Lysyl-tRNA synthetase from Myxococcus xanthus catalyzes the formation of diadenosine penta- and hexaphosphates from adenosine tetraphosphate

Archives of Biochemistry and Biophysics 604 (2016) 152e158

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Lysyl-tRNA synthetase from Myxococcus xanthus catalyzes the formation of diadenosine penta- and hexaphosphates from adenosine tetraphosphate 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

a b s t r a c t

Article history: Received 3 May 2016 Received in revised form 23 June 2016 Accepted 2 July 2016 Available online 5 July 2016

Myxococcus xanthus lysyl-tRNA synthetase (LysS) produces diadenosine tetraphosphate (Ap4A) from ATP in the presence of Mn2þ; in the present study, it also generated Ap4 from ATP and triphosphate. When ATP and Ap4 were incubated with LysS and pyrophosphatase, first Ap4A, Ap5A, and ADP, and then Ap5, Ap6A, and Ap3A were generated. The results suggest that in the first step, LysS can form lysyl-AMP and lysyl-ADP intermediates from Ap4 and release triphosphate and diphosphate, respectively, whereas in the second step, it can produce Ap5 from lysyl-ADP with triphosphate, and Ap6A from lysyl-ADP with Ap4. In addition, in the presence of Ap4 and pyrophosphatase, but absence of ATP, LysS also generates diadenosine oligophosphates (ApnAs: n ¼ 3e6). These results indicate that LysS has the ability to catalyze the formation of various ApnAs from Ap4 in the presence of pyrophosphatase. Furthermore, the formation of Ap4A by LysS was inhibited by tRNALys in the presence of 1 mM ATP. To the best of our knowledge, this is the first report of Ap5A and Ap6A synthesis by lysyl-tRNA synthetase. © 2016 Elsevier Inc. All rights reserved.

Keywords: Lysyl-tRNA synthetase Diadenosine pentaphosphate Diadenosine hexaphosphate Adenosine tetraphosphate Myxococcus xanthus

1. Introduction Aminoacyl-tRNA synthetases are essential enzymes found in all living organisms [1], which catalyze the attachment of amino acids to their cognate tRNA molecules in a highly specific, two-step reaction [2]. The first step involves activation of an amino acid concomitant with ATP hydrolysis to form an aminoacyl-AMP intermediate, while in the second step, the activated amino acid is transferred to the free hydroxyl of the ribose at the 30 end of tRNA. Based on their structure, the existing 20 aminoacyl-tRNA synthetases are divided into two classes [3]. Class I enzymes are mostly monomeric and catalyze the coupling of the aminoacyl group to the 20 -hydroxyl group of tRNA, whereas class II enzymes are multimeric and prefer the 30 -hydroxyl site. Moreover, the majority of class II aminoacyl-tRNA synthetases can catalyze the formation of diadenosine tetraphosphate (Ap4A) in the absence of cognate tRNAs [4]. Ap4A composed of two adenosine moieties joined by the 50 e50 linkage of four phosphates is present in both prokaryotic and eukaryotic cells. It was found that in prokaryotes, the intracellular

* Corresponding author. E-mail address: [email protected] (Y. Kimura). http://dx.doi.org/10.1016/j.abb.2016.07.002 0003-9861/© 2016 Elsevier Inc. All rights reserved.

concentration of Ap4A is increased by heat and oxidative stress [5,6]. Further studies have implicated Ap4A metabolism in prokaryotes in the regulation of stress response, biofilm formation, and pathogenesis of bacterial infections [7e9]. Lysyl-tRNA synthetases are unique among the aminoacyl-tRNA synthetases because they comprise both class I and class II enzymes. In eukaryotes and most prokaryotes, lysyl-tRNA synthetases belong to the class II enzymes, and class II lysyl-tRNA synthetases efficiently produce Ap4A [10]. Class II lysyl-tRNA synthetase contains a smaller N-terminal tRNA-binding domain and a larger Cterminal catalytic domain, which contains three characteristic sequence motifs [11]. Motif 1 is involved in formation of the interface between two catalytic domains during enzyme dimerization, whereas motif 2 binds ATP, and motif 3 interacts with nucleotide phosphates and Mg2þ ions, thus stabilizing the bent conformation of ATP [12]. Lysyl-tRNA synthetases produce Ap4A during a two-step enzymatic reaction: first, lysyl-AMP is synthesized from lysine and ATP with the release of pyrophosphate, and then, Ap4A is generated from lysyl-AMP and ATP; the first step requires Mg2þ, while the second needs Zn2þ, which is shown to inhibit tRNA aminoacylation activity of lysyl-tRNA synthetase from Escherichia coli [13e15].

