Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus

FEBS Letters 588 (2014) 3395–3402

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Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus Masashi Sasaki 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 11 June 2014 Revised 8 July 2014 Accepted 24 July 2014 Available online 6 August 2014 Edited by Miguel De la Rosa Keywords: ApaH-like phosphatase Diadenosine polyphosphate hydrolase ApnA Protein phosphatase Myxococcus xanthus

a b s t r a c t We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn2+, PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co2+ PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn2+ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co2+ was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Myxococcus xanthus is a gram-negative bacterium that lives in soil and preys on other bacteria [1]. Under starvation conditions, roughly 100,000 vegetative cells aggregate to form a structure called a fruiting body. Inside this structure, rod-shaped cells differentiate into spherical spores. Since M. xanthus demonstrates complex social behavior, signal transduction systems in this bacterium have been studied widely. This bacterium has nearly 100 eukaryotic-like serine (Ser)/threonine (Thr) and tyrosine (Tyr) protein kinase homologs [2]. Eukaryotic protein phosphatases catalyze dephosphorylation reactions of Ser- and Thr-phosphorylated, or Tyr-phosphorylated residues. Serine/threonine protein phosphatases are divided into two major families, the phosphoprotein phosphatases (PPPs), and the metal ion-dependent protein phosphatases (PPMs) [3]. The PPP family consists of the PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7 groups, while the PPM family includes the PP2C group. The protein tyrosine phosphatase (PTP) family consists of both tyrosinespecific phosphatases, as well as dual-specificity phosphatases. In

⇑ Corresponding author. Fax: +81 87 891 3021. E-mail address: [email protected] (Y. Kimura).

addition, several members of the haloacid dehalogenase (HAD) superfamily have been demonstrated to display serine or tyrosine phosphatase activity [4,5]. Alignment analysis studies have estimated that M. xanthus contains 25 PPPs, 4 PPMs, and 3 dualspecificity PTPs [2]. However, our studies have shown that some proteins of the M. xanthus PPP group are class III cAMP-phosphodiesterases, which also catalyze the hydrolysis of ATP, ADP, and AMP [6]. One group of bacterial PPPs shares homology with diadenosine tetraphosphate (Ap4A) hydrolases (ApaHs), and is termed ApaHlike phosphatases [7–9]. The Ap4A is composed of two adenosine moieties joined in a 5–50 linkage by a chain of four phosphates that is ubiquitous in prokaryotic and eukaryotic cells [10]. The concentration of Ap4A is increased after exposure of cells to various stresses such as heat, oxidative, nutritional, and DNA damage [11]. In bacteria, ApnA hydrolases can be classified into two distinct families, namely, Nudix (nucleoside diphosphate linked to X)asymmetrical hydrolases and ApaH-symmetrical hydrolases [12,13]. There is a shared sequence homology between ApaH and PPP. Recently, Jiang et al. identified a novel member of the metallophosphoesterase superfamily from Streptococcus pneumoniae, namely, SapH, which catalyzes the hydrolysis of ApnA, phosphodiesters and ATP [14]. Additionally, SapH shares a low sequence homology with ApaH.

http://dx.doi.org/10.1016/j.febslet.2014.07.031 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Alignment analysis suggested the possibility that M. xanthus contains two ApaH-like phosphatases. Since there are few reports to date on the enzymatic studies of ApaH-like phosphatases, we investigated the enzymatic characterization of two of these enzymes, MXAN_1509 and MXAN_7163, designated PrpA and ApaH, respectively. 2. Materials and methods 2.1. Expression and purification of PrpA and ApaH DNA fragments containing the prpA and apaH genes were amplified by PCR using primers (Supplementary Table S1). The products were inserted into a cold shock expression vector, pCold vector (Takara Bio). These plasmids were used for transformation of Escherichia coli. The cells were incubated in lysogeny 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. Proteins, produced in the soluble fraction from E. coli, with an N-terminal hexahistidine tag, were purified by affinity chromatography on a Talon column (Clontech).

