. Biochem. 126 (1982) 135–139. [9] S. Blanquet, P. Plateau, S. Onesti, Class II Lysyl-tRNA Synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), The Aminoacyl-tRNA Synthetases, Eurekah, Georgetown, TX, 2005, pp. 227–240. [10] G. Desogus, F. Todone, P. Brick, S. Onesti, Biochemistry 39 (2000) 8418–8425. [11] F. Lévéque, P. Plateau, P. Dessen, S. Blanquet, Nucleic Acids Res. 18 (1990) 305– 312. [12] F. Lévéque, M. Gazeau, M. Fromant, S. Blanquet, P. Plateau, J. Bacteriol. 173 (1991) 7903–7910. [13] A. Brevet, J. Chen, F. Lévéque, S. Blanquet, P. Plateau, J. Biol. Chem. 270 (1995) 14439–14444. [14] M. Wright, N. Boonyalai, J.A. Tanner, A.D. Hindley, A.D. Miller, FEBS J. 273 (2006) 3534–3544. [15] G. Eriani, M. Delarue, O. Poch, J. Gangloff, D. Moras, Nature 347 (1990) 203– 206. [16] M. Ibba, D. Söll, Annu. Rev. Biochem. 69 (2000) 617–650. [17] M. Guo, M. Ignatov, K. Musier-Forsyth, P. Schimmel, X.-L. Yang, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 2331–2336. [18] X. Chen, N. Boonyalai, C. Lau, C. Thipayang, Y. Xu, M. Wright, M.D. Miller, FEBS J. 280 (2013) 102–114. [19] S. Blanquet, P. Plateau, A. Brevet, Mol. Cell. Biochem. 52 (1983) 3–11. [20] X. Chen, X. Yuhong, Acta Biophys. Sin. 29 (2013) 35–45. [21] L. Dibbelt, U. Pachmann, H.G. Zachau, Nucleic Acid Res. 8 (1980) 4021–4039. [22] R.-T. Guo, Y.E. Chong, M. Guo, X.-L. Yang, J. Biol. Chem. 284 (2009) 28968– 28976.