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Talon affinity column according to previously published protocols [18]. The LysS concentration was determined by the Bradford method using bovine serum albumin as the standards [21]. All compounds, except for Ap4 and Ap6A, were obtained from Sigma Aldrich. Ap4 and Ap6A were obtained from Jena Bioscience.

Myxococcus xanthus is a soil bacterium that displays a variety of complex and highly coordinated behavioral reactions, including social motility and fruiting body formation [16,17]. In this microorganism, the role of Ap4A is unknown; however, we previously reported that M. xanthus class II lysyl-tRNA synthetase (LysS) and diadenosine polyphosphate hydrolase (ApaH) can synthesize and degrade, respectively, diadenosine oligophosphates (ApnAs) [18,19]. Although M. xanthus LysS belongs to the class II aminoacyl-tRNA synthetases, it requires only Mn2þ for Ap4A synthesis, while Zn2þ inhibits Ap4A production [18]. M. xanthus LysS first produces Ap4A from ATP in the presence of pyrophosphatase (Fig. 1A). After Ap4A is converted to ATP and enzyme-bound lysyl-AMP, LysS efficiently utilizes inorganic phosphate cleaved from pyrophosphate (PPi) by pyrophosphatase, and lysyl-AMP to produce ADP, which is combined with lysyl-AMP to generate Ap3A. The Ap3A produced is converted to lysyl-AMP and ADP-the end product of the reaction. In addition, AMP can be gradually generated through a 240-min reaction, suggesting that LysS may hydrolyze lysyl-AMP to yield AMP and lysine (Fig. 1C). In the absence of pyrophosphatase, LysS also produces Ap4A, which reaches the equilibrium with ATP after 90 min, indicating that a reverse reaction (lysyl-AMP þ PPi / ATP þ lysine) is taking place or that the released PPi inhibits the first step, i.e., lysyl-AMP formation (Fig. 1B). The yields of Ap3A and ADP in the reaction are low. M. xanthus vegetative cells contain the following concentrations of ribonucleoside triphosphates and ribonucleoside diphosphates: 1.9 nmol ATP, 1.1 nmol GTP, 0.4 nmol UTP, 0.3 nmol ADP and 0.8 nmol CDP per 1  109 cells [20]. LysS can probably produce various ApnA(N)s in vivo using these NTPs and NDPs. In this study, we investigated whether M. xanthus LysS can produce ApnAs in the presence of ATP together with other ribonucleotides.

2.2. Enzyme assay

2. Materials and methods

To analyze the time dependence of the synthesis of ApnAs and other nucleotides, the assay was performed in a total volume of 30 ml of 5 mM substrate, 5 mM MnSO4, 2 mM lysine, and 12 mM LysS in the absence and presence of 0.6 units (1.2 mg) of inorganic pyrophosphatase from Saccharomyces cerevisiae (Sigma-Aldrich) at 37  C for 4 h. Aliquots (4 ml) were removed at successive time intervals and immediately applied onto a resource Q column (GE health). 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, 73% at 0.5 min, 61% at 10 min, 25% at 26 min, and 0% at 28 min for analysis of the Ap4G, Ap5A, or Ap6A product; or 90% at 0 min, 70% at 0.5 min, 45% at 16 min, and 0% at 17 min for analysis of the Ap3A, Ap3G, Ap4, or Ap4A product. The contents of the post-elution fractions were identified by their retention time compared with standards. To determine the kinetic parameters (Km) for substrates, Ap4A synthetase activity was measured using 1e10 mM ATP. Additionally, Ap3A, Ap4, or Ap5A synthetase activity was measured using 5 mM ATP and 0.5e10 mM ADP, 5 mM ATP and 0.05e2 mM triphosphate, or 5 mM ATP and 0.1e2 mM Ap4, respectively, as substrates for the determination of Km. LysS (5e10 mM) was incubated with substrates, 2 mM lysine, and 5 mM MnCl2 in the presence or absence of inorganic pyrophosphatase (0.6 U) at 37  C for 30 min. Km was estimated from the Michaelis-Menten curve.

