Journal of Bioscience and Bioengineering VOL. 119 No. 2, 123e130, 2015 www.elsevier.com/locate/jbiosc
Characterization of glycerophosphoethanolamine ethanolaminephosphodiesterase from Streptomyces sanglieri Shingo Mineta,1 Kazutaka Murayama,2 and Daisuke Sugimori1, * Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan1 and Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo, Aoba, Sendai 980-8575, Japan2 Received 18 June 2014; accepted 9 July 2014 Available online 15 August 2014
Streptomyces sanglieri extracellularly produces a glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP). The gene encoding the enzyme was found to consist of a 2124-bp ORF, which codes for an N-terminal 48 residue signal peptide required for secretion and a 660 amino acid mature protein with a calculated molecular mass of 72,918 Da. The maximum activity for sn-glycero-3-phosphoethanolamine (GPE) was found at pH 8.4 and 65 C in the presence of 0.1% (w/v) Triton X-100. The enzyme was activated in the presence of 2 mM EDTA; however, Zn2D remarkably inhibited activity. During the hydrolysis of GPE at 65 C and pH 8.4, the apparent Vmax, turnover number (kcat) and Km were determined to be 0.430 mmol minL1 mg-proteinL1, 522 sL1 and 0.785 mM, respectively. The enzyme exhibited specificity toward GPE and hydrolyzed ethanolamine-type substrates such as 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, lysophosphatidylethanolamine and ethanolamine lysoplasmalogen, but not 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine. Moreover, the enzyme showed no activity toward other phospholipids, such as glycerophospholipids and plasmalogens, and sn-glycero-3-phosphodiesters except for sn-glycero-3-phosphoglycerol, suggesting that GPE-EP is not a phospholipase C (PLC). However, the amino acid sequence of GPE-EP shows 86% identity to that of PLC from Streptomyces sp. SirexAA-E (UniProt accession no. G2NFN1). Recombinant GPE-EP was functionally expressed in Escherichia coli using pET-24a(D). GPE hydrolysis by GPE-EP may represent a new pathway for phosphatidylethanolamine metabolism. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycerophosphoethanolamine ethanolaminephosphodiesterase; Purification; Characterization; Molecular cloning; Expression; Streptomyces sanglieri]
sn-Glycero-3-phosphodiesters (GPX), i.e., sn-glycero-3-phosphocholine (GPC), sn-glycero-3-phosphoethanolamine (GPE), snglycero-3-phosphoserine (GPS), sn-glycero-3-phosphate (GPA), snglycero-3-phosphoinositol (GPI) and sn-glycero-3-phosphoglycerol (GPG), are an important intermediate in glycerophospholipid metabolism. Nevertheless, there is only few limited information on the metabolic pathway and key enzyme. We recently reported a new glycerophosphocholine cholinephosphodiesterase (GPC-CP14; EC 3.1.4.38) from Streptomyces sanglieri strain A14 (1). Furthermore, we detected GPE-specific enzyme, i.e., glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP), from strain A14. Both two enzymes are very similar to phospholipase C (PLC; EC 3.1.4.3) that cleaves PE into diacylglycerol and phosphoethanolamine. In contrast, GPE-EP cleaves GPE into glycerol and phosphoethanolamine (Fig. 1). The difference in the substrate recognition mechanism between GPE-EP, GPC-CP14, and PLC remains unknown. The identification of GPE-EP may help to realize a new pathway for phosphatidylethanolamine metabolism. Moreover, the characterization of GPE-EP may allow prediction of the
* Corresponding author. Tel./fax: þ81 24 548 8206. E-mail address: [email protected] (D. Sugimori).
physiological role of PLC as well as those phosphodiesterases in actinomycete. Here, we report the purification, characterization, molecular cloning, functional expression, structure modeling, and the catalytic mechanism prediction of a novel GPE-EP from S. sanglieri strain A14.
MATERIALS AND METHODS Materials Tryptic soy Broth (TSB), Bacto Tryptone, Bacto peptone, Bacto Casamino acid and Malt extract were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Yeast extract and alkaline phosphatase from calf intestine (CIAP) were purchased from Oriental yeast Co., Ltd. (Tokyo, Japan). Soybean lecithin and polyoxyethylene laurylether (Brij 35) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). BIOMOL Green Reagent and L-a-phosphatidylinositol (PI) were purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). 1,2-Dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-dipalmitoyl-sn-glycero3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DHPE), 1,2dibutyryl-sn-glycero-3-phosphocholine (DBPC), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), L-a-phosphatidylserine (PS), L-a-lysophosphatidylcholine (LPC), L-a-lysophosphatidylserine (LPS), L-a-lysophosphatidylglycerol (LPG), 1stearoyl-2-hydroxy-sn-glycerol-3-phosphate (LPA), L-a-lysophosphatidylinositol (LPI), 1-O-10 -(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine (LPlsPC; choline lysoplasmalogen), 1-O-10 -(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPlsPE; ethanolamine lysoplasmalogen), 1-(1Z-octadecenyl)-2arachidonoyl-sn-glycero-3-phosphocholine (PlsPC; choline plasmalogen) and 1-
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.07.005
124
MINETA ET AL.
FIG. 1. Hydrolytic reaction of GPE by GPE-EP.
(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE; ethanolamine plasmalogen) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). L-a-phosphatidylglycerol (PG) and L-a-lysophosphatidylethanolamine (LPE) were purchased from Doosan Serdary Research Laboratories (Toronto, ON, Canada). GPC was purchased from Bachem (Torrance, CA, USA). HiTrap DEAE FF, HiTrap Q HP, HiTrap Phenyl HP, HisTrap HP, Resource PHE and Mono Q columns were purchased from GE Healthcare Japan (Tokyo, Japan). Toyopearl PPG-600M and Butyl-650M were purchased from Tosoh (Tokyo, Japan). All other chemicals were of the highest or analytical grade. Glycero-3-phosphodiesters Recombinant phospholipase A1 (SaPLA1) from Streptomyces albidoflavus strain NA297 was extracellularly produced by Streptomyces lividans and was used for the synthesis of GPX after ammonium sulfate purification (2). GPX such as GPE, GPS, GPI, GPA and GPG, except GPC, were prepared as indicated below. The corresponding lysophospholipids (40 mM) were completely hydrolyzed in 20 mM TriseHCl buffer (pH 7.2), 0.5% (w/v) Triton X-100 and 1.15 U of the recombinant SaPLA1 (115 U/mL, 217 U/mg) at 37 C overnight. Complete conversion of lysophospholipid into the corresponding GPX was confirmed by the concentration of free fatty acids released by SaPLA1. The concentration of free fatty acids was determined with a NEFA C Kit (Wako Pure Chemical Industries Ltd., Osaka, Japan) using oleic acid as the standard and according to the instructions of the manufacturer. The reaction mixture was boiled at 100 C for 5 min and extracted with chloroformemethanol (2:1, v/v), followed by centrifugation (18,300 g for 5 min). The upper layer was collected and the solvent was removed by evaporation for 5 min using a vacuum evaporator. The resulting aqueous solution was used for the investigation of substrate specificity. Bacterial strains and culture conditions Approximately 1500 strains were isolated from soil samples from Fukushima, Japan, using a humic acid-vitamin agar plate culture (3). Strain A14 exhibiting GPE-EP activity as well as GPC-CP activity was selected (1). Strain A14 colonies from ISP2 (10 g malt extract, 4 g yeast extract and 4 g glucose per liter, pH 7.2) agar plates were scraped and inoculated into a test tube (18 mm, 180 mm) containing 5 mL seed medium of ISP2. These cultures were incubated with shaking (160 strokes per min) at 28 C. After 48 h cultivation, a 1% (v/v) inoculum was transferred into a 500-mL flask containing 50 mL of the fermentation medium of ISP2 supplemented with 0.41 mM Brij 35 and cultivated with shaking (180 rpm) at 28 C for 72 h. Escherichia coli HST08 premium competent cells (Takara Bio Inc., Shiga, Japan) were used as a host for molecular cloning. The pGEM-T Easy Vector (Promega) and the pMD20-T vector (Takara Bio) were used as cloning vectors. Recombinant E. coli cells were cultured on LuriaeBertani (LB) agar plates at 37 C; if necessary, ampicillin, isopropyl-b-Dthiogalactopyranoside and X-Gal were used as supplements in the agar. A pUC702 carrying the promoter, signal peptide sequence and the terminator region of phospholipase D (PLD) from Streptoverticillium cinnamoneum (4) and pET24a(þ) (Merck KGaA, Darmstadt, Germany) were used as expression vectors. S. lividans 1326 (NBRC15675), which was obtained from NITE Biological Resource Center (Chiba, Japan), and SHuffle T7 Express Competent E. coli (New England Biolabs Japan, Tokyo) were used as a host for the expression of proteins. Recombinant S. lividans cells were cultured in 3% (w/v) TSB containing 5 mg/mL thiostrepton at 28 C. Wild-type enzyme purification All procedures were performed at 4 C. The culture supernatant was obtained by centrifugation (18,800 g for 20 min) after 72 h of culturing. The supernatant was placed in a saturated ammonium sulfate solution ((NH4)2SO4 mass fractionation ¼ 80%) and was centrifuged at 18,800 g for 30 min. The precipitate was suspended in 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0). The enzyme sample was loaded onto a PPG-600M column (2.5 3.5 cm) equilibrated with the same buffer. The column was washed with 3 column volumes (CV) of the same buffer at a flow rate of 2 cm/min, and the protein was eluted with a linear gradient (10 CV) of 1e0 M (NH4)2SO4 in the same buffer at 6 mL/min (1.22 cm/min). The active fractions were pooled and then replaced by 20 mM TriseHCl buffer (pH 9.0) using Vivaspin 20e30,000 MWCO (Vivaspin 20e30 k; GE Healthcare Japan) followed by loading the sample onto a HiTrap Q HP column (5 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer at a flow rate of 5 mL/min, and the protein was eluted with a linear gradient (20 CV) of 0e1 M NaCl in the same buffer at 4 mL/min (2 cm/min). The active fractions were pooled and then replaced by 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20e30 k concentrators followed by application to a HiTrap Phenyl HP column (5 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer at a flow rate of 5 mL/min, and the protein was eluted with a linear gradient (16 CV) of 1 to 0 M (NH4)2SO4 in the same buffer and 8 CV of 20 mM TriseHCl buffer