2.2. Site-directed mutagenesis The apaH gene cloned into the expression vector pCold was used as a template for PCR. Site-directed mutations were generated with the PrimeSTAR mutagenesis basal kit (Takara Bio) using the primer pairs described in Supplementary Table S1. The PCR products were rather used for transformation of E. coli. After confirmation of the desired mutations by DNA sequencing, the mutant enzymes were expressed and purified by the methods described above. 2.3. Assay of phosphatase activity The phosphatase activity assays, performed using p-nitrophenyl phosphate (pNPP) as a substrate, were carried out in a total volume of 25 ll containing 50 mM Tris–HCl (pH 8.0), 50 mM pNPP, 1 mM MnCl2, and 10 pmol PrpA or 1 pmol ApaH at 50 °C. The reaction was stopped by the addition of 20 ll of 3 M NaOH. The release of p-nitrophenol (pNP) was determined spectrophotometrically at 415 nm in a 96-well plate reader. Control reactions containing no enzyme were used to measure the background level of pNP.

Fig. 1. Multiple sequence alignment of PrpA and ApaH from Myxococcus xanthus (Mx_PrpA and Mx_ApaH, respectively) with Bacillus subtilis PrpE (Bs_PrpE), Escherichia coli ApaH (Ec_ApaH) and Homo sapiens PP2B (Hs_PP2B). The four conserved motifs are enclosed in boxes. Identical amino acid residues are indicated white letters on a black background and homologous residues are indicated by white letters on a shaded background.

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A

Table 2 Effects of potential inhibitors on PrpA and ApaH activity with pNPP.

140

Inhibitor

Relative activity (%)

120 100 80 60 40 20 2

+ M n 2+ Fe 3 + + Co 2+

N

N 2 i + Co

on e Cu 2+ Fe 2 + M n 2+ Zn

2+

2+

M g 2+ Ca

0

B

Concentration

140

None Iodoacetamide Sodium orthovanadate N-ethylmaleimide Sodium fluoride Okadaic acid Iodoacetic acid Glycerol phosphate Trisodium phosphate EDTA Ammonium molybdate

1 mM 1 mM 1 mM 1 mM 1 lM 10 mM 20 mM 20 mM 5 mM 50 mM

Relative activity (%) PrpA

ApaH

100 ± 1.3 98.7 ± 5.2 90.9 ± 3.8 73.8 ± 6.2 95.4 ± 11.0 102.9 ± 1.3 58.0 ± 4.5 46.1 ± 2.4 24.8 ± 0.5 13.2 ± 0.4 96.3 ± 14.2

100 ± 2.1 90.4 ± 8.3 6.7 ± 3.9 62.6 ± 9.1 111.9 ± 5.4 88.3 ± 0.6 93.0 ± 11.9 33.4 ± 2.1 6.2 ± 1.2 2.8 ± 0.4 88.8 ± 3.7

The assays were performed in 50 mM Tris–HCl buffer (pH 8.0), 50 mM pNPP, and 1 mM MnCl2 at 50 °C for 30 min. Data are mean ± SEM values of three independent measurements.

Relative activity (%)

120 100 80 60 40 20 2+

M

n 2+ Fe 3 + + N 2 i +

2+

g 2+ Ca

M

2+

N 2 i + Co

2+

Fe 2 + M n 2+ Zn

Cu

N

on

e

0

Fig. 2. Effects of metal ions on PrpA (A) and ApaH (B) phosphatase activities for pnitrophenyl phosphate. Reactions were carried out with 50 mM pNPP in 50 mM Tris–HCl buffer, pH 8.0, and PrpA or ApaH in the presence of 1 mM metal ions at 50 °C. Activities are expressed relative to the activity measured in the presence of 1 mM MnCl2, set as 100%. Values are presented as mean ± SEM, determined from the average of three independent measurements.

