Journal of Bioscience and Bioengineering VOL. 117 No. 4, 422e430, 2014 www.elsevier.com/locate/jbiosc
Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri Daisuke Sugimori,1, * Junki Ogasawara,1 Koki Okuda,1 and Kazutaka Murayama2 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 2 September 2013; accepted 4 October 2013 Available online 7 November 2013
A novel metal ion-independent glycerophosphocholine cholinephosphodiesterase (GPC-CP) of Streptomyces sanglieri was purified 53-fold from culture supernatant with 1.1% recovery (583 U/mg-protein). The enzyme functions as a monomer with a molecular mass of 66 kDa. The gene encoding the enzyme consists of a 1941-bp ORF that produces a signal peptide of 38 amino acids for secretion and a 646 amino acid mature protein with a calculated molecular mass of 70,447 Da. The maximum activity was found at pH 7.2 and 40 C. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37e60 C) and within a narrow pH range near pH 7. The enzyme was stable at 50 C for 30 min and between pH 5e10.5. The enzyme exhibited specificity toward GPC and glycerol-3-phosphoethanolamine and hydrolyzed glycerol-3-phosphate and lysophosphatidylcholine. However, the enzyme showed no activity toward any diacylglycerophospholipids and little activity toward other glycerol-3-phosphodiesters and lysophospholipids. The enzyme was not inhibited in the presence of 2 mM SDS and Mg2D; however, Cu2D, Zn2D, and Co2D remarkably inhibited activity. Enzyme activity was also slightly enhanced by Ca2D, NaD, EDTA, DTT, and 2-mercaptoethanol. During the hydrolysis of GPC at 37 C and pH 7.2, apparent Vmax and turnover number (kcat) were determined to be 24.7 mmol minL1 mg-proteinL1 and 29.0 sL1, respectively. The apparent Km and kcat/Km values were 1.41 mM and 20.6 mML1 sL1, respectively. GPC hydrolysis by GPC-CP might represent a new metabolic pathway for acquisition of a phosphorus source in actinomycetes. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycerophosphocholine cholinephosphodiesterase; Purification; Characterization; Molecular cloning; Expression; New metabolic pathway; Streptomyces]
Glycero-3-phosphodiester is an important intermediate in glycerophospholipid metabolism. Nevertheless, there is only few limited information on the metabolic pathway and key enzyme. Glycerophosphocholine cholinephosphodiesterase (GPC-CP; EC 3.1.4.38) cleaves sn-glycerol-3-phosphocholine (GPC) into glycerol and phosphocholine (Fig. 1). GPC-CP has been found in the brains of mammals such as Bos taurus, Mus musculus, Rattus norvegicus, and Homo sapiens (BRENDA, http://www.brenda-enzymes.info/php/ result_flat.php4?ecno¼3.1.4.38). Janzen et al. reported that GPCCP activity is reduced in multiple sclerosis plaques (1). It has also been reported that GPC exhibits physiological functions such as enhancing the secretion of growth hormone and alleviating the cognitive symptoms of Alzheimer’s disease (2,3). Therefore, GPC-CP is useful for investigating the biological functions of GPC. The known GPC-CPs can be further divided into groups based on cellular location, i.e., membrane-bound and cytosolic enzymes. These are metal
* Corresponding author. Tel./fax: þ81 24 548 8206. E-mail address: [email protected] (D. Sugimori).
ion-dependent enzymes requiring Ca2þ, Co2þ, Cu2þ, Mn2þ, or Zn2þ for their activity. However, this enzyme has not been purified and characterized in detail, and to date, no information is available concerning the gene and amino acid sequence of GPC-CP. During our screening study of phospholipase C (PLC), we unexpectedly detected a novel metal ion-independent GPC-CP (GPCCP14) secreted by Streptomyces sanglieri strain A14. Although Streptomyces PLCs have been reported (4e6), no previous information is available regarding a bacterial GPC-CP; therefore, its significance in the metabolism of GPC, glycerophospholipids, or phosphorus is unknown. Moreover, finding of GPC-CP14 will help to identify the missing link in the glycerophospholipid metabolism in other organisms as well as actinomycetes. Here, we report the purification, characterization, molecular cloning, and extracellular production of this novel GPC-CP14. We also describe the kinetics for the hydrolytic reaction and the substrate specificity of the enzyme. To our knowledge, this represents the first report of purified GPCCP as novel-type enzyme. Further characterization of GPC-CP may allow prediction of the role of PLC, phosphatase, or phosphodiesterase superfamily proteins with unknown function.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.10.004
VOL. 117, 2014
NOVEL GLYCEROPHOSPHOCHOLINE CHOLINEPHOSPHODIESTERASE
423
FIG. 1. Hydrolytic reaction of GPC by GPC-CP.
MATERIALS AND METHODS Materials Bacto tryptic soy broth (TSB), Bacto tryptone, Bacto peptone, Bacto casamino acids and Bacto 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-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-snglycero-3-phosphate (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1-palmitoyl-2-oleoylsn-glycero-3-phospho-rac-(1-glycerol) (POPG), L-a-lysophosphatidylcholine (LPC), L-a-lysophosphatidylserine (LPS), L-a-lysophosphatidylglycerol (LPG), 1-stearoyl-2hydroxy-sn-glycerol-3-phosphate (LPA), L-a-lysophosphatidylinositol (LPI), 1-O-10 (Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine (LPlsPC), 1-O-10 -(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPlsPE), 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (PlsPC), and 1-(1Zoctadecenyl)-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). L-a-Lysophosphatidylethanolamine (LPE) was purchased from Doosan Serdary Research Laboratories (Toronto, ON, Canada). Sphingomyelin (SM) from chicken egg yolk and b-acetyl-gsn-hexadecyl-L-a-phosphatidylcholine (PAF) were purchased from SigmaeAldrich Japan Co., LLC. (Tokyo, Japan). sn-Glycerol-3-phosphocholine (GPC) was purchased from Bachem (Torrance, CA, USA). Recombinant phospholipase A1 (PLA1) from Streptomyces albidoflavus strain NA297 was extracellularly produced by Streptomyces lividans and was used for synthesis of sn-glycerol-3-phosphodiesters (GPXs) after ammonium sulfate purification (7). GPXs such as sn-glycerol-3-phosphoethanolamine (GPE), sn-glycerol-3phosphoserine (GPS), sn-glycerol-3-phosphoinositol (GPI), sn-glycerol-3-phosphate (GPA), and sn-glycerol-3-phosphoglycerol (GPG), except GPC, were prepared using the recombinant PLA1 as mentioned below. DEAE Sepharose Fast Flow, HiTrap Butyl FF, HiTrap Q HP, Mono Q, Mono P, RESOURCE ISO, HisTrap HP, Superdex 200 10/300 GL columns, and Polybuffer 74 were purchased from GE Healthcare Japan (Tokyo, Japan). All other chemicals were of the highest or analytical grade. GPXs were prepared using the recombinant PLA1 as follows. Lysophospholipids (8 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 PLA1 (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 PLA1. The concentration of free fatty acids was determined with a NEFA C Kit (Wako Pure Chemical Industries) 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 (water) layer was collected and the solvent was removed by evaporation for 5 min using vacuum evaporator. The resulting aqueous solution was used for investigation of substrate specificity. Bacterial strains and culture conditions Approximately 1500 strains as unidentified actinomycetes were isolated from soil samples obtained from Fukushima, Japan, using humic acidevitamin agar plate culture (8). Strains exhibiting PLC activity were screened using lecithin-emulsion medium (9). PLC activity toward PE or LPC was assessed in an assay mixture, comprising 10% (v/v) culture supernatant, 50 mM HEPESeNaOH buffer (pH 7.2), 0.8 mM PE or LPC, 1% (w/v) Triton X-100 (no addition for LPC). The enzyme activity was determined using a method described in the following enzyme activity assays. Most of strains exhibited very little PLC activity toward PE. Strain A14, which showed the highest hydrolytic activity toward LPC in a 5-mL culture, was selected for further investigation. Moreover, we found that GPC was the preferred substrate over LPC. Consequently, GPC-CP from strain A14 was investigated. Strain A14 was maintained on ISP2 (10 g malt extract, 4 g yeast extract, and 4 g glucose per liter, pH 7.2) agar plates. Several colonies from the agar plate were scraped and inoculated into a test tube (18 mm, 180 mm) containing 5 mL of the seed medium of ISP2. The mixtures 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 was cultivated with shaking (180 rpm) at 28 C for 48 h. The