2.1. General

2.3. Preparation of tRNALys Two tRNALys genes, MXAN_2011 and MXAN_3185, were

Recombinant LysS was expressed in E. coli, and purified using a

Concentration (mM)

A.

B.

+ ATP, + IPP

5

5

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4

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3 ATP

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Ap3A AMP

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Reaction time (min)

+ ATP, + IPP

Ap3A ADP

ADP

ATP

lysyl-AMP

ATP PPi IPP 2Pi

Pi

H2O

Ap4A ATP

AMP ADP

Fig. 1. LysS catalyzed the conversion of ATP to Ap4A, Ap3A or ADP in the presence (A) or absence (B) of inorganic pyrophosphatase. () ATP, (C) Ap4A, (:) ADP, (-) Ap3A, (;) AMP. (C) Diagram of the reaction mechanism occurring in A. PPi, pyrophosphate; Pi, monophosphate; IPP, inorganic pyrophosphatase. The substrate is indicated in bold text, and the major products are enclosed in squares.

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amplified by PCR using, as primers, 50 - CGACTCACTATAGGGGGGAGCGTAGCTCAATTGGC-30 and 50 -GGTACCCGGGGATCCTGAGAGCGGAGGGAATCGAAC-30 , and 50 CGACTCACTATAGGGGAGTCGCTAGCTCAGCTGGTAG-30 and 50 GGTACCCGGGGATCCTGAGTCGCGGGGGGCTCGAAC-30 , respectively, and then cloned into pT7 blue vector (Takara Bio). For linearization of the DNA template, two pT7 blue-tRNALys were amplified by PCR, using primers 50 -AATAGCGAAGAGGCCCGC-30 and 50 -TGAGAGCGGAGGGAATCG-30 , or 50 -AATAGCGAAGAGGCCCGC-30 and 50 TGAGTCGCGGGGGGCTCG-30 , respectively, and then purified by phenol/chloroform extraction. The PCR-generated fragments were used in transcription reactions (T7 RiboMAX Express large-scale RNA production system, Promega). After the in vitro transcription reaction was performed, transcripts were purified using a Nick column (GE healthcare). The concentrations of the transcripts were estimated by measuring the absorbance at 260 nm. The mixture of two tRNALys was used for the enzyme assay. 2.4. Site-directed mutagenesis The lysS gene cloned into the expression vector pCold was used as a template for PCR. Site-directed mutations were generated with PrimeStar Max DNA polymerase (Takara Bio) using the primer pairs described in Table S1. The PCR products were used for transformation of E. coli. After confirmation of the desired mutations by DNA sequencing, the mutant enzymes were expressed and purified by a previously described method (18). 3. Results and discussion M. xanthus LysS efficiently produces Ap4A, Ap3A, and ADP from ATP in the presence of Mn2þ and pyrophosphatase [18]. The Km value of LysS for ATP with regard to Ap4A synthesis with or without pyrophosphatase was 3.8 mM or 1.3 mM, respectively. It has been reported that the inhibition of catalytic activity with high substrate concentrations is a general phenomenon observed for more than 20% of existing enzymes [22]. Analysis of LysS kinetics also revealed strong inhibition by ATP at concentrations higher than 8 mM (data not shown). We previously reported that LysS catalyzed Ap4A hydrolysis to yield mainly ATP and AMP or ADP in the absence or presence of pyrophosphatase, respectively, whereas in the absence of pyrophosphatase, it generated ATP and Ap3A from Ap4A and ADP [18]. To further evaluate LysS activity, we determined the products generated by LysS in the presence of ATP together with other ribonucleotides. 3.1. LysS produced Ap3G from ATP and GDP, and Ap4G from ATP and GTP First, LysS was incubated with 5 mM ATP and 1e10 mM GTP at 37  C for 30 min (Fig. 2A). The conversion of ATP to Ap4A decreased with the addition of GTP in a concentration-dependent manner; 50% inhibition was observed in the presence of 5 mM GTP. The addition of 5 mM CTP caused even higher inhibition (80%) of Ap4A production by LysS (data not shown), suggesting that GTP or CTP may act as a competitive inhibitor of LysS in the first- and/or second-step reactions. LysS did not produce Gp4G when incubated with 5 mM GTP alone. When LysS was incubated with 5 mM ATP and 5 mM ADP in the presence of pyrophosphatase, the kinetics of the Ap4A formation was similar to that with ATP alone, suggesting that ADP does not affect Ap4A formation by LysS (Fig. 2B). We previously reported that the LysS enzymatic activity that produces Ap4A was not inhibited by AMP (18). In the absence of pyrophosphatase, Ap4A formation was slower