J. BIOSCI. BIOENG., (pH 8.0) at 4 mL/min (2 cm/min). The active fractions were pooled and then replaced by 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20e30 k followed by application to a Resource PHE column (1 mL) equilibrated with the same buffer. The sample was loaded at a flow rate of 1 mL/min. The flow-through fraction was pooled. The buffer was exchanged with 20 mM TriseHCl buffer (pH 9.0) by using the same method mentioned above. The enzyme solution was applied to a Mono Q column (1 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer at a flow rate of 0.5 mL/min (2.55 cm/min) and the protein was eluted with a linear gradient (80 CV) of 0e1 M NaCl at the same flow rate. Fractions exhibiting high specific activity were pooled and used for further investigation. Enzyme activity assays For characterizing the GPE-EP activity, a standard assay mixture: 10% (v/v) enzyme sample solution, 50 mM TriseHCl buffer (pH 8.4), 4 mM GPE and 0.1% (w/v) Triton X-100 was incubated at 65 C for 5 min. The reaction was stopped by incubation at 100 C for 5 min. The sample was subsequently centrifuged at 21,600 g for 5 min. Five microliter of the supernatant was subsequently added to 95 ml of 50 mM glycineeNaOH buffer (pH 9.6) containing 1 mM MgCl2, 0.1 mM ZnCl2 and 1.6 U of CIAP followed by incubation at 56 C for 10 min. Inorganic phosphate released by the dephosphorylation of phosphoethanolamine was quantified with BIOMOL Green Reagent according to the manufacturer’s instructions (5). The rates of soluble phosphate release from the enzyme reaction mixtures were calculated, and one unit (U) of enzyme activity was defined as the amount of enzyme that hydrolyzed 1 mmol of substrate per minute. Since GPE used as a substrate was prepared by hydrolysis of 40 mM LPE containing 0.5% (w/v) Triton X-100 using SaPLA1, at most 0.05% (w/v) Triton X-100 was contaminated in the GPE-EP reaction mixture. In addition to, substrate specificity of phospholipids was investigated using mixed micelle substrates with 0.1% (w/v) Triton X-100. Furthermore, the effect of Triton X-100 concentration on the GPE-EP activity was investigated. GPE was prepared by hydrolysis of 40 mM LPE without Triton X-100 using SaPLA1. The enzyme activity was assayed under standard assay conditions with the same assay buffer containing 4 mM GPE in the absence or presence of Triton X-100. The highest activity was found in the presence of 0.1% (w/ v) Triton X-100 (Fig. S1). With considering these conditions, the standard assay mixture containing 0.1% (w/v) Triton X-100 was determined. Substrate specificity The substrate specificity of the purified GPE-EP was assessed by assaying enzyme activity, as described above, using alternative GPX substrates or phospholipid substrates such as diacylglycerophospholipids, lysophospholipids and plasmalogens (4 mM final concentration) in the assay. Effect of pH, temperature, and chemicals on enzyme activity Each buffer (sodium acetate, BisTriseHCl, TriseHCl, and glycineeNaOH) was used to identify the pH that led to optimum GPE-EP activity, and to determine the pH stability of the purified enzyme. The optimum pH was examined by incubation at 37 C for 5 min with 4 mM GPE in 50 mM of each buffer. The pH stability was assayed by incubating the purified enzyme at 4 C for 3 h in 50 mM of each buffer solution. The remaining activity was assayed under the standard assay conditions. The GPE-EP activity was determined at each temperature under the standard assay conditions. Thermal stability was determined by incubating the purified enzyme in 50 mM TriseHCl (pH 8.4) at each temperature for 30 min, and then the residual activity was measured by incubation (65 C, 5 min) under the assay mixture: 10% (v/v) enzyme sample solution, 50 mM TriseHCl buffer (pH 8.4), 4 mM GPE and 0.1% (w/v) Triton X-100. The effect of chemicals such as metal ions and SDS on the enzyme activity was investigated. The purified enzyme was assayed under standard assay conditions with the same assay buffer containing 2 mM of each chemical or inhibitor. Inhibitors assessed were EDTA, 2-mercaptoethanol, dithiothreitol (DTT), iodoacetamide (IAA) and phenylmethanesulfonyl fluoride (PMSF). Additionally, for the EDTA inhibition experiment, the purified enzyme (w1 mM) was assayed under standard assay conditions following pre-incubation at 37 C for 30 min with 50 mM TriseHCl (pH 8.4) containing 10 mM EDTA. PMSF was dissolved in dimethyl sulfoxide (DMSO) and added to the enzyme sample. In the case of IAA, the purified enzyme samples (4.33 mg/mL, 59.4 nM) were incubated at 37 C for 60 min in the same buffer containing 2 mM IAA before use in enzyme assays. Protein analysis The protein concentration was determined with the Pierce BCA protein assay kit (Takara Bio) and BSA as the standard. Protein samples were analyzed by 12% SDS-PAGE according to Laemmli (6). Proteins on the SDS-PAGE gel were stained with Coomassie brilliant blue R-250 (CBB) or silver staining (Silver stain KANTO III; Kanto Chemical Co., Inc., Tokyo, Japan). The molecular mass of the purified enzyme was estimated by gel filtration. Gel filtration was performed using a Superdex 200 10/300 GL column (1.0 30 cm) at a flow rate of 0.5 mL/ min (0.64 cm/min) with 20 mM TriseHCl (pH 8.0) containing 0.15 M NaCl. The column was calibrated with a gel filtration calibration kit (GE Healthcare) before and after the enzyme was subjected to chromatography. Peptide sequencing The purified protein was resolved by SDS-PAGE and then electroblotted onto a PVDF membrane (Immobilon-PSQ transfer membrane, Millipore Co., Billerica, MA, USA). The PVDF membrane was stained with CBB, and the transferred 70-kDa band was excised and subjected to N-terminal amino acid sequence analysis (Procise 494 HT Protein Sequencing System; Applied Biosystems, Foster City, CA, USA). For an internal terminal amino-acid sequencing, an SDS-PAGE gel was stained using CBB, the 70-kDa band was excised and then decolorized with 30% acetonitrile containing 25 mM (NH4)2HCO3. The in-gel digestion was performed by the method described by Shevchenko et al. (7). Briefly, the excised 70-kDa band
VOL. 119, 2015
GLYCEROPHOSPHOETHANOLAMINE ETHANOLAMINEPHOSPHODIESTERASE
was digested with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation, Madison, WI, USA) for 45 h at 4 C. The sample (1 ml) of extracted peptides were separated using a nanoAcquity UPLC system (Waters Corporation, Milford, MA, USA) equipped with a nano ACQUITY UPLC BEH130 C18 column (Waters, 75 mm 150 mm, 1.7 mm) and were analyzed using a Xevo QTOF MS, according to a previously reported method (2). De novo sequencing was performed with the ProteinLynx Global SERVER, version 2.3 (Waters). Steady-state kinetics For hydrolysis of GPE, the initial velocity (v) of the enzymatic reaction was determined at each concentration of the substrate under standard assay conditions. The concentration of GPE ([GPE]) was calculated using a molecular weight of 214.15 Da. The purified enzyme concentration in the reaction mixture was constant at 0.336 mg mL1 (4.61 nM). The corresponding [GPE]/v vs. [GPE] plot was treated according to a MichaeliseMenten equation. Kinetic constants were determined by extrapolation using the HaneseWoolf plot by linear regression (KaleidaGraph, Synergy Software, Reading, PA, USA). The Km and Vmax were determined from the x-intercept and slope of the regression line, respectively. The turnover number (kcat) was calculated using a molecular weight of 72,918 for a monomeric protein with a single catalytic site. From the sequence inforHomology modeling of GPE-EP and GPC-CP14 mation, GPE-EP and GPC-CP14 have phosphoesterase family domain and exhibited similarity to structure of acid phosphatase from Francisella tularensis (AcpA; Protein Data Bank code: 2D1G). The structural features of AcpA have been studied (8). Based on a template (AcpA, 2D1G), the homology models of GPE-EP and GPC-CP14 were created using an HHPRED search (http://toolkit.tuebingen.mpg.de/hhpred) (9) and Modeller 9.11 (http://toolkit.tuebingen.mpg.de/modeller) (10). The N-terminal region of the GPE-EP amino acid sequence from 49D to 460P (GPE-EP: 49e460) and that of the GPC-CP14 amino acid sequence from 39G to 471D (GPC-CP14: 39e471) were used to create the models. VERIFY3D (http://nihserver.mbi.ucla.edu/Verify_ 3D/) (11) was used to assess the quality of the predicted models. Cloning of GPE-EP gene Chromosomal DNA of S. sanglieri was purified according to Kieser et al. (12). Oligonucleotides were synthesized based on the Nterminal and internal amino acid sequences of the enzyme for use in PCR with sense primer S1 (50 -gacggcctsggcgcsatcaagcac-30 ) and antisense primer A1-4 (50 scgccagtcsswgatgttsggctc-30 ). The PCR reaction mixture (25 mL) contained: 1 KOD FX Neo buffer, 7.5 pmol of each primer, 10 nmol of dNTPs, 0.5 U of KOD FX Neo DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) and w50 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 98 C for 10 s and 68 C for 45 s. The obtained PCR fragment was purified and cloned into pGEM-T Easy vector and the resulting recombinant plasmid was called pGPE. Sequencing of the partial GPE-EP gene on pGPE was performed with the BigDye Terminator cycle sequencing kit and analyzed in an ABI Prism 3100 genetic analyzer (Life Technologies Corporation, Carlsbad, CA, USA). To reveal the nucleotide sequence of the 50 -upstream and 30 downstream region of the gene, inverse PCR was carried out using IV-S primer (50 -gctacgtctgctccgaggtcttcgac-30 ) and IV-AS primer (50 -gtactggaggtccttcttctgcg tctcg-30 ) designed based on the partial sequence of GPE-EP gene of pGPE. The genomic DNA (1 mg) was digested with Sma I and self-ligated. The inverse PCR amplification was completed with the reaction mixture (25 ml) containing 1 KOD FX Neo buffer, 7.5 pmol of each primer, 10 nmol of dNTPs, 0.5 U of KOD FX Neo DNA polymerase and w25 ng (above the 0.4-ml ligation mixture) of the Sma I-digested and self-ligated DNA. The PCR program was 94 C for 2 min, followed by 30 cycles of 98 C for 10 s and 69 C for 160 s. The obtained w2.5 kbp DNA fragment was cloned into the pGEM-T Easy vector and the resulting recombinant plasmid was called pIVgpe. To clone the complete GPE-EP gene from the chromosomal DNA of strain A14, two cloning primers were used: the sense primer GPE-NS (50 -gaggctcaacttgaccacggacatttcacg-30 ) and the antisense primer GPE-CA (50 -cagaaaccgaactcccgtgcgaaggaac-30 ) designed based on the sequence of the GPE-EP gene revealed by the inverse PCR, as described above. The genomic PCR amplification was carried out as follows. The PCR reaction mixture (20 mL) contained: 1 PCR buffer, 6 pmol of each primer, 4 nmol of dNTPs, 24 nmol MgSO4, 3% (vol/vol) of DMSO, 0.4 U of KOD Plus DNA polymerase (Toyobo) and w100 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 2 min. The resultant DNA fragment was cloned into the pGEM-T Easy vector and sequenced. Nucleotide and peptide sequence accession number The nucleotide sequences of the 16S rDNA of S. sanglieri strain A14 and the GPE-EP gene, designated gpe-ep, were deposited in the DDBJ/GenBank/EMBL database under the accession numbers AB735535 and AB771750, respectively. Expression and purification of recombinant GPE-EP S. lividans 1326 (NBRC15675), possessing no GPE-EP activity, was used as a host for GPE-EP expression. Two recombinant plasmid vectors, pUC702/pld-sig-gpeep and pUC702/ gpeep-sig, were constructed for extracellular production of active GPE-EP as follows: the recombinant vector pUC702/pld-sig-gpeep used the signal peptide sequence (pld-sig) of phospholipase D (PLD) from S. cinnamoneum, while pUC702/gpeep-sig, was used the signal peptide sequence (gpeep-sig) of GPE-EP itself. For construction of pUC702/pld-sig-gpeep, the nucleotide of the mature GPE-EP enzyme was joined to 30 -downstream of pld-sig in pUC702 (2).