Table 1 Kinetic data of PrpA and ApaH. Enzyme

Substrate

PrpA

pNPP tyrosine phosphopeptide Ap4A Ap5A ATP

ApaH

pNPP tyrosine phosphopeptide Ap4A Ap5A ATP

Kcat (S1)

Kcat/Km (S1/mM)

7.50 ± 0.69 0.21 ± 0.01

27.11 ± 1.03 0.017 ± 0.001

3.63 ± 0.28 0.085 ± 0.005

0.14 ± 0.06 0.19 ± 0.03 0.056 ± 0.029

0.055 ± 0.003 0.035 ± 0.003 0.003 ± 0.001

0.40 ± 0.03 0.18 ± 0.02 0.065 ± 0.049

14.29 ± 2.87 ND

244.6 ± 13.6 ND

17.45 ± 2.57 ND

0.86 ± 0.07 0.49 ± 0.01 1.25 ± 0.20

7.12 ± 0.25 24.01 ± 0.26 0.029 ± 0.003

8.29 ± 0.40 48.57 ± 1.34 0.023 ± 0.004

Km (mM)

addition of 50 ll of Biomol Green reagent, the absorbance of each well was read at 630 nm in a 96-well plate reader. The Km values were determined using the Hanes-Woolf linearization method [15]. Protein concentrations were measured using the Bradford method [16]. Tyrosine-phosphorylated bacterial tyrosine kinase BtkB was incubated with 57 pmol PrpA or 84 pmol ApaH in the presence of 1 mM MnCl2 for 0–90 min at 37 °C, to determine if the ApaH-like phosphatases dephosphorylated the Tyr-phosphorylated protein. The Tyr-phosphorylated BtkB was then detected by Western blotting with an antibody (PY-20) against the phosphorylated tyrosines. 2.5. Assay of ApnA hydrolase activity The ApnA hydrolase activities were measured in a total volume of 25 ll, made up as follows: 50 mM Tris–HCl, pH 8.0; 1 mM MnCl2 or 1 mM CoCl2; 1 mM Ap3A, Ap4A or Ap5A; and 13 pmol PrpA or 0.3 pmol ApaH. The mixtures were incubated at 50 °C for 10–30 min. The concentrations of the substrate and the reaction products in the above assays were detected at 260 nm with a reversed-phase HPLC system under the following conditions: an ODS-80Ts column (0.46  15.0 cm, Tosoh); a mobile phase, solvent A (5 mM tetrabutylammonium bromide in 20 mM potassium phosphate buffer, pH 3.5); and solvent B (solvent A in 60% acetonitrile) [6]. The gradient elution for solvent A, with a flow rate of 1 ml min1, was 95% (v/v) at 0 min, 60% at 15 min, and 0% at 18 min for analyses of Ap3A and Ap4A hydrolysis, or 95% at 0 min, 60% at 10 min, and 0% at 20 min for analysis of Ap5A hydrolysis. 3. Results

ND, not detected. The assays of PrpA were performed in the presence of 1 mM Mn2+. In ApaH, the assays of hydrolase against Ap4A and Ap5A were performed in the presence of 1 mM Co2+, and other assays were performed in the presence of 1 mM Mn2+. Data are mean ± SEM values of three independent measurements.

2.4. Assay of protein phosphatase activity The protein phosphatase activity assays, determined using phosphopeptides as substrates, were performed in 25 ll of an assay buffer, made up of the following components: 50 mM Tris–HCl (pH 8.0); 1 mM MnCl2; 38 pmol PrpA or 260 pmol ApaH; and 0.1 mM RRLIEDAEpYAARG, RKRpSRAE, or RRApTVA. After

3.1. Comparison of the amino acid sequences of PrpA and ApaH A blastP analysis of the eukaryotic protein phosphatases from the M. xanthus genome indicated that M. xanthus has two PPPs that share homology with ApaH. The open reading frames of MXAN_1509 (named prpA) and MXAN_7163 (named apaH) encode proteins of 235 and 221 amino acids, with calculated molecular weights of 26.7 and 24.2 kDa, respectively. The PPP superfamily contains conserved four-sequence motifs, GDXHG, GDXXDRG, GNHE and HGG, where X is any residue in the N-terminal subdomain of about 100 amino acids [8,17]. M. xanthus PrpA contains the three core signature motifs (GDXHG, GDXXDRG, and GNHE); however, M. xanthus ApaH possesses modified motifs 2 and 3, i.e., GDXXAKG and GNHD sequences,