than that with ATP as the only nucleotide substrate (Fig. 2C). Ap3A formation in the presence of ATP and ADP progressed faster than with ATP alone. In Ap3A synthetic reaction, the Km value of LysS for ADP with or without pyrophosphatase was 1.5 mM or 1.3 mM, respectively. Kinetic analysis indicated that LysS also exhibited reduced ability for Ap3A synthesis at ADP concentrations higher than 5 mM (data not shown). The addition of 1e10 mM GDP together with 5 mM ATP caused much weaker inhibition of LysS-catalyzed production of Ap4A in the presence of pyrophosphatase compared with the same concentrations of GTP (Fig. 2A), while 5 mM CDP did not exert any effect (data not shown). It is known that E. coli LysRS catalyzes the synthesis of Ap4A and Ap3N or Ap4N in the presence of ATP together with other NDPs or NTPs, respectively [14]. M. xanthus LysS produced Ap3G from 5 mM ATP and 5 mM GDP, and Ap4G from 5 mM ATP and 5 mM GTP in the presence of pyrophosphatase (Fig. 2D&E). In these reactions, LysS prioritized the production of Ap4A, and then produced Ap3G or Ap4G. The formation rate of Ap3G was higher than that of Ap4G, which may be a result of the inhibition of Ap4A synthesis by GTP, because both Ap3G and Ap4G were produced after conversion of Ap4A to lysyl-AMP and ATP. These results suggest that GTP and GDP can be LysS substrates in the second but not in the first step. 3.2. LysS produced Ap4 from ATP and triphosphate When LysS was incubated with 5 mM ATP and 5 mM triphosphate (PPPi) in the presence of pyrophosphatase for 240 min, it first synthesized Ap4A from ATP; however, there was a 10-min delay in Ap4A production and the maximum Ap4A concentration was about 1.5-fold lower than that with ATP alone, or with ATP and ADP (Fig. 3A). Subsequently, LysS produced Ap4 from lysyl-AMP with PPPi and ADP from lysyl-AMP with phosphate (Pi). After 120 min of reaction, low amounts of Ap5A were generated from lysyl-AMP with Ap4, suggesting that in these conditions, LysS had low catalytic activity to synthesize Ap5A via conjugation of lysyl-AMP intermediate with Ap4 (Fig. 3A&B). However, in the absence of pyrophosphatase, the ability of LysS to produce Ap4A from 5 mM ATP and 5 mM PPPi dramatically decreased (Fig. 3C), suggesting that the presence of both PPi and PPPi synergistically inhibits the formation of lysyl-AMP or Ap4A. The Km value of M. xanthus LysS for PPPi in Ap4 formation with or without pyrophosphatase was 0.82 mM or 0.22 mM, respectively. 3.3. LysS produced Ap5, Ap5A, and Ap6A from ATP and Ap4 When LysS was incubated with 2 mM ATP and 2 mM Ap4 in the presence of pyrophosphatase, the enzyme first generated Ap4A, Ap5A, and ADP, and then Ap5, Ap6A, and Ap3A (Fig. 4A&B). The formation of Ap5A or Ap6A was also a reversible reaction, and they were finally converted to ADP as the end product (Fig. 4B); in this reaction, adenosine was not generated. These results suggest that lysyl-AMP and lysyl-ADP intermediates may be produced from Ap4 in the first reaction so that LysS can synthesize Ap5 from the reaction. LysS may convert Ap4 to lysyl-AMP and PPPi, and then produce Ap5 from lysyl-ADP with PPPi (Fig. 4C). In addition, the reduction of the Ap4 concentration was slower than that of the ATP concentration, suggesting that LysS prefers ATP to Ap4 as a substrate. The absence of pyrophosphatase decreased the reaction rate (Fig. 4D), which may be due to the reversal of the first step in which ATP was formed from lysyl-AMP with PPi or Ap4 was formed from lysyl-ADP with PPi and from lysyl-AMP with PPPi. The Km value of M. xanthus LysS for Ap4 in the synthesis of Ap5A with or without pyrophosphatase was 0.56 mM or 0.15 mM, respectively.