125
The PCR was performed using the following primers: 50 -tgctagcgacgggctcg gagccatcaag-30 (Nhe I-F) containing a first codon (Nhe I, italic; Asp, underlined) of mature GPE-EP and 50 -gacagatcttcagcccgagacgctcgccc-30 (Bgl II-RV; Bgl II, italic). The PCR reaction mixture (20 ml) contained: 1 KOD Plus buffer, 6 pmol of each primer set (Nhe I-F and Bgl II-RV), 4 nmol of dNTPs, 3% (vol/vol) of DMSO, 24 nmol MgSO4, 0.4 U of KOD Plus DNA polymerase (Toyobo), and 100 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 2 min; 70.2 C for 1 min after completion of the 30 cycles. The obtained fragment was purified and digested with Nhe I and Bgl II, and then subcloned into the Nhe I and Bgl II sites of pUC702, i.e., between the signal peptide sequence and the terminator region of PLD. The constructed expression plasmid was sequenced and designated as pUC702/pld-sig-gpeep. For construction of pUC702/gpeep-sig, the nucleotide of gpe-ep containing GPE-EP-signal peptide and the mature enzyme was joined to downstream of the promoter region of PLD in pUC702. To replace SphI site in gpeep, gpe-ep was amplified from the chromosomal DNA by a two-step PCR. The first PCR was performed using the following primer sets: 50 -cagcatgctcacgga catttcacggcgacgg-30 (Sph I-F1) containing a first codon (Sph I, italic; Met, underlined) of GPE-EP-signal peptide and 50 -gcacgcGCATACcgagaccggtgggcttg-30 of Sph I-repair F1 (amplification product size, approximately 1.3 kbp); 50 ccaccggtctcgGTATGCgcgtgccgatg-30 (Sph I-repair F2) and Bgl II-repair RV (amplification product size, 870 bp). The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 80 s. Each amplification product was purified and used for the second PCR as a template. The second PCR was performed using forward primer (Sph I-F1) and reverse primer (Bgl II-RV). The PCR reaction mixture (20 ml) contained: 1 KOD Plus buffer, 6 pmol of each primer set (Sph I-F1 and Bgl II-RV), 4 nmol of dNTPs, 3% (vol/vol) of DMSO, 24 nmol MgSO4, 0.4 U of KOD Plus DNA polymerase, and 50 ng each of the first amplification products as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 72 C for 2 min. The amplification product was purified and used for the additional PCR as a template. The PCR reaction mixture (20 ml) contained: 1 KOD Plus buffer, 6 pmol of each primer set (Sph I-F1 and Bgl II-RV), 4 nmol of dNTPs, 3% (vol/vol) of DMSO, 24 nmol MgSO4, 0.4 U of KOD Plus DNA polymerase, and 100 ng of the 2.1-kbp amplification product as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 2 min. The obtained fragment was purified and digested with Sph I and Bgl II, and then subcloned into the Sph I and Bgl II sites in the pUC702, i.e., between the promoter and terminator region of PLD (13). The constructed expression plasmid was sequenced and designated as pUC702/gpeep-sig. The transformation techniques of Kieser et al. were followed for S. lividans (12). Transformants were screened by 5-mL culturing. Moreover, GPE-EP expression was attempted to produce GPE-EP using E. coli as a host. Two recombinant plasmid vectors, pET24a(þ)/gpeep and pET24a(þ)/ gpeepþHis, were constructed for production of active GPE-EP as follows. To clone gpe-ep into pET24a(þ), gpe-ep was amplified from the chromosomal DNA of S. sanglieri strain A14 by PCR. The PCR was performed using the following primers: 50 -atagtctacatatgaccacggacatttcacggcgacg-30 (Nde I-F1) containing a first codon (Nde I, italic; Met, underlined) of GPE-EP and 50 -tttaagctttcagcccgagacgctcgccc-30 (Hind III-RV1) containing Hind III site (italic). The PCR reaction mixture (25 ml) contained: 1 KOD Plus buffer ver. 2 (Toyobo), 7.5 pmol of each primer set (Nde IF1 and Hind III-RV1), 5 nmol of dNTPs, 3% (vol/vol) of DMSO, 37.5 nmol MgSO4, 0.5 U of KOD Plus DNA polymerase (Toyobo), and 150 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 98 C for 10 s and 72 C for 2 min. The obtained fragment was purified and digested with Nde I and Hind III, and then subcloned into the Nhe I and Hind II sites of pET24a(þ). The constructed expression plasmid was sequenced and designated as pET24a/gpeep. To add His tag to pET24a/gpeep, inverse PCR was carried out using IVS primer (50 -caccaccaccaccaccactgagatccgg-30 ) and IVAS primer (50 -gcccgagacgctcgcccggcc-30 ) designed based on the nucleotide sequence of gpe-ep on pET24a/gpeep. The inverse PCR amplification was completed with the reaction mixture (25 ml) contained 1 KOD Plus buffer ver. 2, 7.5 pmol of each primer, 5 nmol of dNTPs, 3% (vol/vol) of DMSO, 37.5 nmol MgSO4, 0.5 U of KOD Plus DNA polymerase, and 150 ng of pET24a/gpeep as a template. The PCR program was 94 C for 2 min, followed by 10 cycles of 98 C for 10 s and 68 C for 8 min. The obtained 7.5 kbp DNA fragment was self-ligated, and the resulting recombinant plasmid was called pET24a/gpeepþHis. SHuffle T7 Express Competent E. coli was transformed using pET24a/gpeep and pET24a/gpeepþHis. The transformed E. coli cells were incubated in 100 mL LB broth containing 30 mg/mL kanamycin at 30 C for 6 h with shaking (180 rpm). The expression of recombinant GPE-EP was induced with 0.4 mM IPTG at 16 C for 24-h incubation. The cells of the 500-mL culture were resuspended in 1.5 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) and sonicated. The cell-free extracts (cfe, 70 mL) were obtained by centrifugation (21,600 g for 10 min). The obtained supernatant was loaded onto a Butyl-650M column (2.5 by 5 cm) equilibrated with the same buffer. The column was washed with 3 CV, and the protein was eluted with a linear gradient as described above. The active fractions were pooled and followed by application to a HiTrap Q HP column (5 mL) as described above. The active fractions were applied to a HisTrap HP column (5 mL) equilibrated with 5 mM imidazole, 0.5 M NaCl/50 mM TriseHCl buffer (pH 8.0). The
126
MINETA ET AL.
J. BIOSCI. BIOENG., TABLE 1. Purification of GPE-EP from S. sanglieri A14.
Purification step 72-h culture supernatant 0e80% ammonium sulfate PPG-600M Hitrap Q HP Hitrap Phenyl HP Resource PHE Mono Q a
Sample (mL)
a
Activity (U/mL)
Protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification (fold)
% Recovery
480 57 98 24 35 1.5 1
3.66 29.5 11.0 23.7 8.82 18.5 10.8
369 336 99.3 13.2 5.16 0.331 0.104
4.75 4.99 10.8 43.1 59.8 83.8 103
1755 1679 1075 568 309 27.7 10.8
1 1.05 2.28 9.06 12.6 17.6 21.7
100 95.7 61.3 32.4 17.6 1.58 0.613
GPE-EP activity was assayed using the reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100 and 4 mM GPE at 65 C.
column was washed with 3 CV of the same buffer at a flow rate of 4 mL/min (2 cm/ min) and the protein was eluted with a linear gradient (20 CV) of 5 to 0.5 M imidazole at the same flow rate. Fractions exhibiting high specific activity were pooled and used for further investigation.
RESULTS Isolation and identification of strain A14 Strain A14 was assigned as S. sanglieri by morphological, physiological and biochemical characterizations, and 16S rDNA sequence analysis. S. sanglieri strain A14 was deposited as NITE BP-1392 in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan). The 16S rDNA sequence of strain A14 was deposited in the DDBJ database under the accession number AB735535. Purification of GPE-EP The enzyme was purified to electrophoretic homogeneity from the culture supernatant by ammonium sulfate precipitation, anion exchange chromatography and hydrophobic interaction chromatography. A summary of the purification is presented in Table 1. The overall purification of the enzyme from the culture supernatant was 21.7-fold, with an activity yield of 0.613%. The total amount of purified GPE-EP with a specific activity of 103 U/mg-protein was 0.104 mg of protein. Protein analysis The purified enzyme was subjected to SDSPAGE analysis. A single band with an apparent molecular mass of w70 kDa was visualized by silver staining. The result of gel filtration chromatography analysis demonstrated that native GPE-EP functions as a monomeric protein. The N-terminal amino acid sequence was determined as DGLGAIKHVV. LCeMS/MS analysis obtained a number of peptide sequences and internal amino acid
sequences (WMNGWVSAK, GTLADEFAK, TGVAEPNISDWR, TGDLTSAFDFSHAR and YAGEFPVPQHR) were selected as PCR primers for isolating the enzyme gene. Effect of pH, temperature and chemicals on GPE-EP activity As shown in Fig. 2, the highest activity for GPE hydrolysis was found at pH 8.4 and 65 C. The enzyme was stable from pH 6 to 10.2 and between 4 C and 60 C. The thermal stability of GPE-EP was w15 C higher than that of GPC-CP14 (1). Table 2 summarizes the effects of chemicals and inhibitors on GPE-EP activity for GPE hydrolysis. The enzyme activity was inhibited by 2 mM SDS, 2-mercaptoethanol, DTT, Mn2þ, Ca2þ and Fe2þ. Zn2þ ion remarkably inhibited the activity; while PMSF, IAA, Mg2þ, Cu2þ and Co2þ had almost no effect on the enzyme activity. The enzyme activity was found to increase in the presence of 2 mM EDTA. Substrate specificity Table 3 shows the substrate specificity of GPE-EP. The highest hydrolytic activity was recorded with GPE. DHPE, LPE, and LPlsPE were hydrolyzed by GPE-EP; however, only minimal activity was observed with GPG and CDP-choline as substrates. GPE-EP showed no activity with other GPX substrates of GPC, GPA, GPI and GPS. No activity was also observed for phospholipid substrates such as diacylglycerophospholipids (DBPC, POPC, DMPA, DPPE, POPE, PG, PI, PS), lysophospholipids (LPC, LPI, LPS, LPG, LPA) and plasmalogens (PlsPC, PlsPE, LPlsPC). Moreover, GPE-EP exhibited no phosphatase and glycerophosphodiester phosphodiesterase (EC 3.1.4.46) activity, i.e., no release of ethanolamine. CIAP used for the assay of GPE-EP activity was found to have no activity toward GPX.
FIG. 2. Effect of pH (A) and temperature (B) on the activity (open symbols) and stability (closed symbols) of purified GPE-EP. (A) The enzyme activity was assayed at 37 C for 5 min with 4 mM GPE in 50 mM of each buffer. The buffers were: sodium acetate (pH 4.1e5.6; open circles), BisTriseHCl (pH 5.6e7.2; open triangles), TriseHCl (pH 7.2e9.0; open squares) and glycineeNaOH (pH 9.0e10.5; open diamonds). To determine pH stability, the enzyme sample was incubated at 4 C for 3 h in 50 mM of each buffer: sodium acetate (pH 4.1e5.6; closed circles), BisTriseHCl (pH 5.6e7.2; closed triangles), TriseHCl (pH 7.2e9.0; closed squares) and glycineeNaOH (pH 9.0e10.5; closed diamonds). The residual activity was assayed by incubation at 65 C in 50 mM TriseHCl buffer (pH 8.4). (B) The enzyme activity (open circles) was assayed at each temperature in 50 mM TriseHCl buffer (pH 8.4). To determine thermal stability, the enzyme sample was incubated at each temperature for 30 min in 50 mM TriseHCl buffer (pH 8.4) and the residual activity (closed circles) was assayed by incubation at 65 C in 50 mM TriseHCl buffer (pH 8.4). Data are the means of experiments performed in triplicate. Error bars represent the standard deviation.
VOL. 119, 2015
GLYCEROPHOSPHOETHANOLAMINE ETHANOLAMINEPHOSPHODIESTERASE
TABLE 2. Effect of chemicals and inhibitors (2 mM) on the GPE-EP activity for GPE hydrolysis.a Relative activity (%)b
Chemical Control KCl NaCl MgCl2 CoCl2 CuCl2 CaCl2 FeCl2 MnCl2 ZnCl2 EDTA SDS IAA 2-Mercaptoethanol DTT PMSF
100 106 105 101 100 98.9 92.0 92.0 54.0 0.575 113 92.7 101 80.5 64.6 98.8
a The purified enzyme was assayed by using reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100, 4 mM GPE, and 2 mM chemicals or inhibitors at 65 C. b The relative activity is expressed as a percentage of the control activity without chemicals or inhibitors.
Enzyme kinetics Linear regression analysis was performed using a HaneseWoolf plot (data not shown). For the hydrolysis of GPE by GPE-EP at 65 C and pH 8.4, the apparent Vmax and kcat values were determined to be 0.430 mmol min1 mg-protein1 and 522 s1, respectively. The apparent Km and kcat/Km values were 0.785 mM and 665 mM1 s1, respectively. Intriguingly, during hydrolysis of GPE, competitive inhibition was observed in the presence of 1 mM ethanolamine or 8 mM glycerol, but not in the presence of 8 mM GPC (data not shown). For the hydrolysis of GPE at pH 8.4, activation energy was calculated to be 19.8 kJ mol1 using Arrhenius plot (data not shown). Gene cloning The partial nucleotide sequence of the gene encoding GPE-EP (gpe-ep) was determined by standard PCR using primer sets designed from N-terminal and internal amino acid sequences (TGVAEPNISDWR). The 1254-bp determined nucleotide sequence encoded a protein that is 418 amino acids in length. The 447-bp nucleotide sequence of the 50 -upstream region and 1010-bp nucleotide sequence of the 30 -downstream region were determined from a w2.5-kb inverse PCR product. Consequently, a 2711-bp nucleotide sequence was determined. The ORF of gpe-ep was determined to consist of 2124 nucleotides encoding a 708 residue protein. The complete nucleotide sequence of gpe-ep has been deposited in the DDBJ/GenBank/EMBL database under accession number AB771750. The putative TTG translational start codon was preceded at a spacing of 6 bp by a potential ribosome binding site (ggagg). A possible promoter region and terminator region were not found. As shown in Fig. S2, the N-terminal sequence of the mature enzyme starts at Asp49 of the deduced amino acid sequence, indicating that the preceding 48 amino acids are a TABLE 3. Substrate specificity of GPE-EP. Substrate GPE DHPE LPE LPlsPE GPG CDP-choline
Relative specific activity (%)a 100 4 82.8 3.4 31.2 30.1 5.11 0.955 0.413
a The purified enzyme was assayed by using the reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100, 4 mM GPX or 4 mM other phospholipids at 65 C. The relative specific activity is expressed as a percentage of the specific activity for GPE (137 U/mg protein). Results are represented as means standard deviation of experiments performed in triplicate.