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PrpA

50

Specific activity (nmol/min/mg)

Specific activity (nmol/min/mg)

A

40 30 20 10 0 1

B

2

5

30

0.8 0.6 0.4 0.2

3.2. Phosphatase activity of PrpA and ApaH

0

3

PrpA 0

respectively (Fig. 1). In motif 4, M. xanthus ApaH and PrpA contain a HGG or a HAG sequence, respectively. The deduced amino acid sequences of PrpA and ApaH showed that these enzymes had a 23% and a 33% identical sequence, respectively, with that of E. coli ApaH. Both PrpA and ApaH had a 23% identical sequence with that of human Ser/Thr-protein phosphatase 2B. On the other hand, Bacillus subtilis PrpE shared homology with both PPPs and ApaH, given that PrpA and ApaH had a 26% and 24% identical sequence, respectively, with PrpE [18].

ApaH

1.0

1

2

3

ApaH 90

5

30

90 (min) Phosphorylated BtkB

Fig. 3. Protein phosphatase activities of PrpA and ApaH. (A) Substrate specificities of PrpA and ApaH. Protein phosphatase activities were determined using the following substrates: serine phosphopeptide, RKRSpRAE (1); threonine phosphopeptide, RRATpVA (2); and tyrosine phosphopeptide, RRLIEDAEYpAARG (3). (B) In vitro dephosphorylation of Tyr-phosphorylated bacterial tyrosine kinase (BtkB) by PrpA and ApaH. Tyr-phosphorylated BtkB was incubated with PrpA or ApaH in the presence of 1 mM MnCl2 for 5, 30, and 90 min at 37 °C.

A

120

Relative activity (%)

100 80

3.3. Inhibitors of the phosphatase activities of PrpA and ApaH

60 40 20 0 Ap3A

Relative activity (%)

B

Firstly, phosphatase activities of purified, recombinant PrpA and ApaH were assayed with pNPP, commonly used as the substrate in the phosphatase assay in vitro, by measuring the release of pNP. Fig. 2 shows the effect of metal ions on the phosphatase activity of PrpA and ApaH. Phosphatase activity of PrpA was significantly activated by 1 mM Mn2+. Furthermore, there was a 2- to 3-fold increase in PrpA phosphatase activity toward pNPP in the presence of 1 mM Fe2+, Co2+, Ca2+, or Fe3+ compared with the result for the control (no metal ions added). Similarly, Mn2+ was the most effective ion in stimulating the phosphatase activity of ApaH. In addition, the activity of ApaH was increased 3- to 8-fold by the addition of 1 mM Ni2+, Co2+, Mg2+, Ca2+, or Fe3+. The optimum temperatures for PrpA and ApaH phosphatase activities against pNPP were identified at 50 and 55 °C, respectively (data not shown). The relative activities of PrpA and ApaH at 40 °C against those at 50–55 °C were about 40–45%. Both enzymes showed a broad optimal pH range [5–10]. The PrpA and ApaH were the most active at a pH of 8, using 0.1 M Tris–HCl, and they retained more than 80% of their activities within a pH range of 6–9, using 0.1 M maleic acid and Tris–HCl. The Km values of PrpA and ApaH for pNPP, at pH 8.0 and 50 °C, were 7.50 ± 0.69 mM and 14.29 ± 2.87 mM, respectively (Table 1). The Kcat value of ApaH for pNPP was about 9-fold higher than that of PrpA.

Ap4A

Ap5A

120 100 80 60

As shown in Table 2, both the phosphatase activities of PrpA and ApaH were inhibited by a competitive inhibitor, trisodium phosphate (Na3PO4), and a divalent ion chelator, EDTA. The IC50 values of PrpA and ApaH for trisodium phosphate were 8.1 ± 0.7 mM and 3.2 ± 0.3 mM, respectively. Sodium orthovanadate (Na3VO4) is a commonly used general inhibitor of protein phosphotyrosyl phosphatases, alkaline phosphatases, and ATPases [19]. Sodium orthovanadate, at 1 mM, did not significantly inhibit the phosphatase activity of PrpA, whereas it inhibited the phosphatase activity of ApaH by 93%. The IC50 value of ApaH for sodium orthovanadate was 82.7 ± 4.6 lM. A concentration of 1 lM okadaic acid, which is an inhibitor specific for the PPP family, did not have any significant inhibitory effect on PrpA or ApaH activity. 3.4. Protein phosphatase activity toward phosphopeptides and tyrosine-phosphorylated protein