M. Oka et al. / Archives of Biochemistry and Biophysics 604 (2016) 152e158

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A.

+ ATP, + ADP, + IPP ADP

7

Concentration (mM)

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Fig. 2. (A) Effects of GTP and GDP on LysS ability to synthesize Ap4A. Reactions containing 4 mM or 11 mM LysS, 5 mM ATP, 2 mM lysine, and 5 mM MnCl2 in 50 mM HEPES (pH 8.0) were performed in the presence of 0, 1, 2.5, 5, 7.5, or 10 mM GTP (C) or GDP () at 37  C for 30 min. Relative activity was calculated as Ap4A production in each reaction normalized to that without GTP or GDP set as 100%. The data represent the means ± SEM of independent experiments performed in triplicate. (B&C) Time-dependent changes in the concentrations of reaction products generated by LysS from 5 mM ATP and 5 mM ADP in the presence (B) or absence (C) of pyrophosphatase. () ATP, (C) Ap4A, (:) ADP, (-) Ap3A, (;) AMP. (D & E) HPLC profiles of reaction products formed by LysS (24 mM) with 5 mM ATP and 5 mM GDP (D) or 5 mM ATP and 5 mM GTP (E) after 1 h and 2 h of incubation at 37  C. The Ap3G and Ap4G products were analyzed by HPLC analysis, using two different gradient programs as described in the Materials and Methods section.

A. 5

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+ ATP, + PPPi, + IPP

+ ATP, + PPPi, + IPP

ATP

Concentration (mM)

ADP

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IPP

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Ap4

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Fig. 3. Time-dependent changes in the concentrations of reaction products generated by LysS from ATP and PPPi. LysS (12 mM) was incubated with 5 mM ATP, 5 mM PPPi, 2 mM lysine, and 5 mM MnCl2 in the presence (A) or absence (C) of 0.6 U inorganic pyrophosphatase at 37  C for 240 min () ATP, (C) Ap4A, (:) ADP, (-) Ap3A, (;) AMP, (A) Ap4, ( ) Ap5A. (B) Mechanism of the reaction described in A. The substrates are indicated in bold text, and the major products are enclosed in squares.

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Fig. 4. (A) HPLC profiles of the reaction products generated by LysS from ATP and Ap4. (B & D) Time-dependent formation of reaction products from 2 mM ATP and 2 mM Ap4 in the presence (B) or absence (D) of pyrophosphatase. A 30-ml reaction mixture containing 50 mM HEPES (pH 8.0), 5 mM LysS, 5 mM MnCl2, 0.6 U inorganic pyrophosphatase, 2 mM ATP, and 2 mM Ap4 was incubated at 37  C for 240 min () ATP, (C) Ap4A, (:) ADP, (-) Ap3A, (;) AMP, (A) Ap4, (7) Ap5, ( ) Ap5A, ( ) Ap6A. (C) Mechanism of the reaction described in B. The substrates are indicated in bold text, and the major products are enclosed in squares.