127
signal sequence for secretion. A twin arginine translocation (Tat) pathway motif (RRRLF) was used for the signal sequence (14). The molecular weight of the gene product (i.e., the 660-amino-acid protein) without the signal sequence is calculated to be 72,918 Da in molecular weight, in agreement with that of the purified enzyme estimated by SDS-PAGE analysis. The isoelectric point (pI) of GPE-EP was calculated to be 6.17 using Genetyx-Mac version 16.0.8 (Genetyx Corporation, Tokyo, Japan). Comparative sequence analysis of GPE-EP A homology search performed using the BLAST algorithm indicated that the amino acid sequence of GPE-EP shares 86% and 85% identity with that of PC-specific phospholipase C (PC-PLC) from Streptomyces sp. SirexAA-E (UniProt accession no. G2NFN1) (Fig. S2) and putative non-hemolytic PLC (nhPLC) from Streptomyces sp. W007 (UniProt accession no. H0BLP6) (Fig. S3). Moreover, GPE-EP showed high identity to numerous Streptomyces PLCs, however, most of them remain to be biochemically and functionally uncharacterized. As shown in Fig. S3, a distance-based phylogenetic analysis of those protein amino acid sequences revealed that GPE-EP belongs to PLC/acid phosphatase (APase) superfamily, and it is clearly separated from a group containing GPC-CP14, Pseudomonas fluorescens protein (CGDEase) of the PLC/APase superfamily with CDP-ethanolamine and DHPE hydrolase activity (15), and AcpA. However, RGD motif conserved in the PLC/APase superfamily did not detected in GPE-EP (16). Intriguingly, we have reported previously that S. sanglieri A14 extracellularly produces GPC-CP14 (1). The amino acid sequence of mature GPE-EP shows 37.3% identity (79.5% similarity) to that of GPC-CP14. Furthermore, Pfam analysis (17) suggested that the N-terminal region (54e458) of GPE-EP is assigned as phosphoesterase family domain and shares structural similarities with AcpA (8), and two small domains of unknown function tandemly exist in the C-terminal region (520e602 and 603e698) of GPE-EP. Secondary structure analysis with PSIpred (18) predicted that the tandem domains are composed primarily of b-strands. Homology modeling analysis The structure models of GPEEP: 49e460 and GPC-CP14: 39e471 based on AcpA are shown in Fig. 3. VERIFY3D showed that the homology models of GPE-EP: 49e460 and GPC-CP14: 39e471 had 112.8 and 160.1 of 3De1D total score (i.e., modeling quality), respectively, indicating that the structural models drawn using AcpA as a template by Modeller are sufficient for comparative structural discussions. However, the C-terminal half region of GPE-EP (461N to 708G) and that of GPC-CP14 (472R to 684V) resulted in failure to create models. The putative catalytic residue was observed at T187 of GPE-EP (Fig. 3). Moreover, E63, N64, T187, D389, and E390 were conserved in GPE-EP as metal ion binding site. In addition, histidine residues (H129 and H352) surrounding phosphate of substrate were observed in the active site of GPE-EP. GPE-EP can form a disulfide bond between C176 and C438. Expression and efficient purification of recombinant GPEEP S. lividans cells transformed with pUC702 carrying gpe-ep resulted in failure to express the recombinant GPE-EP. E. coli cells transformed using pET24a/gpeep and pET24a/gpeepþHis were efficiently produced the recombinant GPE-EP. The recombinant enzyme (6.92 mg-protein) with a high specific activity (363 U/mgprotein) and total activity (2516 U) was purified to electrophoretic homogeneity from the cfe (5419 U) of E. cells carrying pET24a/ gpeepþHis using simple purification steps (Table 4). DISCUSSION We purified and characterized GPE-EP from S. sanglieri strain A14. The molecular mass (72,918 Da) of GPE-EP is very similar to that of GPC-CP14 (70,447 Da) (1) or that of CGDEase (75 kDa) from
128
MINETA ET AL.
J. BIOSCI. BIOENG.,
FIG. 3. Structures of AcpA, GPE-EP and GPC-CP14. (A) Overall structure. Three enzyme structures are represented in ribbon model from the same view. GPE-EP and GPC-CP14 are predicted by homology modeling. (B) Active site structures. Amino acid residues in the active site are depicted by stick model.
P. fluorescens CECT7229 (15), which are monomeric and extracellular proteins. However, AcpA is a homodimer of 57 kDa subunits and periplasmic enzyme (8). Costas et al. (15) reported that CGDEase is a Mg2þ-dependent enzyme and is inhibited by EDTA. AcpA appears to be a Ca2þ-dependent enzyme (8). In contrast, GPE-EP was not inhibited by EDTA, and the enzyme activity was not stimulated by tested metal ions such as Ca2þ and Mg2þ. However, the metal ion binding site (E63, N64, T187, D389, and E390) was observed in the predicted active center of GPE-EP, suggesting that a metal ion may be tightly bind to the enzyme. In fact, 1 mol of Ca2þ was detected in 1 mol of GPC-CP14 (unpublished data). Zn2þ showed similar inhibition of GPE-EP, GPC-CP14, and CGDEase (15). The highest activity of GPE-EP was observed at 65 C, in accord with the cloud point of 1% (w/v) Triton X-100 (63.7 C) (19). Besides, the enzyme activity was influenced by the concentration of Triton X-100 in the reaction mixture, and the highest activity was found in the presence of 0.1% (w/v) Triton X100 above critical micelle concentration (0.25e0.27 mM) (20). On the other hand, since GPE is a water-soluble and hydrophilic compound, Triton X-100 has little direct effect on the substrate; rather, a conformational change enhancing the enzyme activity might be caused by Triton X-100. Alternatively, the detergent may just keep the protein from binding to tube walls. In fact, the concentrated GPE-EP sample (>4 mg-protein/mL) led to the aggregation, suggesting that GPE-EP is a hydrophobic protein. Consequently, GPE-EP may be dispersed or solubilized such as membrane-bound protein by Triton X-100. For these reason, we have presumed the optimum Triton X-100 concentration exists for the enzyme activity. Further studies are required to elucidate activation mechanism of the enzyme by Triton X-100. GPE-EP was
stable across a broad pH range (pH 6e10), and the stability is very similar to that of GPC-CP14 (pH 5e10); although the maximal pH (8.4) of GPE-EP was different from that (pH 7.2) of GPC-CP14 and those of the mammalian GPC-CPs (pH 9.5 and 10), suggesting that the physiological function of GPE-EP may be essentially different from GPC-CP14 and mammalian GPC-CPs. The apparent Km (0.785 mM) of GPE-EP was somewhat higher than that of CGDEase (0.14 mM) (15) and those of mammalian GPCCPs (21e23), and one-half of that (1.41 mM) of GPC-CP14 (1). The apparent Vmax of GPE-EP (0.430 mmol min1 mg-protein1) was considerably higher than the Vmax, i.e., 24.7 mmol1 min1 mgprotein1, of GPC-CP14 (1) and that 2.4 mmol1 min1 mg-protein1 of CGDEase (1). In addition, kcat and kcat/Km values (522 s1 and 665 mM1 s1) of GPE-EP are much higher than those of GPC-CP14 (29 s1 and 20.6 mM1 s1), suggesting that GPE-EP possesses the higher substrate affinity and much higher turnover number as compared to GPC-CP14 and CGDEase. Namely, GPE-EP can treat rapidly even at lower concentration of GPE. GPC-CP14 exhibited almost equal enzyme activity toward GPC and GPE (1). However, the catalytic efficiency (kcat/Km) of GPE-EP toward GPE is much higher than that of GPC-CP14. Strain A14 produces GPE-EP as well as GPC-CP14. Therefore, we conclude GPE-EP is specialized to degrade GPE. The kcat and kcat/Km values of GPE-EP are higher than those of previously reported PLCs (BRENDA, http://www.brenda-enzymes. info/php/result_flat.php4?ecno¼3.1.4.3), yet lower than those of general bacterial enzymes, suggesting that the binding affinity of GPE-EP toward substrate GPE is higher than those of bacterial enzymes, and the turnover number (kcat) and the catalytic efficiency (kcat/Km) may be somewhat lower than the vast majority of enzymes. The activation energy (19.8 kJ mol1) for the hydrolysis of
TABLE 4. Purification of recombinant GPE-EP. Purification step cfe of 24-h culture Butyl-650M Hitrap Q HP Histrap HP a
Sample (mL)
a
Activity (U/mL)
Protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification (fold)
% Recovery
70 40 25 6
77.4 98.9 133 419
84.6 34.7 10.9 6.92
64.0 114 306 363
5419 3957 3333 2516
1 1.8 4.8 5.7
100 73.0 61.5 46.4
GPE-EP activity was assayed using the reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100 and 4 mM GPE at 65 C.