40 20 0 Ap3A

Ap4A

Ap5A

Fig. 4. Diadenosine polyphosphate (ApnA) hydrolase activities of PrpA (A) and ApaH (B). The assays were carried out using 1 mM ApnA in the presence of 1 mM Mn2+ (blank bar) or 1 mM Co2+ (solid bar). Activity in the presence of 1 mM Ap4A, 1 mM Mn2+ and PrpA (A) or 1 mM Ap5A, 1 mM Co2+ and ApaH (B) was taken as 100%. Values are presented as mean ± SEM, determined from the average of three independent measurements.

The dephosphorylation activities of PrpA and ApaH toward three phosphorylated peptides are shown in Fig. 3A. Both PrpA and ApaH demonstrated the highest dephosphorylation activity toward the Tyr-phosphorylated containing peptide, and a weak dephosphorylation activity toward Ser- and Thr-phosphorylated containing peptides. However, the Kcat of ApaH activity toward the Tyr-phosphorylated peptide was about 40-fold lower than that of PrpA (Table 1). The Km value of PrpA, with the Tyr-phosphorylated peptide as a substrate, was 0.21 ± 0.01 mM. The Km value of

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Absorbance at 260 nm

A

Co2+

Mn2+ 1.0

0.5

0

Ap4A

ADP AMP

4

ATP

8 12 16 Retention time (min)

20

4

8 12 16 Retention time (min)

Mn2+

B 1.0

Absorbance at 260 nm

Ap4A

20

Co2+ Ap5A

Ap5A AMP ADP

Ap4

0.5

ATP

0

4

8 12 16 20 Retention time (min)

24

4

8 12 16 20 Retention time (min)

24

Fig. 5. Reversed phase liquid chromatography of hydrolysis products of Ap4A (A) and Ap5A (B) by PrpA in the presence of 1 mM Mn2+ or 1 mM Co2+. 1 mM Ap4A (A) or 1 mM Ap5A (B) was incubated for 30 min with PrpA in the presence of 1 mM Mn2+ or 1 mM Co2+. The Ap4A and Ap5A hydrolysis products were analyzed by HPLC analysis using two different gradient programs as described in Section 2.

ApaH for the peptide could not be determined because the activity of ApaH was too low. The bacterial tyrosine kinase, BtkB, of M. xanthus is autophosphorylated at the tyrosine residues in the C-terminal tyrosine cluster [20]. Incubation of the phosphorylated BtkB with either of the ApaH-like phosphatases, in the presence of 1 mM Mn2+, showed that PrpA but not ApaH dephosphorylated BtkB (Fig. 3B). 3.5. The ApnA hydrolase activity of PrpA and ApaH The ApnA hydrolase activities of recombinant PrpA and ApaH were assayed with Ap3A, Ap4A, or Ap5A as substrates, and the concentrations of the ApnA hydrolysis products were estimated by a reversed-phase HPLC system. The hydrolase activity of PrpA had broad substrate specificity, and exhibited the maximal ApnA activity in the presence of Mn2+ (Fig. 4). The activity of PrpA against Ap4A, with 1 mM Mn2+, was about 2-fold higher than that with 1 mM Co2+. In the presence of Mn2+, PrpA mainly hydrolyzed Ap4A asymmetrically into AMP and ATP, and Ap5A into AMP and Ap4 (Fig. 5). Interestingly, PrpA catalyzed a symmetrical cleavage of Ap4A that resulted in the production of two molecules of ADP in the presence of Co2+. In addition, PrpA, incubated with Co2+, cleaved Ap5A into ADP and ATP. The Km values for the hydrolase activity of PrpA toward Ap4A and Ap5A were several-fold lower than those of ApaH (Table 1). With reference to the ApnA hydrolase activity of ApaH, the most efficient activity occurred in the presence of Co2+. The ApnA hydrolase activities against Ap4A and Ap5A were about 2- to 3-fold higher in the presence of 1 mM Co2+ than in the presence of 1 mM Mn2+ (Fig. 4). The ApaH degraded Ap5A more readily than Ap3A or Ap4A. In the presence of Co2+ or Mn2+, ApaH degraded Ap4A into two molecules of ADP and Ap5A into ADP and ATP. The Kcat values of the ApaH activity toward Ap4A and Ap5A were 127- and 685-fold higher than those of PrpA, respectively (Table 1).

