Purification and characterization of 1-acyl-sn-glycerol-3-phosphate acyltransferase with a substrate preference for polyunsaturated fatty acyl donors from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10

Purification and characterization of 1-acyl-sn-glycerol-3-phosphate acyltransferase with a... Abstract 1-Acyl-sn-glycerol-3-phosphate acyltransferase (designated as PlsC in bacteria) catalyzes the acylation of lysophosphatidic acid and is responsible for the de novo production of phosphatidic acid, a precursor for the synthesis of various membrane glycerophospholipids. Because PlsC is an integral membrane protein, it is generally difficult to solubilize it without causing its inactivation, which has been hampering its biochemical characterization despite its ubiquitous presence and physiological importance. Most biochemical studies of PlsC have been carried out using crude membrane preparations or intact cells. In this study, we succeeded in solubilization and purification of a recombinant PlsC in its active form from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10 using 6-cyclohexyl-1-hexyl-β-d-maltoside as the detergent. We characterized the purified enzyme and found that it has a substrate preference for the acyl donors with a polyunsaturated fatty acyl group, such as eicosapentaenoyl group. These results provide a new method for purification of the PlsC family enzyme and demonstrate the occurrence of a new PlsC with unique substrate specificity. 1-acyl-sn-glycerol-3-phosphate acyltransferase, lipid metabolism, membrane protein, phospholipid, PlsC 1,2-Diacyl-sn-glycerol 3-phosphate, or phosphatidic acid (PA), is a precursor for the synthesis of various glycerophospholipids, in both eukaryotes and bacteria, which constitute biological membranes (1–3). The de novo synthesis of PA involves two distinct acyltransferases that catalyze the sequential acylation of sn-glycerol 3-phosphate (G3P) (4) (Fig. 1). The first step is the acylation of hydroxyl group at the sn-1 position of G3P to produce 1-acyl-sn-glycerol 3-phosphate, or lysophosphatidic acid (LPA), by the action of G3P acyltransferase (GPAT). There are two types of GPATs—PlsB and PlsY—which show no homology in their amino acid sequences. The second acylation step, where a hydroxyl group at the sn-2 position of LPA is acylated to yield PA, is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT; EC number: 2.3.1.51) (5). This enzyme, designated as PlsC in bacteria, is a membrane protein that has four acyltransferase motifs (6). An AGPAT protein homologous to PlsC also occurs in eukaryotes (including yeast, fungi, plants and mammals), and its role in PA production in these organisms is conserved (7, 8). Fig. 1 View largeDownload slide Schematic representation of the de novo glycerophospholipid biosynthesis pathway. R and R′ represent hydrocarbon chains, and X represents a polar head group. ACP stands for acyl carrier protein. Fig. 1 View largeDownload slide Schematic representation of the de novo glycerophospholipid biosynthesis pathway. R and R′ represent hydrocarbon chains, and X represents a polar head group. ACP stands for acyl carrier protein. Despite such ubiquitous presence and an important role in lipid metabolism, purification of AGPATs/PlsCs in their active forms has not been reported until recently (9). The difficulty in purification of these enzymes is supposed to be due to their membrane association: AGPATs/PlsCs tend to be inactivated following solubilization from their native membranous milieu into the detergent micelles. Therefore, biochemical insights into the enzymatic characteristics of AGPATs/PlsCs have been limited. Because AGPATs/PlsCs play a major role in determining the acyl chain composition of membrane phospholipids, the substrate specificity of these enzymes can significantly affect the physicochemical and biochemical properties of biological membrane. Various bacteria possess more than one plsC gene (10–14), and the occurrence of multiple PlsCs, which possibly have different substrate specificities, in a single bacterium could be a considerable advantage in modifying the fatty acyl composition of cell membrane in response to the changes in extracellular environment. So far, although mutagenesis experiments using intact cells or crude extracts provided a clue to estimate their properties (10–13), potential functional redundancy between different PlsC homologs in vivo and lack of in