VOL. 119, 2015
GLYCEROPHOSPHOETHANOLAMINE ETHANOLAMINEPHOSPHODIESTERASE
GPE by GPE-EP was similar to that (18.8 kJ mol1) reported for lecithin, i.e., PC, hydrolysis by the wild-type SaPLA1 (2). Here, we discuss the substrate specificity and substrate recognition of GPE-EP. GPE-EP exhibited GPE-specific activity and no activity toward all diacylglycerophospholipids containing PE, except for DHPE, strongly suggesting that GPE-EP is not originally a PLC. Nevertheless, GPE-EP hydrolyzed DHPE, LPE and LPlsPE with w30e80% relative activity, indicating that the enzyme prefers ethanolamine-type substrates. Triton X-100 molecules can form mixed micelles with diacylglycerophospholipids including DHPE, DPPE and POPE or lysophospholipids including LPE and LPlsPE. PE molecules with two long acyl chains (DPPE and POPE) should be exclusively contained within the micelles, whereas PE molecules with short acyl chains (DHPE) or with single acyl chain (LPE and LPlsPE) may be partly exist as monomers (24e26). Thus, both the physical characteristics of their interfaces would be distinct from each other; consequently, both of them are different from each other in the binding mode for the enzyme and the substrate molecules. George et al. reported that both the surface concentration of lipid and the bulk concentration play critical roles in defining the kinetic parameters of lipid-dependent enzymes (24). Taken together, the difference in the binding mode may affect the activity of GPE-EP. Alternatively, the specificity toward DHPE, LPE and LPlsPE is likely to be controlled (in part) by steric hindrance of the fatty acyl chain of substrates. We believe that GPE-EP possibly recognizes the head group and the acyl part of the alkenylether chain of the substrate. The results of competitive inhibition of GPE-EP activity by ethanolamine or glycerol support our conclusion. Costas et al. reported that CGDEase prefers CDP-ethanolamine (100% relative activity), GPE (96% relative activity) and DHPE (115% relative activity), and showed activity toward CDPcholine (23% relative activity), GPC (7% relative activity), and pNPPC (29% relative activity) (15). The substrate specificity of GPEEP seems to be somewhat similar to that of CGDEase; however, GPE-EP exhibited no activity toward CDP-choline and GPC, demonstrating that the substrate molecule recognition by GPE-EP is more stringent than those of CGDEase and GPC-CP14 (1); the substrate molecule recognition of these enzymes may be distinct from one another. It has also been reported that, in the case of Bacillus cereus PC-PLC, the polar head group and also the associated carbonyl group are important for phospholipid recognition (27). The fatty acyl chains also play a significant role in substrate binding and the PLC appears to prefer greater than six carbons for hydrolysis of phospholipids. In summary, we conclude that GPE-EP may recognize hydroxyl groups of the glycerol backbone as well as the head group and the fatty acyl chain conformation of the substrate molecule. Further studies are required to elucidate the substrate recognition mechanism of GPE-EP. The crystallization of GPE-EP is currently being undertaken. We discuss physiological role of GPE-EP. As shown in Fig. S4, although glycerophosphodiester phosphodiesterase (EC 3.1.4.46) has been known, there is no report of GPE-EP. We therefore propose that GPE hydrolysis by GPE-EP may represent a new metabolic pathway for PE metabolism and acquisition of phosphorus and carbon sources in actinomycetes. Because of the limited availability of environmental phosphorus (
Characterization of glycerophosphoethanolamine ethanolaminephosphodiesterase from Streptomyces sanglieri Shingo Mineta,1 Kazutaka Murayama,2 and Daisuke Sugimori1, * Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan1 and Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo, Aoba, Sendai 980-8575, Japan2 Received 18 June 2014; accepted 9 July 2014 Available online 15 August 2014
Streptomyces sanglieri extracellularly produces a glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP). The gene encoding the enzyme was found to consist of a 2124-bp ORF, which codes for an N-terminal 48 residue signal peptide required for secretion and a 660 amino acid mature protein with a calculated molecular mass of 72,918 Da. The maximum activity for sn-glycero-3-phosphoethanolamine (GPE) was found at pH 8.4 and 65 C in the presence of 0.1% (w/v) Triton X-100. The enzyme was activated in the presence of 2 mM EDTA; however, Zn2D remarkably inhibited activity. During the hydrolysis of GPE at 65 C and pH 8.4, the apparent Vmax, turnover number (kcat) and Km were determined to be 0.430 mmol minL1 mg-proteinL1, 522 sL1 and 0.785 mM, respectively. The enzyme exhibited specificity toward GPE and hydrolyzed ethanolamine-type substrates such as 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, lysophosphatidylethanolamine and ethanolamine lysoplasmalogen, but not 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine. Moreover, the enzyme showed no activity toward other phospholipids, such as glycerophospholipids and plasmalogens, and sn-glycero-3-phosphodiesters except for sn-glycero-3-phosphoglycerol, suggesting that GPE-EP is not a phospholipase C (PLC). However, the amino acid sequence of GPE-EP shows 86% identity to that of PLC from Streptomyces sp. SirexAA-E (UniProt accession no. G2NFN1). Recombinant GPE-EP was functionally expressed in Escherichia coli using pET-24a(D). GPE hydrolysis by GPE-EP may represent a new pathway for phosphatidylethanolamine metabolism. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycerophosphoethanolamine ethanolaminephosphodiesterase; Purification; Characterization; Molecular cloning; Expression; Streptomyces sanglieri]
sn-Glycero-3-phosphodiesters (GPX), i.e., sn-glycero-3-phosphocholine (GPC), sn-glycero-3-phosphoethanolamine (GPE), snglycero-3-phosphoserine (GPS), sn-glycero-3-phosphate (GPA), snglycero-3-phosphoinositol (GPI) and sn-glycero-3-phosphoglycerol (GPG), are an important intermediate in glycerophospholipid metabolism. Nevertheless, there is only few limited information on the metabolic pathway and key enzyme. We recently reported a new glycerophosphocholine cholinephosphodiesterase (GPC-CP14; EC 3.1.4.38) from Streptomyces sanglieri strain A14 (1). Furthermore, we detected GPE-specific enzyme, i.e., glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP), from strain A14. Both two enzymes are very similar to phospholipase C (PLC; EC 3.1.4.3) that cleaves PE into diacylglycerol and phosphoethanolamine. In contrast, GPE-EP cleaves GPE into glycerol and phosphoethanolamine (Fig. 1). The difference in the substrate recognition mechanism between GPE-EP, GPC-CP14, and PLC remains unknown. The identification of GPE-EP may help to realize a new pathway for phosphatidylethanolamine metabolism. Moreover, the characterization of GPE-EP may allow prediction of the
* Corresponding author. Tel./fax: þ81 24 548 8206. E-mail address: [email protected] (D. Sugimori).
physiological role of PLC as well as those phosphodiesterases in actinomycete. Here, we report the purification, characterization, molecular cloning, functional expression, structure modeling, and the catalytic mechanism prediction of a novel GPE-EP from S. sanglieri strain A14.
MATERIALS AND METHODS Materials Tryptic soy Broth (TSB), Bacto Tryptone, Bacto peptone, Bacto Casamino acid and Malt extract were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Yeast extract and alkaline phosphatase from calf intestine (CIAP) were purchased from Oriental yeast Co., Ltd. (Tokyo, Japan). Soybean lecithin and polyoxyethylene laurylether (Brij 35) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). BIOMOL Green Reagent and L-a-phosphatidylinositol (PI) were purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). 1,2-Dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-dipalmitoyl-sn-glycero3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DHPE), 1,2dibutyryl-sn-glycero-3-phosphocholine (DBPC), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), L-a-phosphatidylserine (PS), L-a-lysophosphatidylcholine (LPC), L-a-lysophosphatidylserine (LPS), L-a-lysophosphatidylglycerol (LPG), 1stearoyl-2-hydroxy-sn-glycerol-3-phosphate (LPA), L-a-lysophosphatidylinositol (LPI), 1-O-10 -(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine (LPlsPC; choline lysoplasmalogen), 1-O-10 -(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPlsPE; ethanolamine lysoplasmalogen), 1-(1Z-octadecenyl)-2arachidonoyl-sn-glycero-3-phosphocholine (PlsPC; choline plasmalogen) and 1-
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.07.005
124
MINETA ET AL.
FIG. 1. Hydrolytic reaction of GPE by GPE-EP.
(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE; ethanolamine plasmalogen) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). L-a-phosphatidylglycerol (PG) and L-a-lysophosphatidylethanolamine (LPE) were purchased from Doosan Serdary Research Laboratories (Toronto, ON, Canada). GPC was purchased from Bachem (Torrance, CA, USA). HiTrap DEAE FF, HiTrap Q HP, HiTrap Phenyl HP, HisTrap HP, Resource PHE and Mono Q columns were purchased from GE Healthcare Japan (Tokyo, Japan). Toyopearl PPG-600M and Butyl-650M were purchased from Tosoh (Tokyo, Japan). All other chemicals were of the highest or analytical grade. Glycero-3-phosphodiesters Recombinant phospholipase A1 (SaPLA1) from Streptomyces albidoflavus strain NA297 was extracellularly produced by Streptomyces lividans and was used for the synthesis of GPX after ammonium sulfate purification (2). GPX such as GPE, GPS, GPI, GPA and GPG, except GPC, were prepared as indicated below. The corresponding lysophospholipids (40 mM) were completely hydrolyzed in 20 mM TriseHCl buffer (pH 7.2), 0.5% (w/v) Triton X-100 and 1.15 U of the recombinant SaPLA1 (115 U/mL, 217 U/mg) at 37 C overnight. Complete conversion of lysophospholipid into the corresponding GPX was confirmed by the concentration of free fatty acids released by SaPLA1. The concentration of free fatty acids was determined with a NEFA C Kit (Wako Pure Chemical Industries Ltd., Osaka, Japan) using oleic acid as the standard and according to the instructions of the manufacturer. The reaction mixture was boiled at 100 C for 5 min and extracted with chloroformemethanol (2:1, v/v), followed by centrifugation (18,300 g for 5 min). The upper layer was collected and the solvent was removed by evaporation for 5 min using a vacuum evaporator. The resulting aqueous solution was used for the investigation of substrate specificity. Bacterial strains and culture conditions Approximately 1500 strains were isolated from soil samples from Fukushima, Japan, using a humic acid-vitamin agar plate culture (3). Strain A14 exhibiting GPE-EP activity as well as GPC-CP activity was selected (1). Strain A14 colonies from ISP2 (10 g malt extract, 4 g yeast extract and 4 g glucose per liter, pH 7.2) agar plates were scraped and inoculated into a test tube (18 mm, 180 mm) containing 5 mL seed medium of ISP2. These cultures were incubated with shaking (160 strokes per min) at 28 C. After 48 h cultivation, a 1% (v/v) inoculum was transferred into a 500-mL flask containing 50 mL of the fermentation medium of ISP2 supplemented with 0.41 mM Brij 35 and cultivated with shaking (180 rpm) at 28 C for 72 h. Escherichia coli HST08 premium competent cells (Takara Bio Inc., Shiga, Japan) were used as a host for molecular cloning. The pGEM-T Easy Vector (Promega) and the pMD20-T vector (Takara Bio) were used as cloning vectors. Recombinant E. coli cells were cultured on LuriaeBertani (LB) agar plates at 37 C; if necessary, ampicillin, isopropyl-b-Dthiogalactopyranoside and X-Gal were used as supplements in the agar. A pUC702 carrying the promoter, signal peptide sequence and the terminator region of phospholipase D (PLD) from Streptoverticillium cinnamoneum (4) and pET24a(þ) (Merck KGaA, Darmstadt, Germany) were used as expression vectors. S. lividans 1326 (NBRC15675), which was obtained from NITE Biological Resource Center (Chiba, Japan), and SHuffle T7 Express Competent E. coli (New England Biolabs Japan, Tokyo) were used as a host for the expression of proteins. Recombinant S. lividans cells were cultured in 3% (w/v) TSB containing 5 mg/mL thiostrepton at 28 C. Wild-type enzyme purification All procedures were performed at 4 C. The culture supernatant was obtained by centrifugation (18,800 g for 20 min) after 72 h of culturing. The supernatant was placed in a saturated ammonium sulfate solution ((NH4)2SO4 mass fractionation ¼ 80%) and was centrifuged at 18,800 g for 30 min. The precipitate was suspended in 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0). The enzyme sample was loaded onto a PPG-600M column (2.5 3.5 cm) equilibrated with the same buffer. The column was washed with 3 column volumes (CV) of the same buffer at a flow rate of 2 cm/min, and the protein was eluted with a linear gradient (10 CV) of 1e0 M (NH4)2SO4 in the same buffer at 6 mL/min (1.22 cm/min). The active fractions were pooled and then replaced by 20 mM TriseHCl buffer (pH 9.0) using Vivaspin 20e30,000 MWCO (Vivaspin 20e30 k; GE Healthcare Japan) followed by loading the sample onto a HiTrap Q HP column (5 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer at a flow rate of 5 mL/min, and the protein was eluted with a linear gradient (20 CV) of 0e1 M NaCl in the same buffer at 4 mL/min (2 cm/min). The active fractions were pooled and then replaced by 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20e30 k concentrators followed by application to a HiTrap Phenyl HP column (5 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer at a flow rate of 5 mL/min, and the protein was eluted with a linear gradient (16 CV) of 1 to 0 M (NH4)2SO4 in the same buffer and 8 CV of 20 mM TriseHCl buffer