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3.4. LysS could produce Ap3A, Ap4A, Ap5A, and Ap6A from Ap4 Adenylate kinase from rabbit, acetyl-CoA synthetase from Saccharomyces cerevisiae, and acyl-CoA synthetase from Pseudomonas fragi catalyze Ap4 synthesis from ATP and ADP, ATP and AMP, and ATP and PPPi, respectively [23e25]. In the presence of Ap4 and pyrophosphatase but in the absence of ATP, LysS produced ADP, ATP, Ap3A, Ap4A, Ap5, Ap5A, and Ap6A (Fig. 5A). This reaction was similar to that with ATP and Ap4; however, the level of Ap4A formation by LysS with Ap4 was lower than that with ATP and Ap4. This finding also indicates that LysS converts Ap4 into lysyl-AMP and lysyl-ADP intermediates and releases triphosphate and pyrophosphate, respectively, resulting in the production of ApnAs (n ¼ 3e6) and ADP in the presence of pyrophosphatase (Fig. 5C). In the absence of pyrophosphatase, LysS mainly produced Ap5, Ap5A, and Ap6A from Ap4 (Fig. 5B). This process also reached a state of equilibrium after 60 min, suggesting that a reverse reaction may occur with lysyl-AMP and PPPi or lysyl-ADP and PPi in the first step (Fig. 5D). Adenylate kinase plays a major role in regulating interconversion of adenine nucleotides and energy homeostasis of living cells, and is essential for the growth and survival of E.coli and Streptococcus pneumoniae [26, 27]. Since Ap5A functions as a specific inhibitor of adenylate kinase, its generation by LysS may be important for the regulation of adenine nucleotide homeostasis and energy metabolism in M. xanthus. 3.5. LysS had weak Ap5A hydrolytic activity We have previously reported that LysS degrades Ap4A to yield mainly 2 mol of ADP or ATP and AMP in the presence or absence of pyrophosphatase, respectively [18]. When LysS was incubated with

▫

Ap5A in the presence of pyrophosphatase, it slowly hydrolyzed Ap5A to yield Ap4 and AMP; however, the hydrolysis of Ap5A was lower compared to that of Ap4A (Fig. 6A). 3.6. tRNA inhibited Ap4A formation by LysS For some aminoacyl-tRNA synthetases such as E. coli phenylalanyl- and human glycyl-tRNA synthetase, Ap4A formation is significantly inhibited (2e10-fold) by the addition of cognate tRNAs [28e31]. On the other hand, the activity of arginyl- and tryptophanyl-tRNA synthetase are not affected by tRNA [29]. As shown in Fig. 6B, LysS activity to synthesize Ap4A was inhibited by the addition of in vitro transcribed tRNALys in a concentrationdependent manner; thus, the presence of tRNALys at the 1:1 M ratio to LysS decreased its enzymatic activity by about 5-fold. 3.7. Mutational analysis of LysS Zn2þ has a dramatic effect on the activation of class II lysyl-tRNA synthetases. Although M. xanthus LysS has high amino acid sequence similarity with other class II lysyl-tRNA synthetases (Supplementary Fig. 1S), it does not require Zn2þ for the synthesis of Ap4A. Thus, E264 in motif 2 of E. coli LysU is considered a critical residue for the enhancement of Ap4A synthesis by Zn2þ [32] and is conserved in M. xanthus LysS which, however, is not stimulated by Zn2þ. To identify amino acids defining LysS specificity to metal requirement, we performed sequence alignment of M. xanthus LysS with lysyl-tRNA synthetase from E. coli, S. cerevisiae, and Homo sapiens, and determined the residues unique for LysS catalytic domain of M. xanthus (Fig. 1S). Then, seven LysS mutants were obtained by site-directed mutagenesis, and analyzed for their ability to synthesize Ap4A in the presence of 5 mM Mn2þ. The

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Fig. 5. (A & B) Time-dependent formation of reaction products by 8 mM LysS from 2 mM Ap4 in the presence (A) or absence (B) of pyrophosphatase. () ATP, (C) Ap4A, (:) ADP, (-) Ap3A, (;) AMP, (A) Ap4, (7) Ap5, ( ) Ap5A, ( ) Ap6A. (C & D) Mechanisms of the reactions described in A and B, respectively. The substrate is indicated in bold, and major products are enclosed in squares.

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▫

A.