culture supernatant was obtained by centrifugation (18,800 g for 20 min) and the enzyme was purified. Escherichia coli HST08 premium competent cells (Takara Bio Inc., Shiga, Japan) were used as a host for molecular cloning. A pGEM-T Easy Vector (Promega Corporation, Madison, WI, USA) was used as a cloning vector. A pUC702 carrying the promoter, signal peptide sequence and the terminator region of phospholipase D (PLD) from Streptoverticillium cinnamoneum was used as an expression vector (10). Recombinant E. coli cells were cultured on LuriaeBertani agar plates at 37 C, and when necessary, ampicillin, isopropyl-b-D-thiogalactopyranoside and X-Gal were used as supplements in the agar. Recombinant S. lividans cells were cultured in 3% (w/v) TSB containing 5 mg/mL thiostrepton at 28 C. S. lividans 1326 (NBRC15675), which was obtained from NITE Biological Resource Center (Chiba, Japan), was used as a host for the expression of proteins. 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 48 h of culturing. The enzyme was purified by 30e80% ammonium sulfate fractionation. The obtained precipitate was suspended in 20 mM TriseHCl buffer, pH 9.0 (buffer A) and dialyzed against buffer A. The enzyme sample was loaded onto a DEAE Sepharose column (2.5 3.5 cm) equilibrated with buffer A. The column was washed with 3 column volumes (CV) of buffer A, and protein was eluted with a linear gradient (10 CV) of 0e1 M NaCl in buffer A at 6 mL/min (1.22 cm/min). The active fractions were pooled, and the buffer was replaced by 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20e10,000 MWCO (GE Healthcare Japan) followed by application to a HiTrap Butyl FF column (5 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer, and protein was eluted with a linear gradient (5 CV) of 1 to 0 M (NH4)2SO4 in 20 mM TriseHCl buffer (pH 8.0) and an isocratic elution (20 CV) of the same buffer at 2.5 mL/min (1.25 cm/ min). The active fractions were pooled, and the buffer was exchanged with buffer A using Vivaspin 20 followed by application to a HiTrap Q HP column (5 mL) equilibrated with buffer A. After the column washing (3 CV), the protein was eluted with a linear gradient (20 CV) of 0e1 M NaCl in buffer A at 2.5 mL/min (1.25 cm/min). The active fractions were pooled, and the buffer was exchanged with buffer A using Vivaspin 20 followed by application to a Mono Q column (1 mL) equilibrated with buffer A. After the column washing (3 CV), the protein was eluted with a linear gradient (20 CV) of 0e1 M NaCl in buffer A at 0.5 mL/min (2.5 cm/min). The active fractions were pooled and then exchanged with 0.15 M NaCl/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20. The enzyme solution was applied to a Superdex 200 10/ 300 GL column (24 mL) equilibrated with the same buffer, and the protein was eluted at 1 mL/min (1.27 cm/min). The active fractions were pooled and then exchanged with 25 mM BisTriseHCl buffer (pH 6.3) using Vivaspin 20. The enzyme solution was applied to a Mono P column (1 mL) equilibrated with the same buffer. After the column washing (3 CV), the protein was eluted with a linear pH gradient (20 CV) of Polybuffer 74-HCl (pH 4) at 0.5 mL/min (2.5 cm/min). Fractions exhibiting high specific GPC-CP activity were pooled and used for subsequent investigation. Enzyme activity assays For GPC-CP activity, a standard assay mixture, containing 5 mL of enzyme solution, 12.5 mL of 0.2 M TriseHCl buffer (pH 7.2), 5 mL of 80 mM GPC and 27.5 mL of distilled water was incubated at 37 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 microliters of the supernatant was 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 phosphorylcholine was quantified with BIOMOL Green Reagent according to the manufacturer’s instructions (11). 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. Substrate specificity The substrate specificity of purified GPC-CP was assessed by assaying enzyme activity as described above using alternative GPX substrates (0.8 mM final concentration) in the assay. In the case of phospholipid substrates such as diacylglycerophospholipids, lysophospholipids, and plasmalogens, the hydrolytic activity was assessed in an assay mixture, comprising 5 mL of purified enzyme solution, 12.5 mL of 0.2 M HEPESeNaOH buffer (pH 7.2), 5 mL of 8 mM phospholipid substrates/5% (w/v) Triton X-100, and 27.5 mL of distilled water. Effect of pH, temperature, and chemicals on enzyme activity Each buffer (sodium acetate, BisTriseHCl, TriseHCl, and glycineeNaOH) was used to identify
424
SUGIMORI ET AL.
optimum pH and pH stability of the purified enzyme. The optimum pH was examined by incubation at 37 C for 5 min with 8 mM GPC 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 standard assay conditions. GPC-CP activity was determined at each temperature point under standard assay conditions. Thermal stability was determined by incubating the purified enzyme in 0.16 M TriseHCl (pH 7.2) at each temperature for 30 min, and then residual activity was measured by incubation under standard assay conditions. The effect of chemicals such as metal ions and SDS on the enzyme activity was also investigated. The purified enzyme was assayed under standard assay conditions following pre-incubation at 37 C for 5 min with the same assay buffer containing 2 mM of chemicals or inhibitors. Inhibitors assessed were EDTA, 2-mercaptoethanol, dithiothreitol (DTT), iodoacetamide (IAA), and PMSF. PMSF was dissolved in dimethyl sulfoxide and added to the enzyme sample. In the case of IAA, the purified enzyme samples (4.17 mg/mL, 59.2 nM) were incubated at 37 C for 60 min in the same buffer containing 2 mM IAA before use in enzyme assays. Protein analysis Protein concentration was determined with Pierce BCA protein assay kit (Takara Bio Inc.) and BSA as the standard. Protein samples were analyzed by SDS-PAGE according to methods described by Laemmli (12). The molecular mass of purified enzyme was estimated by gel filtration. Gel filtration was performed using the Superdex 200 10/300 GL column (1.0 30 cm) at 0.5 mL/min (0.64 cm/min) with 0.15 M NaCl/20 mM TriseHCl (pH 8.0). 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). The PVDF membrane was stained with Coomassie brilliant blue R-250 (CBB), and the transferred 66-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 internal terminal amino-acid sequencing, the SDS-PAGE gel was stained using CBB, and the 66-kDa band was excised and then decolorized with 30% acetonitrile containing 25 mM (NH4)2HCO3. The in-gel digestion was performed using a method described by Shevchenko et al. (13). Briefly, the excised 66-kDa band was digested with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation) for 45 h at 4 C. The fragments were analyzed using a nano ACQUITY UPLC Xevo QTof MS system (Waters Corp., Milford, MA, USA). The sample solution was transferred to an autosampler vial. One mL of sample was separated on a nano ACQUITY UPLC BEH130 C18 column (75 mm 150 mm, 1.7 mm) and analyzed using a Xevo QTOF MS, according to previously reported methods (7). Steady-state kinetics For hydrolysis of GPC and LPC as substrate, the initial velocity (v) of the enzymatic reaction was determined at each concentration of the substrate under standard assay conditions. The concentration of LPC ([LPC]) was calculated using a molecular weight of 503.33. The purified enzyme concentration was constant at 4.17 mg mL1 (59.2 nM). The corresponding v vs. 1/[GPC] or 1/[LPC] plots was treated according to a MichaeliseMenten equation. Kinetic constants were determined by extrapolation using the LineweavereBurk plot by linear regression (KaleidaGraph, Synergy Software). The Km and Vmax were determined from the xand y-intercepts of the regression line, respectively. The kcat was calculated using a molecular weight of 70,447 for a monomeric protein with a single catalytic site. Chromosomal DNA of S. sanglieri strain A14 was Cloning of GPC-CP14 gene purified according to methods reported by Kieser et al. (14). Oligonucleotides were synthesized based on the N-terminal (GTVQDVEHIVV) and internal amino acid sequences (TVTNAY, PYVDGAAD, and GDQAGAAF) of the enzyme for use in PCR with sense primer N (50 -ccgtscaggacgtsgagcacatcg-30 ) and antisense primer A1 (50 -aasgcsgcgccsgcctggtc-30 ), A2 (50 -tgttgtasgcgttggtsacg-30 ), and A3 (50 gcsgcgccgtcgacgtasg-30 ). The PCR reaction mixture (20 mL) contained: 1 KOD FX buffer, 6 pmol of each primer, 8 nmol of dNTPs, 0.5 U of KOD FX DNA polymerase (Toyobo Co., Ltd., Osaka, Japan), and w14 ng of the chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 1.5 min, and 68 C for 2 min after completion of the 25 cycles. The obtained PCR fragment was cloned into pGEM-T Easy Vector, and the resulting recombinant plasmid was designated pGPC. Sequencing of the partial GPC-CP gene on pGPC was performed with the BigDye Terminator cycle sequencing kit (Applied Biosystems) and analyzed in an ABI Prism 3100 genetic analyzer (Applied Biosystems). To reveal the nucleotide sequence of the 30 downstream region of the gene, gene walking was performed using Takara LA PCR in vitro Cloning kit (Takara Bio) as follows: genomic DNA (3 mg) was digested with PstI and then ligated to PstI Cassette (Takara Bio) and used as a template for PCR. The 1st PCR amplification was carried out in the reaction mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol each of Cassette primer C1 (Takara Bio, 50 -gtacatattgtcgttagaacgcgtaatacgactca-30 ) and gene-specific primer Gw1 (50 caagtacaaactgacgttcagc-30 ), 8 nmol of dNTPs, and 0.5 U of KOD FX Neo DNA polymerase (Toyobo Co., Ltd.). The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 25 cycles. The reaction mixture (0.4 mL) from the 1st PCR was used as a template for the 2nd PCR. The 2nd PCR amplification was carried out in a PCR mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol each of Cassette primer C2 (Takara Bio, 50 -cgttagaacgcgtaatacgactcactatagggaga-30 ) and