J. BIOSCI. BIOENG., (pH 8.0) at 4 mL/min (2 cm/min). The active fractions were pooled and then replaced by 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20e30 k followed by application to a Resource PHE column (1 mL) equilibrated with the same buffer. The sample was loaded at a flow rate of 1 mL/min. The flow-through fraction was pooled. The buffer was exchanged with 20 mM TriseHCl buffer (pH 9.0) by using the same method mentioned above. The enzyme solution was applied to a Mono Q column (1 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer at a flow rate of 0.5 mL/min (2.55 cm/min) and the protein was eluted with a linear gradient (80 CV) of 0e1 M NaCl at the same flow rate. Fractions exhibiting high specific activity were pooled and used for further investigation. Enzyme activity assays For characterizing the GPE-EP activity, a standard assay mixture: 10% (v/v) enzyme sample solution, 50 mM TriseHCl buffer (pH 8.4), 4 mM GPE and 0.1% (w/v) Triton X-100 was incubated at 65 C for 5 min. The reaction was stopped by incubation at 100 C for 5 min. The sample was subsequently centrifuged at 21,600 g for 5 min. Five microliter of the supernatant was subsequently added to 95 ml of 50 mM glycineeNaOH buffer (pH 9.6) containing 1 mM MgCl2, 0.1 mM ZnCl2 and 1.6 U of CIAP followed by incubation at 56 C for 10 min. Inorganic phosphate released by the dephosphorylation of phosphoethanolamine was quantified with BIOMOL Green Reagent according to the manufacturer’s instructions (5). The rates of soluble phosphate release from the enzyme reaction mixtures were calculated, and one unit (U) of enzyme activity was defined as the amount of enzyme that hydrolyzed 1 mmol of substrate per minute. Since GPE used as a substrate was prepared by hydrolysis of 40 mM LPE containing 0.5% (w/v) Triton X-100 using SaPLA1, at most 0.05% (w/v) Triton X-100 was contaminated in the GPE-EP reaction mixture. In addition to, substrate specificity of phospholipids was investigated using mixed micelle substrates with 0.1% (w/v) Triton X-100. Furthermore, the effect of Triton X-100 concentration on the GPE-EP activity was investigated. GPE was prepared by hydrolysis of 40 mM LPE without Triton X-100 using SaPLA1. The enzyme activity was assayed under standard assay conditions with the same assay buffer containing 4 mM GPE in the absence or presence of Triton X-100. The highest activity was found in the presence of 0.1% (w/ v) Triton X-100 (Fig. S1). With considering these conditions, the standard assay mixture containing 0.1% (w/v) Triton X-100 was determined. Substrate specificity The substrate specificity of the purified GPE-EP was assessed by assaying enzyme activity, as described above, using alternative GPX substrates or phospholipid substrates such as diacylglycerophospholipids, lysophospholipids and plasmalogens (4 mM final concentration) in the assay. Effect of pH, temperature, and chemicals on enzyme activity Each buffer (sodium acetate, BisTriseHCl, TriseHCl, and glycineeNaOH) was used to identify the pH that led to optimum GPE-EP activity, and to determine the pH stability of the purified enzyme. The optimum pH was examined by incubation at 37 C for 5 min with 4 mM GPE in 50 mM of each buffer. The pH stability was assayed by incubating the purified enzyme at 4 C for 3 h in 50 mM of each buffer solution. The remaining activity was assayed under the standard assay conditions. The GPE-EP activity was determined at each temperature under the standard assay conditions. Thermal stability was determined by incubating the purified enzyme in 50 mM TriseHCl (pH 8.4) at each temperature for 30 min, and then the residual activity was measured by incubation (65 C, 5 min) under the assay mixture: 10% (v/v) enzyme sample solution, 50 mM TriseHCl buffer (pH 8.4), 4 mM GPE and 0.1% (w/v) Triton X-100. The effect of chemicals such as metal ions and SDS on the enzyme activity was investigated. The purified enzyme was assayed under standard assay conditions with the same assay buffer containing 2 mM of each chemical or inhibitor. Inhibitors assessed were EDTA, 2-mercaptoethanol, dithiothreitol (DTT), iodoacetamide (IAA) and phenylmethanesulfonyl fluoride (PMSF). Additionally, for the EDTA inhibition experiment, the purified enzyme (w1 mM) was assayed under standard assay conditions following pre-incubation at 37 C for 30 min with 50 mM TriseHCl (pH 8.4) containing 10 mM EDTA. PMSF was dissolved in dimethyl sulfoxide (DMSO) and added to the enzyme sample. In the case of IAA, the purified enzyme samples (4.33 mg/mL, 59.4 nM) were incubated at 37 C for 60 min in the same buffer containing 2 mM IAA before use in enzyme assays. Protein analysis The protein concentration was determined with the Pierce BCA protein assay kit (Takara Bio) and BSA as the standard. Protein samples were analyzed by 12% SDS-PAGE according to Laemmli (6). Proteins on the SDS-PAGE gel were stained with Coomassie brilliant blue R-250 (CBB) or silver staining (Silver stain KANTO III; Kanto Chemical Co., Inc., Tokyo, Japan). The molecular mass of the purified enzyme was estimated by gel filtration. Gel filtration was performed using a Superdex 200 10/300 GL column (1.0 30 cm) at a flow rate of 0.5 mL/ min (0.64 cm/min) with 20 mM TriseHCl (pH 8.0) containing 0.15 M NaCl. The column was calibrated with a gel filtration calibration kit (GE Healthcare) before and after the enzyme was subjected to chromatography. Peptide sequencing The purified protein was resolved by SDS-PAGE and then electroblotted onto a PVDF membrane (Immobilon-PSQ transfer membrane, Millipore Co., Billerica, MA, USA). The PVDF membrane was stained with CBB, and the transferred 70-kDa band was excised and subjected to N-terminal amino acid sequence analysis (Procise 494 HT Protein Sequencing System; Applied Biosystems, Foster City, CA, USA). For an internal terminal amino-acid sequencing, an SDS-PAGE gel was stained using CBB, the 70-kDa band was excised and then decolorized with 30% acetonitrile containing 25 mM (NH4)2HCO3. The in-gel digestion was performed by the method described by Shevchenko et al. (7). Briefly, the excised 70-kDa band
VOL. 119, 2015
GLYCEROPHOSPHOETHANOLAMINE ETHANOLAMINEPHOSPHODIESTERASE
was digested with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation, Madison, WI, USA) for 45 h at 4 C. The sample (1 ml) of extracted peptides were separated using a nanoAcquity UPLC system (Waters Corporation, Milford, MA, USA) equipped with a nano ACQUITY UPLC BEH130 C18 column (Waters, 75 mm 150 mm, 1.7 mm) and were analyzed using a Xevo QTOF MS, according to a previously reported method (2). De novo sequencing was performed with the ProteinLynx Global SERVER, version 2.3 (Waters). Steady-state kinetics For hydrolysis of GPE, the initial velocity (v) of the enzymatic reaction was determined at each concentration of the substrate under standard assay conditions. The concentration of GPE ([GPE]) was calculated using a molecular weight of 214.15 Da. The purified enzyme concentration in the reaction mixture was constant at 0.336 mg mL1 (4.61 nM). The corresponding [GPE]/v vs. [GPE] plot was treated according to a MichaeliseMenten equation. Kinetic constants were determined by extrapolation using the HaneseWoolf plot by linear regression (KaleidaGraph, Synergy Software, Reading, PA, USA). The Km and Vmax were determined from the x-intercept and slope of the regression line, respectively. The turnover number (kcat) was calculated using a molecular weight of 72,918 for a monomeric protein with a single catalytic site. From the sequence inforHomology modeling of GPE-EP and GPC-CP14 mation, GPE-EP and GPC-CP14 have phosphoesterase family domain and exhibited similarity to structure of acid phosphatase from Francisella tularensis (AcpA; Protein Data Bank code: 2D1G). The structural features of AcpA have been studied (8). Based on a template (AcpA, 2D1G), the homology models of GPE-EP and GPC-CP14 were created using an HHPRED search (http://toolkit.tuebingen.mpg.de/hhpred) (9) and Modeller 9.11 (http://toolkit.tuebingen.mpg.de/modeller) (10). The N-terminal region of the GPE-EP amino acid sequence from 49D to 460P (GPE-EP: 49e460) and that of the GPC-CP14 amino acid sequence from 39G to 471D (GPC-CP14: 39e471) were used to create the models. VERIFY3D (http://nihserver.mbi.ucla.edu/Verify_ 3D/) (11) was used to assess the quality of the predicted models. Cloning of GPE-EP gene Chromosomal DNA of S. sanglieri was purified according to Kieser et al. (12). Oligonucleotides were synthesized based on the Nterminal and internal amino acid sequences of the enzyme for use in PCR with sense primer S1 (50 -gacggcctsggcgcsatcaagcac-30 ) and antisense primer A1-4 (50 scgccagtcsswgatgttsggctc-30 ). The PCR reaction mixture (25 mL) contained: 1 KOD FX Neo buffer, 7.5 pmol of each primer, 10 nmol of dNTPs, 0.5 U of KOD FX Neo DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) and w50 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 98 C for 10 s and 68 C for 45 s. The obtained PCR fragment was purified and cloned into pGEM-T Easy vector and the resulting recombinant plasmid was called pGPE. Sequencing of the partial GPE-EP gene on pGPE was performed with the BigDye Terminator cycle sequencing kit and analyzed in an ABI Prism 3100 genetic analyzer (Life Technologies Corporation, Carlsbad, CA, USA). To reveal the nucleotide sequence of the 50 -upstream and 30 downstream region of the gene, inverse PCR was carried out using IV-S primer (50 -gctacgtctgctccgaggtcttcgac-30 ) and IV-AS primer (50 -gtactggaggtccttcttctgcg tctcg-30 ) designed based on the partial sequence of GPE-EP gene of pGPE. The genomic DNA (1 mg) was digested with Sma I and self-ligated. The inverse PCR amplification was completed with the reaction mixture (25 ml) containing 1 KOD FX Neo buffer, 7.5 pmol of each primer, 10 nmol of dNTPs, 0.5 U of KOD FX Neo DNA polymerase and w25 ng (above the 0.4-ml ligation mixture) of the Sma I-digested and self-ligated DNA. The PCR program was 94 C for 2 min, followed by 30 cycles of 98 C for 10 s and 69 C for 160 s. The obtained w2.5 kbp DNA fragment was cloned into the pGEM-T Easy vector and the resulting recombinant plasmid was called pIVgpe. To clone the complete GPE-EP gene from the chromosomal DNA of strain A14, two cloning primers were used: the sense primer GPE-NS (50 -gaggctcaacttgaccacggacatttcacg-30 ) and the antisense primer GPE-CA (50 -cagaaaccgaactcccgtgcgaaggaac-30 ) designed based on the sequence of the GPE-EP gene revealed by the inverse PCR, as described above. The genomic PCR amplification was carried out as follows. The PCR reaction mixture (20 mL) contained: 1 PCR buffer, 6 pmol of each primer, 4 nmol of dNTPs, 24 nmol MgSO4, 3% (vol/vol) of DMSO, 0.4 U of KOD Plus DNA polymerase (Toyobo) and w100 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 2 min. The resultant DNA fragment was cloned into the pGEM-T Easy vector and sequenced. Nucleotide and peptide sequence accession number The nucleotide sequences of the 16S rDNA of S. sanglieri strain A14 and the GPE-EP gene, designated gpe-ep, were deposited in the DDBJ/GenBank/EMBL database under the accession numbers AB735535 and AB771750, respectively. Expression and purification of recombinant GPE-EP S. lividans 1326 (NBRC15675), possessing no GPE-EP activity, was used as a host for GPE-EP expression. Two recombinant plasmid vectors, pUC702/pld-sig-gpeep and pUC702/ gpeep-sig, were constructed for extracellular production of active GPE-EP as follows: the recombinant vector pUC702/pld-sig-gpeep used the signal peptide sequence (pld-sig) of phospholipase D (PLD) from S. cinnamoneum, while pUC702/gpeep-sig, was used the signal peptide sequence (gpeep-sig) of GPE-EP itself. For construction of pUC702/pld-sig-gpeep, the nucleotide of the mature GPE-EP enzyme was joined to 30 -downstream of pld-sig in pUC702 (2).