3.6. The ATPase activity of PrpA and ApaH B. subtilis PrpE has an ATPase activity, but E. coli ApaH shows no catalytic activity toward ATP [13,18,21]. Both M. xanthus PrpA and ApaH hydrolyzed ATP. The manganese cation (Mn2+) was the most effective metal cation for the activity of both enzymes (data not shown). The activity of ApaH against ATP, with 1 mM Mn2+, was about 2.5-fold higher than that with 1 mM Co2+. In addition, since PrpA had a low Km value for ATP (Table 1), the ATP produced by the hydrolytic action of PrpA on Ap4A may be hydrolyzed to ADP. The Kcat values of PrpA and ApaH for ATP hydrolysis were 5% and 0.4% of the value for Ap4A hydrolysis, respectively (Table 1). On the other hand, neither PrpA nor ApaH had cAMP phophodiesterase activity (data not shown). 3.7. Mutational analysis of ApaH PrpA and ApaH have similar amino acid sequences (37% identical sequence); however, comparison of the enzymatic activity of PrpA and ApaH revealed a difference in their substrate specificity. To identify the amino acids that confer the substrate specificity, ApaH mutants were constructed by site-directed mutagenesis. In motif 2, PPPs contain a conserved motif DRG, whereas M. xanthus ApaH possesses an AKG sequence at the homologous position. An ApaH mutant, with Ala39 replaced by aspartate, showed both a reduced phosphatase activity against pNPP and hydrolase activity against Ap4A and Ap5A, whereas this mutant did not display a reduced hydrolase activity against ATP (Fig. 6). A further decrease in hydrolase activity was observed in a double mutant (A39D/ K40R), which had a reduced ATP hydrolase activity. However, these mutants showed an almost 4- to 5-fold increase in protein phosphatase activity against the Tyr-phosphorylated peptide. The specific activity of the ApaH mutant, with a DRG sequence in motif 2, against the Tyr-phosphorylated peptide, was 23% of that of PrpA (data not shown).

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A

B

Fig. 6. (A) Comparison of motifs 2 and 3 of ApaH-like phosphatases. The phosphatases compared were as follows: Myxococcus xanthus ApaH (Mx_ApaH); Escherichia coli ApaH (Es_ApaH); Salmonella enterica ApaH (Se_ApaH); M. xanthus PrpA (Mx_PrpA); and Bacillus subtilis PrpE (Bs_PrpE). Conserved amino acid residues of phosphoprotein phosphatases, in motifs 2 and 3, are shown in bold type. The locations of A39, K40, D64, and H66 residues of mutated ApaH are indicated by triangles. (B) pNPP phosphatase, ApnA hydrolase, ATPase, and protein phosphatase activities of wild-type ApaH and its variant mutants. The substrates used were 50 mM p-nitrophenyl phosphate (pNPP), 1 mM Ap4A, 1 mM Ap5A, 1 mM ATP, and 0.1 mM tyrosine (Tyr)-phosphorylated peptide.