J. BIOSCI. BIOENG., gene-specific primer Gw2 (50 -aagcccgtctccgacagctggaac-30 ), 8 nmol of dNTPs, and 0.5 U of KOD FX Neo DNA polymerase. Amplification was completed using the above-mentioned thermal cycling parameters. To reveal the nucleotide sequence of the 50 -upstream region of the gene, inverse PCR was carried out using Inv-S primer (50 -caccaggccagctttttcggcaaggaaacg-30 ) and Inv-AS primer (50 gaagccccggacgcccttcatcgacccgaagt-30 ) designed based on the 50 -region of the partial sequence of GPC-CP gene on pGPC. The genomic DNA (5.5 mg) was digested with SphI and self-ligated. The inverse PCR amplification was completed with a reaction mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol of each primer, 0.5 U of KOD FX Neo DNA polymerase, and w55 ng (above the 0.4-mL ligation mixture) of the SphI-digested and self-ligated DNA. Amplification was completed using the above-mentioned thermal cycling parameters. The generated fragment (w1.7 kbp) was cloned into the pGEM-T Easy Vector and sequenced. To clone the complete GPC-CP14 gene from the chromosomal DNA of strain A14, sense primer GPC-NS (50 -atgaccgaaatcaaccggcgtcgcttcc-30 ) and antisense primer GPC-CA (50 tcacacggtcgcgatgcccgggtcactcac-30 ) were designed based on the sequence of the GPC-CP14 gene revealed by gene walking and inverse PCR as described above. The genomic PCR amplification was completed with a reaction mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol of each primer, 8 nmol of dNTPs, 0.5 U of KOD FX Neo DNA polymerase, and the chromosomal DNA (w14 ng) using the above-mentioned thermal cycling parameters. 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 GPC-CP14 gene, designated gpc-cp, were deposited in the DDBJ/GenBank/EMBL database under the accession numbers AB735535 and AB771744, respectively. Expression and purification of recombinant GPC-CP14 S. lividans 1326 (NBRC15675), possessing no GPC-CP activity, was used as a host for GPC-CP14 expression. Two recombinant plasmid vectors, pUC702/pld-sig-gpccp and pUC702/ gpccp-sig, were constructed for extracellular production of active GPC-CP14 as follows: the recombinant vector pUC702/pld-sig-gpccp utilized the signal peptide sequence (pld-sig) of PLD from S. cinnamoneum, while pUC702/gpccp-sig used the signal peptide sequence (gpccp-sig) of GPC-CP14 itself. For construction of pUC702/ pld-sig-gpccp, the nucleotide of the mature GPC-CP14 enzyme was joined at the 30 downstream end of pld-sig in pUC702 (7). To replace the BglII site in gpc-cp, gpc-cp was amplified from the chromosomal DNA of S. sanglieri strain A14 by a two-step PCR. The first PCR was performed using the following primers: 50 aaagctagcggaaccgtccaggacgtcg-30 (Nhe I-F1) containing a first codon (Nhe I, italic; Gly, underlined) of mature GPC-CP14 and 50 -ggcgatccaggATATCTtcggcaatctg-30 (Bgl II-repair RV1); 50 -cagattgccgaAGATATcctggatcgcc-30 (Bgl II-repair F2) and 50 ataagatctcacggtcgcgatgcccgg-30 (Bgl II-RV2; BglII, italic). The PCR reaction mixture (20 mL) contained 1 KOD Plus buffer (Toyobo), 5 pmol of each primer set (Nhe I-F1 and Bgl II-repair RV1; Bgl II-repair F2 and Bgl II-RV2), 8 nmol of dNTPs, 0.5 U of KOD Plus DNA polymerase (Toyobo), and 51.6 ng of the chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 25 cycles. The second PCR was performed using each first-step amplification product as a template with forward primer (Nhe I-F1) and reverse primer (Bgl IIRV2). The second PCR was carried out in a reaction mixture (20 mL) containing w300 ng of the products (w800 bp) amplified using the primer set of Nhe I-F1 and Bgl II-repair RV1, and w300 ng of the products (w1.1 kbp) amplified using the primer set of Bgl II-repair F2 and Bgl II-RV2. Amplification was done under the above-mentioned conditions. The obtained fragment was purified and digested with NheI and BglII, and then sub-cloned into the NheI and BglII 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/pldsig-gpccp. For construction of pUC702/gpccp-sig, the nucleotide of gpc-cp containing the GPC-CP-signal peptide and the mature enzyme were joined downstream of the promoter region of PLD in pUC702. To replace SphI and BglII sites in gpc-cp, gpc-cp was amplified from the chromosomal DNA by a three-step PCR. The first PCR was performed using the following primer sets: 50 aaagcatgcttgaaatcaaccggcgtc-30 (Sph I-F1) containing a first codon (Sph I, italic; Met, underlined) of GPC-CP-signal peptide and Bgl II-RV2 (amplification product size, w2 kbp); 50 -gacgacctggGAATGCagttcatcgcag-30 (Sph I-repair F2) and Bgl IIrepair RV1 (amplification product size, w580 bp). Amplification was done under the above-mentioned conditions. Each amplification product was purified and additional PCR with 5 cycles was performed using each first-step amplification product as a template without primer. The second PCR was performed using the additional amplification product as a template with forward primers (Sph I-F1 and Sph I-repair F2) and reverse primers (Bgl II-repair RV1 and Bgl II-RV2). Amplification was done under the above-mentioned conditions. Each amplification product was purified and additional PCR with 5 cycles was performed using the second PCR products as a template without primer. The thermal cycling parameters were 94 C for 2 min, followed by 5 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 5 cycles. The 3rd PCR was performed using the additional amplification product as a template with forward primer (Sph I-F1) and reverse primer (Bgl II-RV2). The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 25 cycles. The obtained fragment was purified and digested with SphI and BglII, and then sub-cloned into
VOL. 117, 2014
NOVEL GLYCEROPHOSPHOCHOLINE CHOLINEPHOSPHODIESTERASE
425
TABLE 1. Purification of GPC-CP from S. sanglieri A14. Purification step 48-h Culture supernatant 30e80% Ammonium sulfate DEAE Sepharose HiTrap Butyl FF HiTrap Q HP Mono Q Superdex 200 Mono P a b
Activitya (U/ml) 1.45 5.81 6.58 5.94 10.2 12.3 9.75 7.31
Protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification (fold)
% Recovery
11.0 8.02 18.8 78.2 164 91.8 594 583b
665 436 395 297 205 55.4 24.4 7.31
1 0.728 1.70 7.11 14.9 8.34 54.0 53.0
100 65.5 59.4 44.6 30.8 8.33 3.67 1.10
60.3 54.3 21.0 3.80 1.26 0.603 4.10 102 1.25 102
The enzyme activity was assayed using the reaction mixture containing 50 mM TriseHCl buffer (pH 7.2) and 0.8 mM GPC at 37 C. One ml of the purified enzyme sample was obtained the 460-ml culture supernatant.
the SphI and BglII sites in the pUC702, i.e., between the promoter and terminator regions of PLD (15). The constructed expression plasmid was sequenced and designated pUC702/gpccp-sig. The transformation techniques of Kieser et al. were followed for S. lividans (14). Transformants were screened by 5-mL cultivation, and those exhibiting the highest GPC-CP activity were selected. Moreover, His-tag (His 6) was joined to the Cterminal amino acid sequence of GPC-CP14 by inverse PCR using pUC702/gpccp-sig as a template and 2 primers, His-InvSense (50 -caccaccactgagacgactgagcgcccggacg30 ) and His-InvAnti (50 -gtggtggtgcacggtcgcgatgcccgggtc-30 ), followed by phosphorylation and self-ligation using KOD-Plus-Mutagenesis Kit (Toyobo). Amplification was done under the above-mentioned conditions. Recombinant GPC-CP14 produced by the transformed S. lividans was purified from 72-h culture supernatant. Ammonium sulfate precipitation (80% sat.) was
done by the above-mentioned experimental procedures. The resulting precipitate was suspended in 20 mM TriseHCl buffer (pH 8.0). The enzyme sample was loaded onto a HisTrap HP column (5 mL) equilibrated with 20 mM HEPESeNaOH buffer (pH 7.4) containing 0.5 M NaCl and 20 mM imidazole. The column was washed with 5 CV of the same buffer, and the protein was eluted with a linear gradient (20 CV) of 20e500 mM imidazole in the same buffer at 2.5 mL/min (1.25 cm/min). The active fractions were pooled. The buffer was exchanged with 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20 and then applied to a Resource ISO column (1 mL) equilibrated with the same buffer. After the column washing (3 CV), the protein was eluted with a linear gradient (20 CV) of 1 to 0 M (NH4)2SO4 in 20 mM TriseHCl buffer (pH 8.0) at 0.5 mL/min (1.6 cm/ min). Fractions exhibiting high specific activity were pooled and used for subsequent investigation.