125
The PCR was performed using the following primers: 50 -tgctagcgacgggctcg gagccatcaag-30 (Nhe I-F) containing a first codon (Nhe I, italic; Asp, underlined) of mature GPE-EP and 50 -gacagatcttcagcccgagacgctcgccc-30 (Bgl II-RV; Bgl II, italic). The PCR reaction mixture (20 ml) contained: 1 KOD Plus buffer, 6 pmol of each primer set (Nhe I-F and Bgl II-RV), 4 nmol of dNTPs, 3% (vol/vol) of DMSO, 24 nmol MgSO4, 0.4 U of KOD Plus DNA polymerase (Toyobo), and 100 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 2 min; 70.2 C for 1 min after completion of the 30 cycles. The obtained fragment was purified and digested with Nhe I and Bgl II, and then subcloned into the Nhe I and Bgl II sites of pUC702, i.e., between the signal peptide sequence and the terminator region of PLD. The constructed expression plasmid was sequenced and designated as pUC702/pld-sig-gpeep. For construction of pUC702/gpeep-sig, the nucleotide of gpe-ep containing GPE-EP-signal peptide and the mature enzyme was joined to downstream of the promoter region of PLD in pUC702. To replace SphI site in gpeep, gpe-ep was amplified from the chromosomal DNA by a two-step PCR. The first PCR was performed using the following primer sets: 50 -cagcatgctcacgga catttcacggcgacgg-30 (Sph I-F1) containing a first codon (Sph I, italic; Met, underlined) of GPE-EP-signal peptide and 50 -gcacgcGCATACcgagaccggtgggcttg-30 of Sph I-repair F1 (amplification product size, approximately 1.3 kbp); 50 ccaccggtctcgGTATGCgcgtgccgatg-30 (Sph I-repair F2) and Bgl II-repair RV (amplification product size, 870 bp). The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 80 s. Each amplification product was purified and used for the second PCR as a template. The second PCR was performed using forward primer (Sph I-F1) and reverse primer (Bgl II-RV). The PCR reaction mixture (20 ml) contained: 1 KOD Plus buffer, 6 pmol of each primer set (Sph I-F1 and Bgl II-RV), 4 nmol of dNTPs, 3% (vol/vol) of DMSO, 24 nmol MgSO4, 0.4 U of KOD Plus DNA polymerase, and 50 ng each of the first amplification products as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 72 C for 2 min. The amplification product was purified and used for the additional PCR as a template. The PCR reaction mixture (20 ml) contained: 1 KOD Plus buffer, 6 pmol of each primer set (Sph I-F1 and Bgl II-RV), 4 nmol of dNTPs, 3% (vol/vol) of DMSO, 24 nmol MgSO4, 0.4 U of KOD Plus DNA polymerase, and 100 ng of the 2.1-kbp amplification product as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 94 C for 15 s and 70.2 C for 2 min. The obtained fragment was purified and digested with Sph I and Bgl II, and then subcloned into the Sph I and Bgl II sites in the pUC702, i.e., between the promoter and terminator region of PLD (13). The constructed expression plasmid was sequenced and designated as pUC702/gpeep-sig. The transformation techniques of Kieser et al. were followed for S. lividans (12). Transformants were screened by 5-mL culturing. Moreover, GPE-EP expression was attempted to produce GPE-EP using E. coli as a host. Two recombinant plasmid vectors, pET24a(þ)/gpeep and pET24a(þ)/ gpeepþHis, were constructed for production of active GPE-EP as follows. To clone gpe-ep into pET24a(þ), gpe-ep was amplified from the chromosomal DNA of S. sanglieri strain A14 by PCR. The PCR was performed using the following primers: 50 -atagtctacatatgaccacggacatttcacggcgacg-30 (Nde I-F1) containing a first codon (Nde I, italic; Met, underlined) of GPE-EP and 50 -tttaagctttcagcccgagacgctcgccc-30 (Hind III-RV1) containing Hind III site (italic). The PCR reaction mixture (25 ml) contained: 1 KOD Plus buffer ver. 2 (Toyobo), 7.5 pmol of each primer set (Nde IF1 and Hind III-RV1), 5 nmol of dNTPs, 3% (vol/vol) of DMSO, 37.5 nmol MgSO4, 0.5 U of KOD Plus DNA polymerase (Toyobo), and 150 ng of S. sanglieri chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 30 cycles of 98 C for 10 s and 72 C for 2 min. The obtained fragment was purified and digested with Nde I and Hind III, and then subcloned into the Nhe I and Hind II sites of pET24a(þ). The constructed expression plasmid was sequenced and designated as pET24a/gpeep. To add His tag to pET24a/gpeep, inverse PCR was carried out using IVS primer (50 -caccaccaccaccaccactgagatccgg-30 ) and IVAS primer (50 -gcccgagacgctcgcccggcc-30 ) designed based on the nucleotide sequence of gpe-ep on pET24a/gpeep. The inverse PCR amplification was completed with the reaction mixture (25 ml) contained 1 KOD Plus buffer ver. 2, 7.5 pmol of each primer, 5 nmol of dNTPs, 3% (vol/vol) of DMSO, 37.5 nmol MgSO4, 0.5 U of KOD Plus DNA polymerase, and 150 ng of pET24a/gpeep as a template. The PCR program was 94 C for 2 min, followed by 10 cycles of 98 C for 10 s and 68 C for 8 min. The obtained 7.5 kbp DNA fragment was self-ligated, and the resulting recombinant plasmid was called pET24a/gpeepþHis. SHuffle T7 Express Competent E. coli was transformed using pET24a/gpeep and pET24a/gpeepþHis. The transformed E. coli cells were incubated in 100 mL LB broth containing 30 mg/mL kanamycin at 30 C for 6 h with shaking (180 rpm). The expression of recombinant GPE-EP was induced with 0.4 mM IPTG at 16 C for 24-h incubation. The cells of the 500-mL culture were resuspended in 1.5 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) and sonicated. The cell-free extracts (cfe, 70 mL) were obtained by centrifugation (21,600 g for 10 min). The obtained supernatant was loaded onto a Butyl-650M column (2.5 by 5 cm) equilibrated with the same buffer. The column was washed with 3 CV, and the protein was eluted with a linear gradient as described above. The active fractions were pooled and followed by application to a HiTrap Q HP column (5 mL) as described above. The active fractions were applied to a HisTrap HP column (5 mL) equilibrated with 5 mM imidazole, 0.5 M NaCl/50 mM TriseHCl buffer (pH 8.0). The
126
MINETA ET AL.
J. BIOSCI. BIOENG., TABLE 1. Purification of GPE-EP from S. sanglieri A14.
Purification step 72-h culture supernatant 0e80% ammonium sulfate PPG-600M Hitrap Q HP Hitrap Phenyl HP Resource PHE Mono Q a
Sample (mL)
a
Activity (U/mL)
Protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification (fold)
% Recovery
480 57 98 24 35 1.5 1
3.66 29.5 11.0 23.7 8.82 18.5 10.8
369 336 99.3 13.2 5.16 0.331 0.104
4.75 4.99 10.8 43.1 59.8 83.8 103
1755 1679 1075 568 309 27.7 10.8
1 1.05 2.28 9.06 12.6 17.6 21.7
100 95.7 61.3 32.4 17.6 1.58 0.613
GPE-EP activity was assayed using the reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100 and 4 mM GPE at 65 C.
column was washed with 3 CV of the same buffer at a flow rate of 4 mL/min (2 cm/ min) and the protein was eluted with a linear gradient (20 CV) of 5 to 0.5 M imidazole at the same flow rate. Fractions exhibiting high specific activity were pooled and used for further investigation.
RESULTS Isolation and identification of strain A14 Strain A14 was assigned as S. sanglieri by morphological, physiological and biochemical characterizations, and 16S rDNA sequence analysis. S. sanglieri strain A14 was deposited as NITE BP-1392 in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan). The 16S rDNA sequence of strain A14 was deposited in the DDBJ database under the accession number AB735535. Purification of GPE-EP The enzyme was purified to electrophoretic homogeneity from the culture supernatant by ammonium sulfate precipitation, anion exchange chromatography and hydrophobic interaction chromatography. A summary of the purification is presented in Table 1. The overall purification of the enzyme from the culture supernatant was 21.7-fold, with an activity yield of 0.613%. The total amount of purified GPE-EP with a specific activity of 103 U/mg-protein was 0.104 mg of protein. Protein analysis The purified enzyme was subjected to SDSPAGE analysis. A single band with an apparent molecular mass of w70 kDa was visualized by silver staining. The result of gel filtration chromatography analysis demonstrated that native GPE-EP functions as a monomeric protein. The N-terminal amino acid sequence was determined as DGLGAIKHVV. LCeMS/MS analysis obtained a number of peptide sequences and internal amino acid
sequences (WMNGWVSAK, GTLADEFAK, TGVAEPNISDWR, TGDLTSAFDFSHAR and YAGEFPVPQHR) were selected as PCR primers for isolating the enzyme gene. Effect of pH, temperature and chemicals on GPE-EP activity As shown in Fig. 2, the highest activity for GPE hydrolysis was found at pH 8.4 and 65 C. The enzyme was stable from pH 6 to 10.2 and between 4 C and 60 C. The thermal stability of GPE-EP was w15 C higher than that of GPC-CP14 (1). Table 2 summarizes the effects of chemicals and inhibitors on GPE-EP activity for GPE hydrolysis. The enzyme activity was inhibited by 2 mM SDS, 2-mercaptoethanol, DTT, Mn2þ, Ca2þ and Fe2þ. Zn2þ ion remarkably inhibited the activity; while PMSF, IAA, Mg2þ, Cu2þ and Co2þ had almost no effect on the enzyme activity. The enzyme activity was found to increase in the presence of 2 mM EDTA. Substrate specificity Table 3 shows the substrate specificity of GPE-EP. The highest hydrolytic activity was recorded with GPE. DHPE, LPE, and LPlsPE were hydrolyzed by GPE-EP; however, only minimal activity was observed with GPG and CDP-choline as substrates. GPE-EP showed no activity with other GPX substrates of GPC, GPA, GPI and GPS. No activity was also observed for phospholipid substrates such as diacylglycerophospholipids (DBPC, POPC, DMPA, DPPE, POPE, PG, PI, PS), lysophospholipids (LPC, LPI, LPS, LPG, LPA) and plasmalogens (PlsPC, PlsPE, LPlsPC). Moreover, GPE-EP exhibited no phosphatase and glycerophosphodiester phosphodiesterase (EC 3.1.4.46) activity, i.e., no release of ethanolamine. CIAP used for the assay of GPE-EP activity was found to have no activity toward GPX.
FIG. 2. Effect of pH (A) and temperature (B) on the activity (open symbols) and stability (closed symbols) of purified GPE-EP. (A) The enzyme activity was assayed at 37 C for 5 min with 4 mM GPE in 50 mM of each buffer. The buffers were: sodium acetate (pH 4.1e5.6; open circles), BisTriseHCl (pH 5.6e7.2; open triangles), TriseHCl (pH 7.2e9.0; open squares) and glycineeNaOH (pH 9.0e10.5; open diamonds). To determine pH stability, the enzyme sample was incubated at 4 C for 3 h in 50 mM of each buffer: sodium acetate (pH 4.1e5.6; closed circles), BisTriseHCl (pH 5.6e7.2; closed triangles), TriseHCl (pH 7.2e9.0; closed squares) and glycineeNaOH (pH 9.0e10.5; closed diamonds). The residual activity was assayed by incubation at 65 C in 50 mM TriseHCl buffer (pH 8.4). (B) The enzyme activity (open circles) was assayed at each temperature in 50 mM TriseHCl buffer (pH 8.4). To determine thermal stability, the enzyme sample was incubated at each temperature for 30 min in 50 mM TriseHCl buffer (pH 8.4) and the residual activity (closed circles) was assayed by incubation at 65 C in 50 mM TriseHCl buffer (pH 8.4). Data are the means of experiments performed in triplicate. Error bars represent the standard deviation.
VOL. 119, 2015
GLYCEROPHOSPHOETHANOLAMINE ETHANOLAMINEPHOSPHODIESTERASE
TABLE 2. Effect of chemicals and inhibitors (2 mM) on the GPE-EP activity for GPE hydrolysis.a Relative activity (%)b
Chemical Control KCl NaCl MgCl2 CoCl2 CuCl2 CaCl2 FeCl2 MnCl2 ZnCl2 EDTA SDS IAA 2-Mercaptoethanol DTT PMSF
100 106 105 101 100 98.9 92.0 92.0 54.0 0.575 113 92.7 101 80.5 64.6 98.8
a The purified enzyme was assayed by using reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100, 4 mM GPE, and 2 mM chemicals or inhibitors at 65 C. b The relative activity is expressed as a percentage of the control activity without chemicals or inhibitors.
Enzyme kinetics Linear regression analysis was performed using a HaneseWoolf plot (data not shown). For the hydrolysis of GPE by GPE-EP at 65 C and pH 8.4, the apparent Vmax and kcat values were determined to be 0.430 mmol min1 mg-protein1 and 522 s1, respectively. The apparent Km and kcat/Km values were 0.785 mM and 665 mM1 s1, respectively. Intriguingly, during hydrolysis of GPE, competitive inhibition was observed in the presence of 1 mM ethanolamine or 8 mM glycerol, but not in the presence of 8 mM GPC (data not shown). For the hydrolysis of GPE at pH 8.4, activation energy was calculated to be 19.8 kJ mol1 using Arrhenius plot (data not shown). Gene cloning The partial nucleotide sequence of the gene encoding GPE-EP (gpe-ep) was determined by standard PCR using primer sets designed from N-terminal and internal amino acid sequences (TGVAEPNISDWR). The 1254-bp determined nucleotide sequence encoded a protein that is 418 amino acids in length. The 447-bp nucleotide sequence of the 50 -upstream region and 1010-bp nucleotide sequence of the 30 -downstream region were determined from a w2.5-kb inverse PCR product. Consequently, a 2711-bp nucleotide sequence was determined. The ORF of gpe-ep was determined to consist of 2124 nucleotides encoding a 708 residue protein. The complete nucleotide sequence of gpe-ep has been deposited in the DDBJ/GenBank/EMBL database under accession number AB771750. The putative TTG translational start codon was preceded at a spacing of 6 bp by a potential ribosome binding site (ggagg). A possible promoter region and terminator region were not found. As shown in Fig. S2, the N-terminal sequence of the mature enzyme starts at Asp49 of the deduced amino acid sequence, indicating that the preceding 48 amino acids are a TABLE 3. Substrate specificity of GPE-EP. Substrate GPE DHPE LPE LPlsPE GPG CDP-choline
Relative specific activity (%)a 100 4 82.8 3.4 31.2 30.1 5.11 0.955 0.413
a The purified enzyme was assayed by using the reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100, 4 mM GPX or 4 mM other phospholipids at 65 C. The relative specific activity is expressed as a percentage of the specific activity for GPE (137 U/mg protein). Results are represented as means standard deviation of experiments performed in triplicate.