vitro characterization of purified PlsCs have been obscuring their precise enzymatic characteristics, including their fatty acyl chain selectivity. A cold-adapted bacterium Shewanella livingstonensis Ac10, which was originally isolated from the Antarctic seawater, grows in the temperature range of 4–25°C (optimum temperature, 18°C). In response to the low temperature, the bacterium produces glycerophospholipids that are esterified at the sn-2 position with a polyunsaturated fatty acid, namely eicosapentaenoic acid (EPA) (15). The EPA-containing phospholipids play an important role in survival of the bacterium in a cold environment (15). There are five putative plsC genes in the genome of S. livingstonensis Ac10 (the gene products are designated as SlPlsC1, SlPlsC2, SlPlsC3, SlPlsC4 and SlPlsC5). We previously revealed that the deletion of the SlPlsC1 gene caused a marked decrease in the amounts of EPA-containing phospholipids, without significantly altering the composition of other phospholipids (16). Thus, SlPlsC1 appeared to be dedicated for the in vivo acylation of LPA with EPA. To determine its precise biochemical characteristics, we established a method to purify SlPlsC1 in its active form and characterized the purified enzyme, which revealed its broad substrate specificity with a preference for various polyunsaturated fatty acyl donors. Materials and Methods Materials Palmitoleoyl-CoA was purchased from Sigma–Aldrich (St. Louis, MO, USA). LPAs, lysophosphatidylethanolamine (LPE) and lysophosphatidylglycerol (LPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 6-Cyclohexyl-1-hexyl-β-d-maltoside (CYMAL-6) was purchased from Anatrace (Maumee, OH, USA). n-Dodecyl-β-d-maltoside (DDM), n-octyl-β-d-glucoside (OG), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), arachidonic acid and EPA were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals used were of analytical grade. Heterologous expression, solubilization and purification of SlPlsC1 Escherichia coli C43(DE3) was transformed with a pET21a-derived expression vector (pET21a-SlPlsC1) that encoded SlPlsC1 fused with a C-terminal hexahistidine-tag, which we constructed previously (16). The transformant was cultured in M9YG medium (1 L) (17) supplemented with ampicillin (100 mg/L) on a reciprocal shaker (100 min−1) at 37°C. After the optical density at 600 nm reached ∼0.7, the culture was incubated at 18°C for additional 20 h. The cells were collected and stored at −30°C until further use. The following procedure was performed on ice or in a cold room. The collected cells (about 3 g) were disrupted by sonication in buffer A [6 ml; sodium phosphate (20 mM, pH 7.4), NaCl (0.5 M), imidazole (20 mM), dithiothreitol (0.5 mM) and glycerol (10%)]. After centrifugation (10,110 g for 30 min at 4°C), the supernatant was recovered and subjected to ultracentrifugation (150,000 g for 1 h at 4°C). The membrane pellet was resuspended in buffer A (6 ml) containing one of the following detergents present at four times higher concentration than their critical micelle concentrations: CYMAL-6 (2.2 mM), DDM (0.68 mM), OG (100 mM) or CHAPS (32 mM). After incubating with gentle shaking for 2 h, the suspension was subjected to ultracentrifugation (100,000 g for 1 h at 4°C) to collect the detergent-solubilized membrane proteins into the supernatant. The proteins solubilized with either CYMAL-6 or DDM were applied onto the HisTrap FF column (1 ml; GE Healthcare, Buckinghamshire, UK), which was preequilibrated with buffer B [sodium phosphate (20 mM, pH 7.4), NaCl (0.5 M), glycerol (10%), and either CYMAL-6 (1.1 mM) or DDM (0.34 mM)] containing 20 mM imidazole. The column was washed with buffer B containing 60 mM imidazole, and subsequently eluted with buffer B containing 0.5 M imidazole. The eluate was concentrated using the Amicon 10 kDa molecular weight cut-off centrifugal filter (Merck Millipore, Darmstadt, Germany). For long-term storage, glycerol was added to the concentrate at a final concentration of 30%. The fractions collected during purification were analyzed by 12.5% SDS-PAGE. Protein concentration was determined by the Bradford method using the Protein Assay CBB solution (Nacalai Tesque). Western blot analysis The protein samples were separated on a 12.5% SDS-PAGE gel and subsequently transferred onto an Immobilon-P membrane (Merck Millipore). The hexahistidine-tagged SlPlsC1 was specifically labelled with anti 6 × histidine, monoclonal