FIG. 2. Effect of pH and temperature on GPC-CP14 activity (A, B) and stability (C, D). (A) The enzyme activity was assayed at 37 C for 5 min with 8 mM GPC in 50 mM of each buffer. The buffers were sodium acetate (pH 4.1e5.6), BisTriseHCl (pH 5.6e7.2), TriseHCl (pH 7.2e9.0) and glycineeNaOH (pH 9.0e10.5). (B) The enzyme activity was assayed at each temperature in 50 mM TriseHCl buffer (pH 7.2). (C) The enzyme was incubated at 4 C for 3 h in 50 mM of each buffer solution. The remaining activity was assayed by incubation at 37 C in 50 mM TriseHCl buffer (pH 7.2). (D) The enzyme was incubated at each temperature for 30 min in 50 mM TriseHCl buffer (pH 7.2), and the residual activity was assayed by incubation at 37 C in 50 mM TriseHCl buffer (pH 7.2). Data are the means of experiments performed in triplicate. Error bars represent the standard deviation.
426
SUGIMORI ET AL.
J. BIOSCI. BIOENG., RESULTS
Isolation and identification of strain A14 Strain A14 was assigned as S. sanglieri by morphological, physiological, 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 accession number AB735535. Purification of GPC-CP14 The overall purification of GPC-CP14 over the culture supernatant of strain A14 was 53-fold, with an activity yield of 1.10% (Table 1). The purified GPC-CP14 with a specific activity of 583 U/mg-protein was obtained for 12.5 mg of protein. Isoelectric point (pI) of GPC-CP14 was estimated to be w5.8 by Mono P column chromatography, in agreement with pI 5.72 calculated using Genetyx-Mac version 16.0.8 (Genetyx Corporation, Tokyo, Japan). Protein analysis The purified enzyme was subjected to SDSPAGE analysis. A single band with an apparent molecular mass of w66 kDa was visualized by CBB stain (Fig. S1). The molecular mass of the native enzyme was estimated to be w68 kDa by gel filtration chromatography, demonstrating that native GPC-CP14 is a monomeric protein. The N-terminal amino acid sequence was determined as GTVQDVEHIVV. By LCeMS/MS analysis, 13 peptide sequences with high probability values were selected as internal amino acid sequences. Among these, the internal amino acid sequences (TVTNAY, PYVDGAAD, and GDQAGAAF) were selected as PCR primers for the enzyme gene cloning. Effect of pH, temperature, and chemicals on enzyme activity As shown in Fig. 2, the highest activity for GPC hydrolysis was found at pH 7.2 and 40 C. The enzyme maintained high activity of >95% relative activity between 37 C and 60 C. The enzyme was stable from pH 5 to pH 10.2 and from 4 C to 45 C. Table 2 summarizes the effects of chemicals and inhibitors on the enzyme activity for GPC. The enzyme activity was inhibited by Mn2þ and Fe2þ; while Co2þ, Cu2þ, and Zn2þ remarkably inhibited enzyme activity. Magnesium ion, Kþ, IAA and SDS had almost no effect on enzyme activity. Enzyme activity was slightly increased by Ca2þ, Naþ, 2-mercaptoethanol, DTT, and EDTA. Interestingly, enzyme activity appeared to be inhibited by dimethyl sulfoxide, used as solvent, but not PMSF.
TABLE 2. Effect of chemicals and inhibitors (2 mM) on the enzyme activity for GPC hydrolysis. Chemical Control CaCl2 MgCl2 MnCl2 ZnCl2 CoCl2 FeCl2 CuCl2 NaCl KCl EDTA Sodium dodecyl sulfate 2-Mercaptoethanol Dithiothreitol Iodoacetamideb PMSF
Relative activity (%)a 100 114 99.2 79.5 0.380 19.4 50.6 0 111 100 113 104 111 105 94.0 78.8
a The purified enzyme was assayed by using reaction mixture containing 50 mM TriseHCl buffer (pH 7.2), 8 mM GPC, and 2 mM chemicals or inhibitors at 37 C. The relative activity is expressed as a percentage of the control activity without chemicals or inhibitors. b After the incubation for 60 min with IAA, the enzyme activity was assayed by the same above.
Substrate specificity Table 3 summarizes the substrate specificity of GPC-CP14. The highest hydrolytic activity was recorded with GPC, while GPE was also preferred. Both LPC and GPA were hydrolyzed by the enzyme, but only minimal activity was observed with LPlsPC and LPE. Additionally, GPC-CP14 produced only minimal hydrolysis with GPI, GPG, LPA, LPlsE, DPPE, and POPS, and produced no discernible activity with GPS, LPI, LPS, LPG, DPPC, DMPA, POPG, PI, SM, PAF, PlsPC, or PlsPE. Enzyme kinetics Linear regression analysis was performed using a LineweavereBurk plot (Fig. 3). For the hydrolysis of GPC by the purified GPC-CP14 at 37 C and pH 7.2, apparent Vmax and turnover rate (kcat) values were determined to be 24.7 mmol min1 mg-protein1 and 29.0 s1, respectively. The apparent Km and kcat/Km values were 1.41 mM and 20.6 mM1 s1, respectively. Intriguingly, during hydrolysis of LPC, substrate inhibition was observed at >1 mM LPC; however, at LPC [ LPlsPC. GPC-CP14 appeared to prefer the choline-associated substrate; however, no activity was recorded with DPPC, SM, PAF, and PlsPC. Additionally, substrate preference observed the following order: GPE [ LPE [ LPlsPE > DPPE. It is also known that the head group of these choline-associated substrates and GPE must be in the same ionization state (21,22). These results suggest that the enzyme may prefer choline and ethanolamine of the substrate head group, and this substrate specificity results from steric hindrance of the fatty acyl chain of substrates but not the ionization state of the substrate head group. 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 (23). The fatty acyl chains also play a significant role in substrate binding, and B. cereus PC-PLC appears to prefer greater than 6 carbons for hydrolysis of phospholipids. In summary, we concluded that the enzyme may recognize hydroxyl groups of the glycerin backbone as well as the head group and the fatty acyl chain conformation of the substrate
428
SUGIMORI ET AL.
J. BIOSCI. BIOENG.,
FIG. 4. Sequence alignment of the deduced amino acid sequence of GPC-CP14 and SaPLC. Underline and dotted underline indicate the amino acid sequence determined by protein sequencing and LCeMS/MS analysis, respectively. Cleavage sites by signal peptidase and twin arginine translocation (Tat) pathway motif are indicated by the arrow and closed circles, respectively.
VOL. 117, 2014
NOVEL GLYCEROPHOSPHOCHOLINE CHOLINEPHOSPHODIESTERASE
429
FIG. 5. Tree view exhibiting amino acid sequence similarity of GPC-CP14 with the other PLCs and AcpA. The tree was constructed using ClustalW. The 0.1 scale represents a genetic unit reflecting 10% of the amino acid substitutions. The phylogenetic tree, drawn using TreeView included the following: UniprotKB accession number J2A2J5, SaPLC; M3C4W1, nhPLC from Streptomyces mobaraensis NBRC13819; D6AGR3, PLC from S. roseosporus NRRL 15998; L8PNA1, PC-PLC from S. viridochromogenes Tue57; B5HSN8, PC-PLC from S. sviceus ATCC 29083; B1VT12, nhPLC from S. griseus subsp. griseus NBRC13350; G0Q5B4, PC-PLC from S. griseus XylebKG-1; Q82ME5, nhPLC from S. avermitilis ATCC 31267; H2JQK9, nhPLC from S. hygroscopicus subsp. jinggangensis 5008; M1N8D7, nhPLC from S. hygroscopicus subsp. jinggangensis TL01; K4QTB5, nhPLC from S. davawensis JCM4913; D9X784, PC-PLC from S. viridochromogenes DSM40736; L7FEV8, PC-PLC from S. turgidiscabies Car8; E4N3T0, PLC from Kitasatospora setae ATCC 33774; A0Q436, AcpA as the outgroup.