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+Ap5A, + IPP

5

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Concentration (mM)

3 2 Ap4 AMP Ap4A ADP Ap3A ATP

1

Relative activity (%)

Ap5A

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Reaction time (min)

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LysS : tRNALys (molar ratio)

Relative activity (%)

120 100 80 60 40 20 0L

5A

48

46 /T

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42 Y

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92 T2

26 V

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22

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/L 4S 19

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Fig. 6. (A) Time-dependent formation of reaction products by 11 mM LysS from 5 mM Ap5A in the presence of pyrophosphatase. () ATP, (C) Ap4A, (:) ADP, (;) AMP, (A) Ap4, ( ) Ap5A. (B) Effects of tRNALys on LysS ability to synthesize Ap4A. Reaction mixtures containing 2.5 mM LysS, 1 mM ATP, 2 mM lysine, 5 mM MnCl2, and 0.6 U pyrophosphatase in 50 mM HEPES (pH 8.0) were incubated with 0, 0.41, 0.83, 1.65, or 2.5 mM tRNALys at 37  C for 15 min. Relative activity was calculated as the percentage of Ap4A production to that without tRNALys set as 100%. (C) The ability of LysS mutants to synthesize Ap4A in the presence of 5 mM Mn2þ (white bars) or 5 mM Mn2þ and 0.15 mM Zn2þ (black bars). Relative activity was calculated after normalization to that of the wild-type enzyme set at 100%. The data represent the means ± SEM of independent experiments performed in triplicate.

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results indicated that three mutant enzymes (L220I/S222G, V264I, and T292D) exhibited the activity similar to that of the wild-type enzyme, while four mutants (N194S/L196I, Y428F, Q462D/Q463D/ T465A, and M480L) demonstrated a decrease in Ap4A formation by 46, 57, 41, and 18%, respectively (Fig. 6C). On the other hand, all mutant enzymes, similar to the wild type LysS, were inhibited by the addition of Zn2þ, suggesting that the mutated residues do not define the difference between M. xanthus LysS and other lysyl-tRNA synthetases regarding Zn2þ requirement. In conclusion, we demonstrated that class II lysyl-tRNA synthetase from M. xanthus produces Ap3G from ATP and GDP, and Ap4G from ATP and GTP, and can also catalyze Ap4 synthesis from ATP and PPPi. In the presence of pyrophosphatase, LysS catalyzes the formation of various ApnAs (n ¼ 3e6) from Ap4, and then converts these ApnA to ADP. tRNALys inhibited the Ap4A synthetase activity of LysS. Acknowledgments This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16K07667). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.abb.2016.07.002. References [1] G.M. Nagel, R.F. Doolittle, Phylogenetic analysis of the aminoacyl-tRNA synthetases, J. Mol. Evol. 40 (1995) 487e498. €ll, Aminoacyl-tRNA synthesis, Annu. Rev. Biochem. 69 (2000) [2] M. Ibba, D. So 617e650. [3] G. Eriani, M. Delarue, O. Poch, J. Gangloff, D. Moras, Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs, Nature 347 (1990) 203e206. [4] O. Goerlich, R. Foeckler, E. Holler, Mechanism of synthesis of adenosine (50 ) tetraphosphate (50 ) adenosine (AppppA) by aminoacyl-tRNA synthetases, Eur. J. Biochem. 126 (1982) 135e139. [5] P.C. Lee, B.R. Bochner, B.N. Ames, AppppA, heat-shock stress, and cell oxidation, Proc. Natl. Acad. Sci. U. S. A. 80 (1983) 7496e7500. [6] P.C. Lee, B.R. Bochner, B.N. Ames, Diadenosine 50 ,5”’-P1,P4-tetraphosphate and related adenylylated nucleotides in Salmonella typhimurium, J. Biol. Chem. 258 (1983) 6827e6834. [7] B.R. Bochner, P.C. Lee, S.W. Wilson, C.W. Cutler, B.N. Ames, AppppA and related adenylylated nucleotides are synthesized as a consequence of oxidation stress, Cell 37 (1984) 225e232. [8] T.M. Ismail, C.A. Hart, A.G. McLennan, Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells, J. Biol. Chem. 278 (2003) 32602e32607. [9] R.D. Monds, P.D. Newell, J.C. Wagner, J.A. Schwartzman, W. Lu, J.D. Rabinowitz, G.A. O’Toole, Di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens via modulation of c-di-GMPdependent pathways, J. Bacteriol. 192 (2010) 3011e3023.