antibody 9F2 (Wako Chemicals, Kyoto, Japan) and was detected using a peroxidase-conjugated anti-rat IgG antibody (Sigma–Aldrich) and Chemi-Lumi One Ultra (Nacalai Tesque). The membrane was scanned with an imaging system C-DiGit (M&S TechnoSystems, Osaka, Japan). N-Terminal amino acid sequence analysis The N-terminal amino acid sequence was determined using the PPSQ-31 A protein sequencer (Shimadzu, Kyoto, Japan). The presence or absence of an N-terminal signal sequence was analyzed using the PSORTb ver3.0.2 (http://www.psort.org/psortb/) and SOSUIsignal (http://harrier.nagahama-i-bio.ac.jp/sosui/sosuisignal/sosuisignal_submit.html) webservers (18, 19). Qualitative AGPAT assay A reaction mixture (120 µl) for the crude membrane fraction contained palmitoleoyl-CoA (0.5 mM), oleoyl-LPA (0.5 mM), Tris-HCl (10 mM, pH 9.0), MgCl2 (1 mM), CYMAL-6 (1.1 mM) and the membrane fraction (ca. 0.8 µg protein). A reaction mixture (80 µl) for the detergent-solubilized fraction contained palmitoleoyl-CoA (0.5 mM), oleoyl-LPA (0.5 mM), Tris-HCl (10 mM, pH 9.0), MgCl2 (1 mM), the detergent-solubilized fraction (ca. 0.4 µg protein), and one of the following detergents: CYMAL-6 (1.1 mM), DDM (0.34 mM), OG (50 mM) or CHAPS (16 mM). The composition of a reaction mixture (80 µl) for purified SlPlsC1 was the same as for the membrane fraction except that the membrane fraction was replaced with purified SlPlsC1 (2.8 µg). These reaction mixtures were incubated at 20°C for 10 min, and the product was extracted using the Bligh and Dyer method (20). The extracted product was analyzed on the silica gel 60 thin-layer chromatography (TLC) plate (Merck Millipore) that was developed using the chloroform–acetone–methanol–acetic acid–water (5:2:1:1:0.5 v/v) solvent system. The TLC plates were stained with molybdenum blue solution. Also, the extracted product was dissolved in the acetonitrile–methanol (2:1 v/v) solvent system containing 0.1% triethylamine and analyzed using the API 3000 mass spectrometer (SCIEX, Ontario, Canada) equipped with an electrospray ionization source under the following conditions: polarity, negative; scan range, m/z 200–900; nebulizer gas, 8; curtain gas, 8; ionspray voltage, −4500 V; declustering potential, −30 V; focusing potential, −200 V; and entrance potential, −10 V. Quantitative AGPAT assay A mixture containing palmitoleoyl-CoA (0.5 mM), oleoyl-LPA (0.5 mM), Tris-HCl (10 mM, pH 9.0), MgCl2 (1 mM) and CYMAL-6 (1.1 mM) was prepared and aliquoted to each reaction tube (36 µl). After preincubating the mixture at 20°C for 1 min, purified SlPlsC1 (4 µl) was added to a final concentration of 0.11 µM (3.1 µg/ml). The reaction was performed at 20°C for 2–3 min and stopped by adding guanidine-HCl (5 M, 40 µl) in HEPES-NaOH (0.5 M, pH 8.0). All reactions were stopped before ∼20% of the substrates were consumed. Next, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; 2 mM, 20 µl) in HEPES-NaOH (0.5 M, pH 8.0) was added, and the reaction mixture was allowed to stand at about 25°C for ∼10 min. The mixture (90 µl) was transferred to a 96-well microplate and absorption was measured at 412 nm using the SpectraMax 190 (Molecular Devices, Sunnyvale, CA, USA). A standard curve was plotted with cysteine. To characterize the enzymatic properties of SlPlsC1, the above-mentioned standard assay conditions were modified as follows: (i) the buffer was replaced with Tris-HCl (pH range 7.5–9), borate-NaOH (pH range 8.5–10) and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS)-NaOH (pH range 10–11); (ii) the reaction was performed in the range of 4–40°C; (iii) the enzyme solution was preincubated in the range of 0–50°C for 30 min before the reaction; (iv) MgCl2 (1 mM) was replaced with CaCl2 (1 mM), MnCl2 (1 mM), NiCl2 (1 mM) or EDTA (10 mM); (v) palmitoleoyl-CoA was replaced with lauroyl-, myristoyl-, palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, arachidoyl-, arachidonoyl-, or eicosapentaenoyl-CoA; (vi) oleoyl-LPA was replaced with palmitoyl-LPA, palmitoleoyl-LPA, oleoyl-LPE, oleoyl-LPG or G3P. Synthesis of fatty acyl-CoAs Various acyl-CoAs were synthesized via the mixed anhydride method (21). Briefly, to various fatty acids (25 µmol), tetrahydrofuran (0.5 ml) containing ethyl chloroformate (4.8 µl), triethylamine (3.5 µl) and butylhydroxytoluene (5.5 mg) was added. After vigorous shaking at room temperature for 2 h, the solvent was evaporated. The residue was dissolved in tetrahydrofuran (1 ml), and the solution was briefly centrifuged. The supernatant (0.5 ml) was transferred to a fresh tube, to which CoA (0.25 ml, 20 mM) in sodium bicarbonate solution (0.1 M) was added. After vigorous shaking at room temperature for 2 h, the reaction mixture was washed twice with diethyl ether (0.5 ml). The fatty acyl-CoAs were precipitated by adding 1/10 volume of 10% perchloric acid and following centrifugation. The precipitate was washed sequentially with 0.5 ml each of diethyl ether, acetone and diethyl ether. The synthetic acyl-CoAs were purified using the high-performance liquid chromatography system equipped with the CAPCELL PAK C18 column (4.6 × 250 mm; Shiseido, Tokyo, Japan). The acyl-CoAs were eluted using ammonium formate (0.1 M)–methanol solvent system with a linear gradient of 70–95%(v/v) of methanol at a flow rate of 0.5 ml/min over 20 min. The elution was monitored by measuring the absorption at 254 nm. The purity of synthetic acyl-CoAs was checked by normal-phase TLC with the n-butanol–acetic acid–water (5:2:3 v/v) solvent system. Results and Discussion Expression, solubilization and purification of recombinant SlPlsC1 In most cases, overexpression of a membrane protein is toxic to the host cells. Generally, this results in a significantly lower expression of the protein or the formation of inclusion bodies. To circumvent this problem, we constructed an expression system for SlPlsC1 using E. coli C43(DE3) (22) and employed mild expression conditions, in which the recombinant cells were cultured in M9YG medium at 18°C without the addition of isopropyl-β-d-1-thiogalactoside. The attempts to express SlPlsC1 either in Luria–Bertani broth or using a low-copy vector pSTV28 gave lower expression level (data not shown). The cultured cells were disrupted, and the cell-free extract was recovered and subjected to ultracentrifugation to obtain membrane pellets. The SDS-PAGE analysis showed that a ∼31 kDa protein was present in the 150,000 g pellet from the E. coli cells harboring pET21a-SlPlsC1 (calculated mass of tagged SlPlsC1, 28.1 kDa), whereas the same protein was not present in the corresponding fraction of the E. coli cells harboring the empty vector (Fig. 2A, lanes 4 and 8). The protein band was identified to be recombinant SlPlsC1 by western blotting using the anti-hexahistidine antibody (Fig. 2C and D, lane 1) and also by in vitro assay showing that the membrane fraction from E. coli harboring pET21a-SlPlsC1 was capable of producing PA more efficiently than that from the control E. coli cells (Fig. 3A). Fig. 2 View largeDownload slide SDS-PAGE and western blot analyses of the fractions collected during purification. (A) The supernatants and pellets obtained by centrifugation of the disrupted E. coli cells harboring pET21a (lanes 1 and 2, respectively) and those obtained by subsequent ultracentrifugation (lanes 3 and 4, respectively). The corresponding fractions from E. coli cells harboring pET21a-SlPlsC1 are shown in lanes 5–8, respectively. (B) The supernatants and pellets (odd- and even-numbered lanes, respectively) obtained by ultracentrifugation after solubilization with the indicated detergents. Mock indicates the samples prepared in the absence of detergents. (C) and (D) The membrane fraction (lane 1), the supernatant obtained by ultracentrifugation after solubilization with CYMAL-6 (lane 2), and the eluate obtained after the affinity chromatography (lane 3) were visualized by CBB staining (C) and chemiluminescence (D). The arrowheads indicate the position of the SlPlsC1 band. Fig. 2 View largeDownload slide SDS-PAGE and western blot analyses of the fractions collected during purification. (A) The supernatants and pellets obtained by centrifugation of the disrupted E. coli cells harboring pET21a (lanes 1 and 2, respectively) and those obtained by subsequent ultracentrifugation (lanes 3 and 4, respectively). The corresponding fractions from E. coli cells harboring pET21a-SlPlsC1 are shown in lanes 5–8, respectively. (B) The supernatants and pellets (odd- and even-numbered lanes, respectively) obtained by ultracentrifugation after solubilization with the indicated detergents. Mock indicates the samples prepared in the absence of detergents. (C) and (D) The membrane fraction (lane 1), the supernatant obtained by ultracentrifugation after solubilization with CYMAL-6 (lane 2), and the eluate obtained after the affinity chromatography (lane 3) were