molecule. Further studies are needed to elucidate the substrate recognition mechanism of GPC-CP14. GPC-CP14 was stable across a broad pH range (pH 5e10), although optimal pH (7.2) of the purified GPC-CP14 enzyme was remarkably different from those of the mammalian GPC-CPs (pH 9.5 and 10), suggesting that the physiological function of GPC-CP14 is essentially different from mammalian GPC-CPs. In fact, GPC-CP14 is an extracellular enzyme and an inducible protein. Interestingly, GPC-CP14 was induced by Brij 35, which is a polyoxyethylene ether like polyethylene glycol. Results of our study did not provide an explanation as to why Brij 35 is an inducer. The apparent Km (1.41 mM) of GPC-CP14 was similar to those of mammalian GPC-CPs (24e26). The specific activity of GPC-CP14 (24.7 mmol min1 mg-protein1) was considerably higher than that (0.276 mmol min1 mg-protein1) of B. taurus (27). However, the turnover number (kcat ¼ 29 s1) and kcat/Km values (20.6 mM1 s1) of GPC-CP14 was lower than those of general bacterial enzymes, suggesting that the binding affinity of GPC-CP14 toward substrate GPC is not higher than those of bacterial enzymes, and the turnover rate and the catalytic efficiency appears to be lower than the vast majority of enzymes. Due to the limited availability of environmental phosphorus (
Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri Daisuke Sugimori,1, * Junki Ogasawara,1 Koki Okuda,1 and Kazutaka Murayama2 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 2 September 2013; accepted 4 October 2013 Available online 7 November 2013
A novel metal ion-independent glycerophosphocholine cholinephosphodiesterase (GPC-CP) of Streptomyces sanglieri was purified 53-fold from culture supernatant with 1.1% recovery (583 U/mg-protein). The enzyme functions as a monomer with a molecular mass of 66 kDa. The gene encoding the enzyme consists of a 1941-bp ORF that produces a signal peptide of 38 amino acids for secretion and a 646 amino acid mature protein with a calculated molecular mass of 70,447 Da. The maximum activity was found at pH 7.2 and 40 C. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37e60 C) and within a narrow pH range near pH 7. The enzyme was stable at 50 C for 30 min and between pH 5e10.5. The enzyme exhibited specificity toward GPC and glycerol-3-phosphoethanolamine and hydrolyzed glycerol-3-phosphate and lysophosphatidylcholine. However, the enzyme showed no activity toward any diacylglycerophospholipids and little activity toward other glycerol-3-phosphodiesters and lysophospholipids. The enzyme was not inhibited in the presence of 2 mM SDS and Mg2D; however, Cu2D, Zn2D, and Co2D remarkably inhibited activity. Enzyme activity was also slightly enhanced by Ca2D, NaD, EDTA, DTT, and 2-mercaptoethanol. During the hydrolysis of GPC at 37 C and pH 7.2, apparent Vmax and turnover number (kcat) were determined to be 24.7 mmol minL1 mg-proteinL1 and 29.0 sL1, respectively. The apparent Km and kcat/Km values were 1.41 mM and 20.6 mML1 sL1, respectively. GPC hydrolysis by GPC-CP might represent a new metabolic pathway for acquisition of a phosphorus source in actinomycetes. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycerophosphocholine cholinephosphodiesterase; Purification; Characterization; Molecular cloning; Expression; New metabolic pathway; Streptomyces]
Glycero-3-phosphodiester is an important intermediate in glycerophospholipid metabolism. Nevertheless, there is only few limited information on the metabolic pathway and key enzyme. Glycerophosphocholine cholinephosphodiesterase (GPC-CP; EC 3.1.4.38) cleaves sn-glycerol-3-phosphocholine (GPC) into glycerol and phosphocholine (Fig. 1). GPC-CP has been found in the brains of mammals such as Bos taurus, Mus musculus, Rattus norvegicus, and Homo sapiens (BRENDA, http://www.brenda-enzymes.info/php/ result_flat.php4?ecno¼3.1.4.38). Janzen et al. reported that GPCCP activity is reduced in multiple sclerosis plaques (1). It has also been reported that GPC exhibits physiological functions such as enhancing the secretion of growth hormone and alleviating the cognitive symptoms of Alzheimer’s disease (2,3). Therefore, GPC-CP is useful for investigating the biological functions of GPC. The known GPC-CPs can be further divided into groups based on cellular location, i.e., membrane-bound and cytosolic enzymes. These are metal
* Corresponding author. Tel./fax: þ81 24 548 8206. E-mail address: [email protected] (D. Sugimori).
ion-dependent enzymes requiring Ca2þ, Co2þ, Cu2þ, Mn2þ, or Zn2þ for their activity. However, this enzyme has not been purified and characterized in detail, and to date, no information is available concerning the gene and amino acid sequence of GPC-CP. During our screening study of phospholipase C (PLC), we unexpectedly detected a novel metal ion-independent GPC-CP (GPCCP14) secreted by Streptomyces sanglieri strain A14. Although Streptomyces PLCs have been reported (4e6), no previous information is available regarding a bacterial GPC-CP; therefore, its significance in the metabolism of GPC, glycerophospholipids, or phosphorus is unknown. Moreover, finding of GPC-CP14 will help to identify the missing link in the glycerophospholipid metabolism in other organisms as well as actinomycetes. Here, we report the purification, characterization, molecular cloning, and extracellular production of this novel GPC-CP14. We also describe the kinetics for the hydrolytic reaction and the substrate specificity of the enzyme. To our knowledge, this represents the first report of purified GPCCP as novel-type enzyme. Further characterization of GPC-CP may allow prediction of the role of PLC, phosphatase, or phosphodiesterase superfamily proteins with unknown function.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.10.004
VOL. 117, 2014
NOVEL GLYCEROPHOSPHOCHOLINE CHOLINEPHOSPHODIESTERASE
423
FIG. 1. Hydrolytic reaction of GPC by GPC-CP.
MATERIALS AND METHODS Materials Bacto tryptic soy broth (TSB), Bacto tryptone, Bacto peptone, Bacto casamino acids and Bacto 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-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-snglycero-3-phosphate (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1-palmitoyl-2-oleoylsn-glycero-3-phospho-rac-(1-glycerol) (POPG), L-a-lysophosphatidylcholine (LPC), L-a-lysophosphatidylserine (LPS), L-a-lysophosphatidylglycerol (LPG), 1-stearoyl-2hydroxy-sn-glycerol-3-phosphate (LPA), L-a-lysophosphatidylinositol (LPI), 1-O-10 (Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphocholine (LPlsPC), 1-O-10 -(Z)-octadecenyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPlsPE), 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (PlsPC), and 1-(1Zoctadecenyl)-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). L-a-Lysophosphatidylethanolamine (LPE) was purchased from Doosan Serdary Research Laboratories (Toronto, ON, Canada). Sphingomyelin (SM) from chicken egg yolk and b-acetyl-gsn-hexadecyl-L-a-phosphatidylcholine (PAF) were purchased from SigmaeAldrich Japan Co., LLC. (Tokyo, Japan). sn-Glycerol-3-phosphocholine (GPC) was purchased from Bachem (Torrance, CA, USA). Recombinant phospholipase A1 (PLA1) from Streptomyces albidoflavus strain NA297 was extracellularly produced by Streptomyces lividans and was used for synthesis of sn-glycerol-3-phosphodiesters (GPXs) after ammonium sulfate purification (7). GPXs such as sn-glycerol-3-phosphoethanolamine (GPE), sn-glycerol-3phosphoserine (GPS), sn-glycerol-3-phosphoinositol (GPI), sn-glycerol-3-phosphate (GPA), and sn-glycerol-3-phosphoglycerol (GPG), except GPC, were prepared using the recombinant PLA1 as mentioned below. DEAE Sepharose Fast Flow, HiTrap Butyl FF, HiTrap Q HP, Mono Q, Mono P, RESOURCE ISO, HisTrap HP, Superdex 200 10/300 GL columns, and Polybuffer 74 were purchased from GE Healthcare Japan (Tokyo, Japan). All other chemicals were of the highest or analytical grade. GPXs were prepared using the recombinant PLA1 as follows. Lysophospholipids (8 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 PLA1 (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 PLA1. The concentration of free fatty acids was determined with a NEFA C Kit (Wako Pure Chemical Industries) 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 (water) layer was collected and the solvent was removed by evaporation for 5 min using vacuum evaporator. The resulting aqueous solution was used for investigation of substrate specificity. Bacterial strains and culture conditions Approximately 1500 strains as unidentified actinomycetes were isolated from soil samples obtained from Fukushima, Japan, using humic acidevitamin agar plate culture (8). Strains exhibiting PLC activity were screened using lecithin-emulsion medium (9). PLC activity toward PE or LPC was assessed in an assay mixture, comprising 10% (v/v) culture supernatant, 50 mM HEPESeNaOH buffer (pH 7.2), 0.8 mM PE or LPC, 1% (w/v) Triton X-100 (no addition for LPC). The enzyme activity was determined using a method described in the following enzyme activity assays. Most of strains exhibited very little PLC activity toward PE. Strain A14, which showed the highest hydrolytic activity toward LPC in a 5-mL culture, was selected for further investigation. Moreover, we found that GPC was the preferred substrate over LPC. Consequently, GPC-CP from strain A14 was investigated. Strain A14 was maintained on ISP2 (10 g malt extract, 4 g yeast extract, and 4 g glucose per liter, pH 7.2) agar plates. Several colonies from the agar plate were scraped and inoculated into a test tube (18 mm, 180 mm) containing 5 mL of the seed medium of ISP2. The mixtures 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 was cultivated with shaking (180 rpm) at 28 C for 48 h. The