[10] J. Charlier, R. Sanchez, Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product, Biochem. J. 248 (1987) 43e51. [11] G. Desogus, F. Todone, P. Brick, S. Onesti, Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction, Biochemistry 39 (2000) 8418e8425. [12] S. Onesti, A.D. Miller, P. Brick, The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli, Structure 3 (1995) 163e176. [13] 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. 227e240. [14] P. Plateau, S. Blanquet, Zinc-dependent synthesis of various dinucleoside 50 ,50 0 -P1,P3-Tri- or 50 ,50 0 - P1,P4-tetraphosphates by Escherichia coli lysyl-tRNA synthetase, Biochemistry 21 (1982) 5273e5279. [15] M. Wright, N. Boonyalai, J.A. Tanner, A.D. Hindley, A.D. Miller, The duality of LysU, a catalyst for both Ap4A and Ap3A formation, FEBS J. 273 (2006) 3534e3544. [16] L. Jelsbak, L. Søgaard-Andersen, Cell behavior and cell-cell communication during fruiting body morphogenesis in Myxococcus xanthus, J. Microbiol. Methods 55 (2003) 829e839. [17] D.E. Whitworth, Myxobacteria. Multicellularity and Differentiation, ASM Press, Washington, DC, 2008. [18] M. Oka, K. Takegawa, Y. Kimura, Enzymatic characterization of a class II lysyltRNA synthetase, LysS, from Myxococcus xanthus, Arch. Biochem. Biophys. 579 (2015) 33e39. [19] M. Sasaki, K. Takegawa, Y. Kimura, Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus, FEBS Lett. 588 (2014) 3395e3402. [20] C.W. Hanson, M. Dworkin, Intracellular and extracellular nucleotides and related compounds during the development of Myxococcus xanthus, J. Bacteriol. 118 (1984) 486e496. [21] M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248e254. [22] M.C. Reed, A. Lieb, H.F. Nijhout, The biological significance of substrate inhibition: a mechanism with diverse functions, BioEssays 32 (2010) 422e429. [23] V.V. Kupriyanov, J.A. Ferretti, R.S. Balaban, Muscle adenylate kinase catalyzes adenosine 50 -tetraphosphate synthesis from ATP and ADP, Biochim. Biophys. Acta 869 (1986) 107e111. [24] A. Guranowski, M.A. Günther Sillero, A. Sillero, Adenosine 50 -tetraphosphate and adenosine 50 -pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase, J. Bacteriol. 176 (1994) 2986e2990. [25] R. Fontes, M.A. Sillero, A. Sillero, Acyl coenzyme A synthetase from Pseudomonas fragi catalyzes the synthesis of adenosine 50 -polyphosphates and dinucleoside polyphosphates, J. Bacteriol. 180 (1998) 3152e3158. [26] T.T. Thach, T.T. Luong, S. Lee, D.K. Rhee, Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity, FEBS Open Bio 4 (2014) 672e682. [27] G.H.W. Haase, M. Brune, J. Reinstein, E.F. Pai, A. Pingoud, A. Wittinghofer, Adenylate kinases from thermosensitive Escherichia coli strains, J. Mol. Biol. 207 (1989) 151e162. [28] P. Plateau, J.F. Mayaux, S. Blanquet, Zinc(II)-dependent synthesis of diadenosine 50 ,5000 -P1, P4-tetraphosphate by E. coli and yeast phenylalanyl transfer ribonucleic acid synthetases, Biochemistry 20 (1981) 4654e4662. [29] O. Goerlich, R. Foeckler, E. Holler, Mechanism of synthesis of adenosine (50 ) tetraphosphate (50 ) adenosine (AppppA) by aminoacyl-tRNA synthetases, Eur. J. Biochem. 126 (1982) 135e142. [30] R.-T. Guo, Y.E. Chong, M. Guo, X.-L. Yang, Crystal structures and biochemical analyses suggest a unique mechanism and role for human glycyl-tRNA synthetase in Ap4A homeostasis, J. Biol. Chem. 284 (2009) 28968e28976. [31] H. Fraga, R. Fontes, Enzymatic synthesis of mono and dinucleoside polyphosphates, Biochim. Biophys. Acta 1810 (2011) 1195e1204. [32] X. Chen, N. Boonyalai, C. Lau, S. Thipayang, Y. Xu, M. Wright, A.D. Miller, Multiple catalytic activities of Escherichia coli lysyl-tRNA synthetase (LysU) are dissected by site-directed mutagenesis, FEBS J. 280 (2013) 102e114.