The GNHE motif is conserved in PPPs, whereas ApaH contains a GNHD sequence in motif 3. The replacement of aspartate by glutamate in ApaH, led to greatly reduced phosphatase and hydrolase activities; however, the mutant did not reduce the protein phosphatase activity. Furthermore, PrpA and PrpE contain a lysine residue, two residues downstream from the glutamate residue in motif 3, whereas a histidine residue is present in ApaH at the same position. A double mutant, D64E/H66K, exhibited a significant increase in all activities compared with those of the single mutant,

D64E. However, the activities of the D64E/H66K mutant, with the exception of Tyr-phosphorylated peptide phosphatase activity, were lower than those of the wild-type enzyme (ApaH). 4. Discussion M. xanthus has two ApaH-like protein phosphatases, PrpA and ApaH. Our results showed that PrpA had a high protein phosphatase activity against the Tyr-phosphorylated peptide and the

M. Sasaki et al. / FEBS Letters 588 (2014) 3395–3402

Tyr-phosphorylated BtkB protein. In addition, PrpA had ApnA and ATP hydrolase activities. This enzymatic characterization was similar to that of B. subtilis PrpE [18]. B. subtilis PrpE also shares homology with PPP and ApaH, and has protein phosphatase, Ap4A hydrolase, and ATPase activities. M. xanthus PrpA required Mn2+ for these maximum activities; however, the activities of PrpE were dependent on Ni2+, and were absent in the presence of Mn2+. The activity of PrpA catalyzed the asymmetrical cleavage of Ap4A or Ap5A in the presence of Mn2+, whereas this enzyme mainly symmetrically hydrolyzed Ap4A or Ap5A in the presence of Co2+. To date, there have been no studies published reporting an alteration in the cleavage pattern of ApnA by the addition of metal ions. Furthermore, in the presence of Mn2+, general ApnA hydrolases, ApaH, and Nudix hydrolases degraded Ap5A to ADP and ATP [22,23], but PrpA hydrolyzed Ap5A to AMP and Ap4. This cleavage pattern of Ap5A has also been identified in an ATP/ApnA and phosphodiesterase enzyme, SapH, a Saccharomyces cerevisiae Ap6A hydrolase and a novel mitochondrial Arabidopsis thaliana Nudix hydrolase, AtNUDT13 [14,24,25]. We have shown that PrpA may catalyze the cleavage of Ap4A and Ap5A, with AMP and ADP as the common products in the presence of Mn2+ or Co2+, respectively. However, we propose that PrpA may function mainly as a protein phosphatase, since PrpA had a considerably lower Kcat value for ApnA when compared with ApaH. We have previously reported that M. xanthus has a tyrosine phosphatase, PhpA, which belongs to the polymerase and histidinol phosphatase (PHP) families [26]. The Kcat value of PrpA for Tyr-phosphorylated peptide was 4-fold lower than that of PhpA. In this study, we demonstrated that ApaH had a phosphatase activity, an ATPase activity, and a strong ApnA hydrolase activity. In E. coli Co2+ and Mn2+ had the strongest stimulatory effect on ApaH activity against Ap4A and Ap5A, and Ap3A, respectively; however, M. xanthus ApaH required Co2+ for maximal hydrolase activity against these three ApnAs. The Kcat values of ApaH, with Ap4A and Ap5A as substrates, were similar to those found in Salmonella enterica ApaH [27]. PPPs contain a conserved DRG sequence in motif 2, and the arginine residue in the DRG sequence interacts with the phosphate group [17]. In addition, mutation of DRG to NRG in a Ser/Thr protein phosphatase resulted in vastly reduced phosphatase activity toward pNPP and phosphorylated proteins [28,29]. M. xanthus and E. coli ApaHs contain AKG and ARG sequences, respectively, instead of a DRG sequence, and M. xanthus ApaH had a high phosphatase activity toward pNPP. Mutation of AKG to DKG in M. xanthus ApaH decreased the phosphatase activity against pNPP and the hydrolase activity against ApnA, and mutation of AKG to DRG further decreased the hydrolase activity against ATP. In contrast, both mutations led to an increase in the protein phosphatase activity. We suggest that the alanine and lysine residues in the AKG sequence of ApaH may be important for the pNPP phosphatase and ApnA hydrolase activities and the ATP hydrolase activity, respectively, and that the aspartate residue in motif 2 of the ApaH mutant may be required for the protein phosphatase activity. Acknowledgment This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (25440087). 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.febslet.2014.07. 031.

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