culture supernatant was obtained by centrifugation (18,800 g for 20 min) and the enzyme was purified. Escherichia coli HST08 premium competent cells (Takara Bio Inc., Shiga, Japan) were used as a host for molecular cloning. A pGEM-T Easy Vector (Promega Corporation, Madison, WI, USA) was used as a cloning vector. A pUC702 carrying the promoter, signal peptide sequence and the terminator region of phospholipase D (PLD) from Streptoverticillium cinnamoneum was used as an expression vector (10). Recombinant E. coli cells were cultured on LuriaeBertani agar plates at 37 C, and when necessary, ampicillin, isopropyl-b-D-thiogalactopyranoside and X-Gal were used as supplements in the agar. Recombinant S. lividans cells were cultured in 3% (w/v) TSB containing 5 mg/mL thiostrepton at 28 C. S. lividans 1326 (NBRC15675), which was obtained from NITE Biological Resource Center (Chiba, Japan), was used as a host for the expression of proteins. 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 48 h of culturing. The enzyme was purified by 30e80% ammonium sulfate fractionation. The obtained precipitate was suspended in 20 mM TriseHCl buffer, pH 9.0 (buffer A) and dialyzed against buffer A. The enzyme sample was loaded onto a DEAE Sepharose column (2.5 3.5 cm) equilibrated with buffer A. The column was washed with 3 column volumes (CV) of buffer A, and protein was eluted with a linear gradient (10 CV) of 0e1 M NaCl in buffer A at 6 mL/min (1.22 cm/min). The active fractions were pooled, and the buffer was replaced by 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20e10,000 MWCO (GE Healthcare Japan) followed by application to a HiTrap Butyl FF column (5 mL) equilibrated with the same buffer. The column was washed with 3 CV of the same buffer, and protein was eluted with a linear gradient (5 CV) of 1 to 0 M (NH4)2SO4 in 20 mM TriseHCl buffer (pH 8.0) and an isocratic elution (20 CV) of the same buffer at 2.5 mL/min (1.25 cm/ min). The active fractions were pooled, and the buffer was exchanged with buffer A using Vivaspin 20 followed by application to a HiTrap Q HP column (5 mL) equilibrated with buffer A. After the column washing (3 CV), the protein was eluted with a linear gradient (20 CV) of 0e1 M NaCl in buffer A at 2.5 mL/min (1.25 cm/min). The active fractions were pooled, and the buffer was exchanged with buffer A using Vivaspin 20 followed by application to a Mono Q column (1 mL) equilibrated with buffer A. After the column washing (3 CV), the protein was eluted with a linear gradient (20 CV) of 0e1 M NaCl in buffer A at 0.5 mL/min (2.5 cm/min). The active fractions were pooled and then exchanged with 0.15 M NaCl/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20. The enzyme solution was applied to a Superdex 200 10/ 300 GL column (24 mL) equilibrated with the same buffer, and the protein was eluted at 1 mL/min (1.27 cm/min). The active fractions were pooled and then exchanged with 25 mM BisTriseHCl buffer (pH 6.3) using Vivaspin 20. The enzyme solution was applied to a Mono P column (1 mL) equilibrated with the same buffer. After the column washing (3 CV), the protein was eluted with a linear pH gradient (20 CV) of Polybuffer 74-HCl (pH 4) at 0.5 mL/min (2.5 cm/min). Fractions exhibiting high specific GPC-CP activity were pooled and used for subsequent investigation. Enzyme activity assays For GPC-CP activity, a standard assay mixture, containing 5 mL of enzyme solution, 12.5 mL of 0.2 M TriseHCl buffer (pH 7.2), 5 mL of 80 mM GPC and 27.5 mL of distilled water was incubated at 37 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 microliters of the supernatant was 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 phosphorylcholine was quantified with BIOMOL Green Reagent according to the manufacturer’s instructions (11). 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. Substrate specificity The substrate specificity of purified GPC-CP was assessed by assaying enzyme activity as described above using alternative GPX substrates (0.8 mM final concentration) in the assay. In the case of phospholipid substrates such as diacylglycerophospholipids, lysophospholipids, and plasmalogens, the hydrolytic activity was assessed in an assay mixture, comprising 5 mL of purified enzyme solution, 12.5 mL of 0.2 M HEPESeNaOH buffer (pH 7.2), 5 mL of 8 mM phospholipid substrates/5% (w/v) Triton X-100, and 27.5 mL of distilled water. Effect of pH, temperature, and chemicals on enzyme activity Each buffer (sodium acetate, BisTriseHCl, TriseHCl, and glycineeNaOH) was used to identify
424
SUGIMORI ET AL.
optimum pH and pH stability of the purified enzyme. The optimum pH was examined by incubation at 37 C for 5 min with 8 mM GPC 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 standard assay conditions. GPC-CP activity was determined at each temperature point under standard assay conditions. Thermal stability was determined by incubating the purified enzyme in 0.16 M TriseHCl (pH 7.2) at each temperature for 30 min, and then residual activity was measured by incubation under standard assay conditions. The effect of chemicals such as metal ions and SDS on the enzyme activity was also investigated. The purified enzyme was assayed under standard assay conditions following pre-incubation at 37 C for 5 min with the same assay buffer containing 2 mM of chemicals or inhibitors. Inhibitors assessed were EDTA, 2-mercaptoethanol, dithiothreitol (DTT), iodoacetamide (IAA), and PMSF. PMSF was dissolved in dimethyl sulfoxide and added to the enzyme sample. In the case of IAA, the purified enzyme samples (4.17 mg/mL, 59.2 nM) were incubated at 37 C for 60 min in the same buffer containing 2 mM IAA before use in enzyme assays. Protein analysis Protein concentration was determined with Pierce BCA protein assay kit (Takara Bio Inc.) and BSA as the standard. Protein samples were analyzed by SDS-PAGE according to methods described by Laemmli (12). The molecular mass of purified enzyme was estimated by gel filtration. Gel filtration was performed using the Superdex 200 10/300 GL column (1.0 30 cm) at 0.5 mL/min (0.64 cm/min) with 0.15 M NaCl/20 mM TriseHCl (pH 8.0). 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). The PVDF membrane was stained with Coomassie brilliant blue R-250 (CBB), and the transferred 66-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 internal terminal amino-acid sequencing, the SDS-PAGE gel was stained using CBB, and the 66-kDa band was excised and then decolorized with 30% acetonitrile containing 25 mM (NH4)2HCO3. The in-gel digestion was performed using a method described by Shevchenko et al. (13). Briefly, the excised 66-kDa band was digested with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation) for 45 h at 4 C. The fragments were analyzed using a nano ACQUITY UPLC Xevo QTof MS system (Waters Corp., Milford, MA, USA). The sample solution was transferred to an autosampler vial. One mL of sample was separated on a nano ACQUITY UPLC BEH130 C18 column (75 mm 150 mm, 1.7 mm) and analyzed using a Xevo QTOF MS, according to previously reported methods (7). Steady-state kinetics For hydrolysis of GPC and LPC as substrate, the initial velocity (v) of the enzymatic reaction was determined at each concentration of the substrate under standard assay conditions. The concentration of LPC ([LPC]) was calculated using a molecular weight of 503.33. The purified enzyme concentration was constant at 4.17 mg mL1 (59.2 nM). The corresponding v vs. 1/[GPC] or 1/[LPC] plots was treated according to a MichaeliseMenten equation. Kinetic constants were determined by extrapolation using the LineweavereBurk plot by linear regression (KaleidaGraph, Synergy Software). The Km and Vmax were determined from the xand y-intercepts of the regression line, respectively. The kcat was calculated using a molecular weight of 70,447 for a monomeric protein with a single catalytic site. Chromosomal DNA of S. sanglieri strain A14 was Cloning of GPC-CP14 gene purified according to methods reported by Kieser et al. (14). Oligonucleotides were synthesized based on the N-terminal (GTVQDVEHIVV) and internal amino acid sequences (TVTNAY, PYVDGAAD, and GDQAGAAF) of the enzyme for use in PCR with sense primer N (50 -ccgtscaggacgtsgagcacatcg-30 ) and antisense primer A1 (50 -aasgcsgcgccsgcctggtc-30 ), A2 (50 -tgttgtasgcgttggtsacg-30 ), and A3 (50 gcsgcgccgtcgacgtasg-30 ). The PCR reaction mixture (20 mL) contained: 1 KOD FX buffer, 6 pmol of each primer, 8 nmol of dNTPs, 0.5 U of KOD FX DNA polymerase (Toyobo Co., Ltd., Osaka, Japan), and w14 ng of the chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 1.5 min, and 68 C for 2 min after completion of the 25 cycles. The obtained PCR fragment was cloned into pGEM-T Easy Vector, and the resulting recombinant plasmid was designated pGPC. Sequencing of the partial GPC-CP gene on pGPC was performed with the BigDye Terminator cycle sequencing kit (Applied Biosystems) and analyzed in an ABI Prism 3100 genetic analyzer (Applied Biosystems). To reveal the nucleotide sequence of the 30 downstream region of the gene, gene walking was performed using Takara LA PCR in vitro Cloning kit (Takara Bio) as follows: genomic DNA (3 mg) was digested with PstI and then ligated to PstI Cassette (Takara Bio) and used as a template for PCR. The 1st PCR amplification was carried out in the reaction mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol each of Cassette primer C1 (Takara Bio, 50 -gtacatattgtcgttagaacgcgtaatacgactca-30 ) and gene-specific primer Gw1 (50 caagtacaaactgacgttcagc-30 ), 8 nmol of dNTPs, and 0.5 U of KOD FX Neo DNA polymerase (Toyobo Co., Ltd.). The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 25 cycles. The reaction mixture (0.4 mL) from the 1st PCR was used as a template for the 2nd PCR. The 2nd PCR amplification was carried out in a PCR mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol each of Cassette primer C2 (Takara Bio, 50 -cgttagaacgcgtaatacgactcactatagggaga-30 ) and