127
signal sequence for secretion. A twin arginine translocation (Tat) pathway motif (RRRLF) was used for the signal sequence (14). The molecular weight of the gene product (i.e., the 660-amino-acid protein) without the signal sequence is calculated to be 72,918 Da in molecular weight, in agreement with that of the purified enzyme estimated by SDS-PAGE analysis. The isoelectric point (pI) of GPE-EP was calculated to be 6.17 using Genetyx-Mac version 16.0.8 (Genetyx Corporation, Tokyo, Japan). Comparative sequence analysis of GPE-EP A homology search performed using the BLAST algorithm indicated that the amino acid sequence of GPE-EP shares 86% and 85% identity with that of PC-specific phospholipase C (PC-PLC) from Streptomyces sp. SirexAA-E (UniProt accession no. G2NFN1) (Fig. S2) and putative non-hemolytic PLC (nhPLC) from Streptomyces sp. W007 (UniProt accession no. H0BLP6) (Fig. S3). Moreover, GPE-EP showed high identity to numerous Streptomyces PLCs, however, most of them remain to be biochemically and functionally uncharacterized. As shown in Fig. S3, a distance-based phylogenetic analysis of those protein amino acid sequences revealed that GPE-EP belongs to PLC/acid phosphatase (APase) superfamily, and it is clearly separated from a group containing GPC-CP14, Pseudomonas fluorescens protein (CGDEase) of the PLC/APase superfamily with CDP-ethanolamine and DHPE hydrolase activity (15), and AcpA. However, RGD motif conserved in the PLC/APase superfamily did not detected in GPE-EP (16). Intriguingly, we have reported previously that S. sanglieri A14 extracellularly produces GPC-CP14 (1). The amino acid sequence of mature GPE-EP shows 37.3% identity (79.5% similarity) to that of GPC-CP14. Furthermore, Pfam analysis (17) suggested that the N-terminal region (54e458) of GPE-EP is assigned as phosphoesterase family domain and shares structural similarities with AcpA (8), and two small domains of unknown function tandemly exist in the C-terminal region (520e602 and 603e698) of GPE-EP. Secondary structure analysis with PSIpred (18) predicted that the tandem domains are composed primarily of b-strands. Homology modeling analysis The structure models of GPEEP: 49e460 and GPC-CP14: 39e471 based on AcpA are shown in Fig. 3. VERIFY3D showed that the homology models of GPE-EP: 49e460 and GPC-CP14: 39e471 had 112.8 and 160.1 of 3De1D total score (i.e., modeling quality), respectively, indicating that the structural models drawn using AcpA as a template by Modeller are sufficient for comparative structural discussions. However, the C-terminal half region of GPE-EP (461N to 708G) and that of GPC-CP14 (472R to 684V) resulted in failure to create models. The putative catalytic residue was observed at T187 of GPE-EP (Fig. 3). Moreover, E63, N64, T187, D389, and E390 were conserved in GPE-EP as metal ion binding site. In addition, histidine residues (H129 and H352) surrounding phosphate of substrate were observed in the active site of GPE-EP. GPE-EP can form a disulfide bond between C176 and C438. Expression and efficient purification of recombinant GPEEP S. lividans cells transformed with pUC702 carrying gpe-ep resulted in failure to express the recombinant GPE-EP. E. coli cells transformed using pET24a/gpeep and pET24a/gpeepþHis were efficiently produced the recombinant GPE-EP. The recombinant enzyme (6.92 mg-protein) with a high specific activity (363 U/mgprotein) and total activity (2516 U) was purified to electrophoretic homogeneity from the cfe (5419 U) of E. cells carrying pET24a/ gpeepþHis using simple purification steps (Table 4). DISCUSSION We purified and characterized GPE-EP from S. sanglieri strain A14. The molecular mass (72,918 Da) of GPE-EP is very similar to that of GPC-CP14 (70,447 Da) (1) or that of CGDEase (75 kDa) from
128
MINETA ET AL.
J. BIOSCI. BIOENG.,
FIG. 3. Structures of AcpA, GPE-EP and GPC-CP14. (A) Overall structure. Three enzyme structures are represented in ribbon model from the same view. GPE-EP and GPC-CP14 are predicted by homology modeling. (B) Active site structures. Amino acid residues in the active site are depicted by stick model.
P. fluorescens CECT7229 (15), which are monomeric and extracellular proteins. However, AcpA is a homodimer of 57 kDa subunits and periplasmic enzyme (8). Costas et al. (15) reported that CGDEase is a Mg2þ-dependent enzyme and is inhibited by EDTA. AcpA appears to be a Ca2þ-dependent enzyme (8). In contrast, GPE-EP was not inhibited by EDTA, and the enzyme activity was not stimulated by tested metal ions such as Ca2þ and Mg2þ. However, the metal ion binding site (E63, N64, T187, D389, and E390) was observed in the predicted active center of GPE-EP, suggesting that a metal ion may be tightly bind to the enzyme. In fact, 1 mol of Ca2þ was detected in 1 mol of GPC-CP14 (unpublished data). Zn2þ showed similar inhibition of GPE-EP, GPC-CP14, and CGDEase (15). The highest activity of GPE-EP was observed at 65 C, in accord with the cloud point of 1% (w/v) Triton X-100 (63.7 C) (19). Besides, the enzyme activity was influenced by the concentration of Triton X-100 in the reaction mixture, and the highest activity was found in the presence of 0.1% (w/v) Triton X100 above critical micelle concentration (0.25e0.27 mM) (20). On the other hand, since GPE is a water-soluble and hydrophilic compound, Triton X-100 has little direct effect on the substrate; rather, a conformational change enhancing the enzyme activity might be caused by Triton X-100. Alternatively, the detergent may just keep the protein from binding to tube walls. In fact, the concentrated GPE-EP sample (>4 mg-protein/mL) led to the aggregation, suggesting that GPE-EP is a hydrophobic protein. Consequently, GPE-EP may be dispersed or solubilized such as membrane-bound protein by Triton X-100. For these reason, we have presumed the optimum Triton X-100 concentration exists for the enzyme activity. Further studies are required to elucidate activation mechanism of the enzyme by Triton X-100. GPE-EP was
stable across a broad pH range (pH 6e10), and the stability is very similar to that of GPC-CP14 (pH 5e10); although the maximal pH (8.4) of GPE-EP was different from that (pH 7.2) of GPC-CP14 and those of the mammalian GPC-CPs (pH 9.5 and 10), suggesting that the physiological function of GPE-EP may be essentially different from GPC-CP14 and mammalian GPC-CPs. The apparent Km (0.785 mM) of GPE-EP was somewhat higher than that of CGDEase (0.14 mM) (15) and those of mammalian GPCCPs (21e23), and one-half of that (1.41 mM) of GPC-CP14 (1). The apparent Vmax of GPE-EP (0.430 mmol min1 mg-protein1) was considerably higher than the Vmax, i.e., 24.7 mmol1 min1 mgprotein1, of GPC-CP14 (1) and that 2.4 mmol1 min1 mg-protein1 of CGDEase (1). In addition, kcat and kcat/Km values (522 s1 and 665 mM1 s1) of GPE-EP are much higher than those of GPC-CP14 (29 s1 and 20.6 mM1 s1), suggesting that GPE-EP possesses the higher substrate affinity and much higher turnover number as compared to GPC-CP14 and CGDEase. Namely, GPE-EP can treat rapidly even at lower concentration of GPE. GPC-CP14 exhibited almost equal enzyme activity toward GPC and GPE (1). However, the catalytic efficiency (kcat/Km) of GPE-EP toward GPE is much higher than that of GPC-CP14. Strain A14 produces GPE-EP as well as GPC-CP14. Therefore, we conclude GPE-EP is specialized to degrade GPE. The kcat and kcat/Km values of GPE-EP are higher than those of previously reported PLCs (BRENDA, http://www.brenda-enzymes. info/php/result_flat.php4?ecno¼3.1.4.3), yet lower than those of general bacterial enzymes, suggesting that the binding affinity of GPE-EP toward substrate GPE is higher than those of bacterial enzymes, and the turnover number (kcat) and the catalytic efficiency (kcat/Km) may be somewhat lower than the vast majority of enzymes. The activation energy (19.8 kJ mol1) for the hydrolysis of
TABLE 4. Purification of recombinant GPE-EP. Purification step cfe of 24-h culture Butyl-650M Hitrap Q HP Histrap HP a
Sample (mL)
a
Activity (U/mL)
Protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification (fold)
% Recovery
70 40 25 6
77.4 98.9 133 419
84.6 34.7 10.9 6.92
64.0 114 306 363
5419 3957 3333 2516
1 1.8 4.8 5.7
100 73.0 61.5 46.4
GPE-EP activity was assayed using the reaction mixture containing 50 mM TriseHCl buffer (pH 8.4), 0.1% (w/v) Triton X-100 and 4 mM GPE at 65 C.
VOL. 119, 2015
GLYCEROPHOSPHOETHANOLAMINE ETHANOLAMINEPHOSPHODIESTERASE
GPE by GPE-EP was similar to that (18.8 kJ mol1) reported for lecithin, i.e., PC, hydrolysis by the wild-type SaPLA1 (2). Here, we discuss the substrate specificity and substrate recognition of GPE-EP. GPE-EP exhibited GPE-specific activity and no activity toward all diacylglycerophospholipids containing PE, except for DHPE, strongly suggesting that GPE-EP is not originally a PLC. Nevertheless, GPE-EP hydrolyzed DHPE, LPE and LPlsPE with w30e80% relative activity, indicating that the enzyme prefers ethanolamine-type substrates. Triton X-100 molecules can form mixed micelles with diacylglycerophospholipids including DHPE, DPPE and POPE or lysophospholipids including LPE and LPlsPE. PE molecules with two long acyl chains (DPPE and POPE) should be exclusively contained within the micelles, whereas PE molecules with short acyl chains (DHPE) or with single acyl chain (LPE and LPlsPE) may be partly exist as monomers (24e26). Thus, both the physical characteristics of their interfaces would be distinct from each other; consequently, both of them are different from each other in the binding mode for the enzyme and the substrate molecules. George et al. reported that both the surface concentration of lipid and the bulk concentration play critical roles in defining the kinetic parameters of lipid-dependent enzymes (24). Taken together, the difference in the binding mode may affect the activity of GPE-EP. Alternatively, the specificity toward DHPE, LPE and LPlsPE is likely to be controlled (in part) by steric hindrance of the fatty acyl chain of substrates. We believe that GPE-EP possibly recognizes the head group and the acyl part of the alkenylether chain of the substrate. The results of competitive inhibition of GPE-EP activity by ethanolamine or glycerol support our conclusion. Costas et al. reported that CGDEase prefers CDP-ethanolamine (100% relative activity), GPE (96% relative activity) and DHPE (115% relative activity), and showed activity toward CDPcholine (23% relative activity), GPC (7% relative activity), and pNPPC (29% relative activity) (15). The substrate specificity of GPEEP seems to be somewhat similar to that of CGDEase; however, GPE-EP exhibited no activity toward CDP-choline and GPC, demonstrating that the substrate molecule recognition by GPE-EP is more stringent than those of CGDEase and GPC-CP14 (1); the substrate molecule recognition of these enzymes may be distinct from one another. It has also been reported that, in the case of Bacillus cereus PC-PLC, the polar head group and also the associated carbonyl group are important for phospholipid recognition (27). The fatty acyl chains also play a significant role in substrate binding and the PLC appears to prefer greater than six carbons for hydrolysis of phospholipids. In summary, we conclude that GPE-EP may recognize hydroxyl groups of the glycerol backbone as well as the head group and the fatty acyl chain conformation of the substrate molecule. Further studies are required to elucidate the substrate recognition mechanism of GPE-EP. The crystallization of GPE-EP is currently being undertaken. We discuss physiological role of GPE-EP. As shown in Fig. S4, although glycerophosphodiester phosphodiesterase (EC 3.1.4.46) has been known, there is no report of GPE-EP. We therefore propose that GPE hydrolysis by GPE-EP may represent a new metabolic pathway for PE metabolism and acquisition of phosphorus and carbon sources in actinomycetes. Because of the limited availability of environmental phosphorus (