J. BIOSCI. BIOENG., gene-specific primer Gw2 (50 -aagcccgtctccgacagctggaac-30 ), 8 nmol of dNTPs, and 0.5 U of KOD FX Neo DNA polymerase. Amplification was completed using the above-mentioned thermal cycling parameters. To reveal the nucleotide sequence of the 50 -upstream region of the gene, inverse PCR was carried out using Inv-S primer (50 -caccaggccagctttttcggcaaggaaacg-30 ) and Inv-AS primer (50 gaagccccggacgcccttcatcgacccgaagt-30 ) designed based on the 50 -region of the partial sequence of GPC-CP gene on pGPC. The genomic DNA (5.5 mg) was digested with SphI and self-ligated. The inverse PCR amplification was completed with a reaction mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol of each primer, 0.5 U of KOD FX Neo DNA polymerase, and w55 ng (above the 0.4-mL ligation mixture) of the SphI-digested and self-ligated DNA. Amplification was completed using the above-mentioned thermal cycling parameters. The generated fragment (w1.7 kbp) was cloned into the pGEM-T Easy Vector and sequenced. To clone the complete GPC-CP14 gene from the chromosomal DNA of strain A14, sense primer GPC-NS (50 -atgaccgaaatcaaccggcgtcgcttcc-30 ) and antisense primer GPC-CA (50 tcacacggtcgcgatgcccgggtcactcac-30 ) were designed based on the sequence of the GPC-CP14 gene revealed by gene walking and inverse PCR as described above. The genomic PCR amplification was completed with a reaction mixture (20 mL) containing 1 KOD FX Neo buffer, 5 pmol of each primer, 8 nmol of dNTPs, 0.5 U of KOD FX Neo DNA polymerase, and the chromosomal DNA (w14 ng) using the above-mentioned thermal cycling parameters. 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 GPC-CP14 gene, designated gpc-cp, were deposited in the DDBJ/GenBank/EMBL database under the accession numbers AB735535 and AB771744, respectively. Expression and purification of recombinant GPC-CP14 S. lividans 1326 (NBRC15675), possessing no GPC-CP activity, was used as a host for GPC-CP14 expression. Two recombinant plasmid vectors, pUC702/pld-sig-gpccp and pUC702/ gpccp-sig, were constructed for extracellular production of active GPC-CP14 as follows: the recombinant vector pUC702/pld-sig-gpccp utilized the signal peptide sequence (pld-sig) of PLD from S. cinnamoneum, while pUC702/gpccp-sig used the signal peptide sequence (gpccp-sig) of GPC-CP14 itself. For construction of pUC702/ pld-sig-gpccp, the nucleotide of the mature GPC-CP14 enzyme was joined at the 30 downstream end of pld-sig in pUC702 (7). To replace the BglII site in gpc-cp, gpc-cp was amplified from the chromosomal DNA of S. sanglieri strain A14 by a two-step PCR. The first PCR was performed using the following primers: 50 aaagctagcggaaccgtccaggacgtcg-30 (Nhe I-F1) containing a first codon (Nhe I, italic; Gly, underlined) of mature GPC-CP14 and 50 -ggcgatccaggATATCTtcggcaatctg-30 (Bgl II-repair RV1); 50 -cagattgccgaAGATATcctggatcgcc-30 (Bgl II-repair F2) and 50 ataagatctcacggtcgcgatgcccgg-30 (Bgl II-RV2; BglII, italic). The PCR reaction mixture (20 mL) contained 1 KOD Plus buffer (Toyobo), 5 pmol of each primer set (Nhe I-F1 and Bgl II-repair RV1; Bgl II-repair F2 and Bgl II-RV2), 8 nmol of dNTPs, 0.5 U of KOD Plus DNA polymerase (Toyobo), and 51.6 ng of the chromosomal DNA as a template. The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 25 cycles. The second PCR was performed using each first-step amplification product as a template with forward primer (Nhe I-F1) and reverse primer (Bgl IIRV2). The second PCR was carried out in a reaction mixture (20 mL) containing w300 ng of the products (w800 bp) amplified using the primer set of Nhe I-F1 and Bgl II-repair RV1, and w300 ng of the products (w1.1 kbp) amplified using the primer set of Bgl II-repair F2 and Bgl II-RV2. Amplification was done under the above-mentioned conditions. The obtained fragment was purified and digested with NheI and BglII, and then sub-cloned into the NheI and BglII 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/pldsig-gpccp. For construction of pUC702/gpccp-sig, the nucleotide of gpc-cp containing the GPC-CP-signal peptide and the mature enzyme were joined downstream of the promoter region of PLD in pUC702. To replace SphI and BglII sites in gpc-cp, gpc-cp was amplified from the chromosomal DNA by a three-step PCR. The first PCR was performed using the following primer sets: 50 aaagcatgcttgaaatcaaccggcgtc-30 (Sph I-F1) containing a first codon (Sph I, italic; Met, underlined) of GPC-CP-signal peptide and Bgl II-RV2 (amplification product size, w2 kbp); 50 -gacgacctggGAATGCagttcatcgcag-30 (Sph I-repair F2) and Bgl IIrepair RV1 (amplification product size, w580 bp). Amplification was done under the above-mentioned conditions. Each amplification product was purified and additional PCR with 5 cycles was performed using each first-step amplification product as a template without primer. The second PCR was performed using the additional amplification product as a template with forward primers (Sph I-F1 and Sph I-repair F2) and reverse primers (Bgl II-repair RV1 and Bgl II-RV2). Amplification was done under the above-mentioned conditions. Each amplification product was purified and additional PCR with 5 cycles was performed using the second PCR products as a template without primer. The thermal cycling parameters were 94 C for 2 min, followed by 5 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 5 cycles. The 3rd PCR was performed using the additional amplification product as a template with forward primer (Sph I-F1) and reverse primer (Bgl II-RV2). The thermal cycling parameters were 94 C for 2 min, followed by 25 cycles of 98 C for 10 s, 68 C for 2 min, and 68 C for 2 min after completion of the 25 cycles. The obtained fragment was purified and digested with SphI and BglII, and then sub-cloned into
VOL. 117, 2014
NOVEL GLYCEROPHOSPHOCHOLINE CHOLINEPHOSPHODIESTERASE
425
TABLE 1. Purification of GPC-CP from S. sanglieri A14. Purification step 48-h Culture supernatant 30e80% Ammonium sulfate DEAE Sepharose HiTrap Butyl FF HiTrap Q HP Mono Q Superdex 200 Mono P a b
Activitya (U/ml) 1.45 5.81 6.58 5.94 10.2 12.3 9.75 7.31
Protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification (fold)
% Recovery
11.0 8.02 18.8 78.2 164 91.8 594 583b
665 436 395 297 205 55.4 24.4 7.31
1 0.728 1.70 7.11 14.9 8.34 54.0 53.0
100 65.5 59.4 44.6 30.8 8.33 3.67 1.10
60.3 54.3 21.0 3.80 1.26 0.603 4.10 102 1.25 102
The enzyme activity was assayed using the reaction mixture containing 50 mM TriseHCl buffer (pH 7.2) and 0.8 mM GPC at 37 C. One ml of the purified enzyme sample was obtained the 460-ml culture supernatant.
the SphI and BglII sites in the pUC702, i.e., between the promoter and terminator regions of PLD (15). The constructed expression plasmid was sequenced and designated pUC702/gpccp-sig. The transformation techniques of Kieser et al. were followed for S. lividans (14). Transformants were screened by 5-mL cultivation, and those exhibiting the highest GPC-CP activity were selected. Moreover, His-tag (His 6) was joined to the Cterminal amino acid sequence of GPC-CP14 by inverse PCR using pUC702/gpccp-sig as a template and 2 primers, His-InvSense (50 -caccaccactgagacgactgagcgcccggacg30 ) and His-InvAnti (50 -gtggtggtgcacggtcgcgatgcccgggtc-30 ), followed by phosphorylation and self-ligation using KOD-Plus-Mutagenesis Kit (Toyobo). Amplification was done under the above-mentioned conditions. Recombinant GPC-CP14 produced by the transformed S. lividans was purified from 72-h culture supernatant. Ammonium sulfate precipitation (80% sat.) was
done by the above-mentioned experimental procedures. The resulting precipitate was suspended in 20 mM TriseHCl buffer (pH 8.0). The enzyme sample was loaded onto a HisTrap HP column (5 mL) equilibrated with 20 mM HEPESeNaOH buffer (pH 7.4) containing 0.5 M NaCl and 20 mM imidazole. The column was washed with 5 CV of the same buffer, and the protein was eluted with a linear gradient (20 CV) of 20e500 mM imidazole in the same buffer at 2.5 mL/min (1.25 cm/min). The active fractions were pooled. The buffer was exchanged with 1 M (NH4)2SO4/20 mM TriseHCl buffer (pH 8.0) using Vivaspin 20 and then applied to a Resource ISO column (1 mL) equilibrated with the same buffer. After the column washing (3 CV), the protein was eluted with a linear gradient (20 CV) of 1 to 0 M (NH4)2SO4 in 20 mM TriseHCl buffer (pH 8.0) at 0.5 mL/min (1.6 cm/ min). Fractions exhibiting high specific activity were pooled and used for subsequent investigation.
FIG. 2. Effect of pH and temperature on GPC-CP14 activity (A, B) and stability (C, D). (A) The enzyme activity was assayed at 37 C for 5 min with 8 mM GPC in 50 mM of each buffer. The buffers were sodium acetate (pH 4.1e5.6), BisTriseHCl (pH 5.6e7.2), TriseHCl (pH 7.2e9.0) and glycineeNaOH (pH 9.0e10.5). (B) The enzyme activity was assayed at each temperature in 50 mM TriseHCl buffer (pH 7.2). (C) The enzyme was incubated at 4 C for 3 h in 50 mM of each buffer solution. The remaining activity was assayed by incubation at 37 C in 50 mM TriseHCl buffer (pH 7.2). (D) The enzyme was incubated at each temperature for 30 min in 50 mM TriseHCl buffer (pH 7.2), and the residual activity was assayed by incubation at 37 C in 50 mM TriseHCl buffer (pH 7.2). Data are the means of experiments performed in triplicate. Error bars represent the standard deviation.
426
SUGIMORI ET AL.
J. BIOSCI. BIOENG., RESULTS
Isolation and identification of strain A14 Strain A14 was assigned as S. sanglieri by morphological, physiological, 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 accession number AB735535. Purification of GPC-CP14 The overall purification of GPC-CP14 over the culture supernatant of strain A14 was 53-fold, with an activity yield of 1.10% (Table 1). The purified GPC-CP14 with a specific activity of 583 U/mg-protein was obtained for 12.5 mg of protein. Isoelectric point (pI) of GPC-CP14 was estimated to be w5.8 by Mono P column chromatography, in agreement with pI 5.72 calculated using Genetyx-Mac version 16.0.8 (Genetyx Corporation, Tokyo, Japan). Protein analysis The purified enzyme was subjected to SDSPAGE analysis. A single band with an apparent molecular mass of w66 kDa was visualized by CBB stain (Fig. S1). The molecular mass of the native enzyme was estimated to be w68 kDa by gel filtration chromatography, demonstrating that native GPC-CP14 is a monomeric protein. The N-terminal amino acid sequence was determined as GTVQDVEHIVV. By LCeMS/MS analysis, 13 peptide sequences with high probability values were selected as internal amino acid sequences. Among these, the internal amino acid sequences (TVTNAY, PYVDGAAD, and GDQAGAAF) were selected as PCR primers for the enzyme gene cloning. Effect of pH, temperature, and chemicals on enzyme activity As shown in Fig. 2, the highest activity for GPC hydrolysis was found at pH 7.2 and 40 C. The enzyme maintained high activity of >95% relative activity between 37 C and 60 C. The enzyme was stable from pH 5 to pH 10.2 and from 4 C to 45 C. Table 2 summarizes the effects of chemicals and inhibitors on the enzyme activity for GPC. The enzyme activity was inhibited by Mn2þ and Fe2þ; while Co2þ, Cu2þ, and Zn2þ remarkably inhibited enzyme activity. Magnesium ion, Kþ, IAA and SDS had almost no effect on enzyme activity. Enzyme activity was slightly increased by Ca2þ, Naþ, 2-mercaptoethanol, DTT, and EDTA. Interestingly, enzyme activity appeared to be inhibited by dimethyl sulfoxide, used as solvent, but not PMSF.
TABLE 2. Effect of chemicals and inhibitors (2 mM) on the enzyme activity for GPC hydrolysis. Chemical Control CaCl2 MgCl2 MnCl2 ZnCl2 CoCl2 FeCl2 CuCl2 NaCl KCl EDTA Sodium dodecyl sulfate 2-Mercaptoethanol Dithiothreitol Iodoacetamideb PMSF
Relative activity (%)a 100 114 99.2 79.5 0.380 19.4 50.6 0 111 100 113 104 111 105 94.0 78.8
a The purified enzyme was assayed by using reaction mixture containing 50 mM TriseHCl buffer (pH 7.2), 8 mM GPC, and 2 mM chemicals or inhibitors at 37 C. The relative activity is expressed as a percentage of the control activity without chemicals or inhibitors. b After the incubation for 60 min with IAA, the enzyme activity was assayed by the same above.
Substrate specificity Table 3 summarizes the substrate specificity of GPC-CP14. The highest hydrolytic activity was recorded with GPC, while GPE was also preferred. Both LPC and GPA were hydrolyzed by the enzyme, but only minimal activity was observed with LPlsPC and LPE. Additionally, GPC-CP14 produced only minimal hydrolysis with GPI, GPG, LPA, LPlsE, DPPE, and POPS, and produced no discernible activity with GPS, LPI, LPS, LPG, DPPC, DMPA, POPG, PI, SM, PAF, PlsPC, or PlsPE. Enzyme kinetics Linear regression analysis was performed using a LineweavereBurk plot (Fig. 3). For the hydrolysis of GPC by the purified GPC-CP14 at 37 C and pH 7.2, apparent Vmax and turnover rate (kcat) values were determined to be 24.7 mmol min1 mg-protein1 and 29.0 s1, respectively. The apparent Km and kcat/Km values were 1.41 mM and 20.6 mM1 s1, respectively. Intriguingly, during hydrolysis of LPC, substrate inhibition was observed at >1 mM LPC; however, at LPC [ LPlsPC. GPC-CP14 appeared to prefer the choline-associated substrate; however, no activity was recorded with DPPC, SM, PAF, and PlsPC. Additionally, substrate preference observed the following order: GPE [ LPE [ LPlsPE > DPPE. It is also known that the head group of these choline-associated substrates and GPE must be in the same ionization state (21,22). These results suggest that the enzyme may prefer choline and ethanolamine of the substrate head group, and this substrate specificity results from steric hindrance of the fatty acyl chain of substrates but not the ionization state of the substrate head group. 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 (23). The fatty acyl chains also play a significant role in substrate binding, and B. cereus PC-PLC appears to prefer greater than 6 carbons for hydrolysis of phospholipids. In summary, we concluded that the enzyme may recognize hydroxyl groups of the glycerin backbone as well as the head group and the fatty acyl chain conformation of the substrate
428
SUGIMORI ET AL.
J. BIOSCI. BIOENG.,
FIG. 4. Sequence alignment of the deduced amino acid sequence of GPC-CP14 and SaPLC. Underline and dotted underline indicate the amino acid sequence determined by protein sequencing and LCeMS/MS analysis, respectively. Cleavage sites by signal peptidase and twin arginine translocation (Tat) pathway motif are indicated by the arrow and closed circles, respectively.
VOL. 117, 2014
NOVEL GLYCEROPHOSPHOCHOLINE CHOLINEPHOSPHODIESTERASE
429
FIG. 5. Tree view exhibiting amino acid sequence similarity of GPC-CP14 with the other PLCs and AcpA. The tree was constructed using ClustalW. The 0.1 scale represents a genetic unit reflecting 10% of the amino acid substitutions. The phylogenetic tree, drawn using TreeView included the following: UniprotKB accession number J2A2J5, SaPLC; M3C4W1, nhPLC from Streptomyces mobaraensis NBRC13819; D6AGR3, PLC from S. roseosporus NRRL 15998; L8PNA1, PC-PLC from S. viridochromogenes Tue57; B5HSN8, PC-PLC from S. sviceus ATCC 29083; B1VT12, nhPLC from S. griseus subsp. griseus NBRC13350; G0Q5B4, PC-PLC from S. griseus XylebKG-1; Q82ME5, nhPLC from S. avermitilis ATCC 31267; H2JQK9, nhPLC from S. hygroscopicus subsp. jinggangensis 5008; M1N8D7, nhPLC from S. hygroscopicus subsp. jinggangensis TL01; K4QTB5, nhPLC from S. davawensis JCM4913; D9X784, PC-PLC from S. viridochromogenes DSM40736; L7FEV8, PC-PLC from S. turgidiscabies Car8; E4N3T0, PLC from Kitasatospora setae ATCC 33774; A0Q436, AcpA as the outgroup.
molecule. Further studies are needed to elucidate the substrate recognition mechanism of GPC-CP14. GPC-CP14 was stable across a broad pH range (pH 5e10), although optimal pH (7.2) of the purified GPC-CP14 enzyme was remarkably different from those of the mammalian GPC-CPs (pH 9.5 and 10), suggesting that the physiological function of GPC-CP14 is essentially different from mammalian GPC-CPs. In fact, GPC-CP14 is an extracellular enzyme and an inducible protein. Interestingly, GPC-CP14 was induced by Brij 35, which is a polyoxyethylene ether like polyethylene glycol. Results of our study did not provide an explanation as to why Brij 35 is an inducer. The apparent Km (1.41 mM) of GPC-CP14 was similar to those of mammalian GPC-CPs (24e26). The specific activity of GPC-CP14 (24.7 mmol min1 mg-protein1) was considerably higher than that (0.276 mmol min1 mg-protein1) of B. taurus (27). However, the turnover number (kcat ¼ 29 s1) and kcat/Km values (20.6 mM1 s1) of GPC-CP14 was lower than those of general bacterial enzymes, suggesting that the binding affinity of GPC-CP14 toward substrate GPC is not higher than those of bacterial enzymes, and the turnover rate and the catalytic efficiency appears to be lower than the vast majority of enzymes. Due to the limited availability of environmental phosphorus (