visualized by CBB staining (C) and chemiluminescence (D). The arrowheads indicate the position of the SlPlsC1 band. Fig. 3 View largeDownload slide Analysis of the products of the in vitro AGPAT assay. (A) AGPAT assay using the membrane fraction from the E. coli cells harboring pET21a (lane 2) and that from the E. coli cells harboring pET21a-SlPlsC1 (lane 3). Lane 1 shows authentic PA. (B) AGPAT assay using the soluble fractions collected after incubation without detergents (lane 2) or with CYMAL-6 (lane 3), DDM (lane 4), OG (lane 5), or CHAPS (lane 6). Lane 1 shows authentic PA. (C) AGPAT assay of the enzyme purified with CYMAL-6. Lane 1, authentic PA; Lane 2, complete reaction; Lane 3, reaction using heat-denatured SlPlsC1; Lane 4, reaction without oleoyl-LPA; Lane 5, reaction without palmitoleoyl-CoA (ori., origin; s.f., solvent front). (D) Mass spectrometric analysis of PA produced by purified SlPlsC1. Front graph, complete reaction; rear graph, reaction using heat-denatured SlPlsC1. Fig. 3 View largeDownload slide Analysis of the products of the in vitro AGPAT assay. (A) AGPAT assay using the membrane fraction from the E. coli cells harboring pET21a (lane 2) and that from the E. coli cells harboring pET21a-SlPlsC1 (lane 3). Lane 1 shows authentic PA. (B) AGPAT assay using the soluble fractions collected after incubation without detergents (lane 2) or with CYMAL-6 (lane 3), DDM (lane 4), OG (lane 5), or CHAPS (lane 6). Lane 1 shows authentic PA. (C) AGPAT assay of the enzyme purified with CYMAL-6. Lane 1, authentic PA; Lane 2, complete reaction; Lane 3, reaction using heat-denatured SlPlsC1; Lane 4, reaction without oleoyl-LPA; Lane 5, reaction without palmitoleoyl-CoA (ori., origin; s.f., solvent front). (D) Mass spectrometric analysis of PA produced by purified SlPlsC1. Front graph, complete reaction; rear graph, reaction using heat-denatured SlPlsC1. Next, a detergent suitable for solubilizing SlPlsC1 was screened by testing the solubilization efficiency and enzymatic activity of the solubilized protein. The detergents used were CYMAL-6, DDM, OG and CHAPS, which are considered to have low impact on the structural integrity of membrane proteins. The membrane fraction was incubated in a buffer containing one of these detergents. After ultracentrifugation, SlPlsC1 was recovered in the supernatant with either of these detergents (Fig. 2B). However, we noticed that only the recombinant protein that was solubilized with either CYMAL-6 or DDM retained the PA-producing activity (Fig. 3B, lanes 3 and 4). Notably, the sample prepared in the absence of detergents also produced PA (Fig. 3B, lane 2). This was probably because a small amount of SlPlsC1, which was not extracted from the membrane, remained in the supernatant (Fig. 2B, lane 1). Thus, the supernatants obtained from the CYMAL-6- or DDM-treated samples were subjected to Ni–NTA affinity chromatography. As shown in the lane 3 of Fig. 2C and D, SlPlsC1 was purified to homogeneity using CYMAL-6, and the protein yield was approximately ∼0.2 mg/g of wet cells. A comparable result was obtained by purification using DDM (data not shown). The N-terminal residues of the purified protein were identified as 1-MLLIARS-7, which corresponded to the estimated N-terminal sequence of the nascent SlPlsC1. This is consistent with the previous report that the N-terminal Met residue is not removed when the second residue has a bulky side chain (23) and with the computational estimation that a conventional cleavable signal peptide is absent in SlPlsC1. We examined AGPAT activity of the purified recombinant proteins, with palmitoleoyl-CoA and oleoyl-LPA as the acyl donor and acceptor substrates, respectively. Product analysis using normal-phase TLC showed that the CYMAL-6-solubilized SlPlsC1 did remain active (Fig. 3C, lane 2; Supplementary Fig. S1, lane 2). PA production was dependent on the presence of the native enzyme and both substrates. The product was further analyzed using mass spectrometry, which showed product ions of m/z 671.8 (Fig. 3D). This m/z value was similar to the theoretical mass of PA with palmitoleoyl and oleoyl groups (calculated mass of [M–H]− ion, 671.5). These results demonstrated that SlPlsC1 retained AGPAT activity after solubilization and purification when CYMAL-6 was used as the detergent. In contrast to the CYMAL-6-solubilized enzyme, the DDM-solubilized enzyme was inactive after purification (Supplementary Fig. S1, lane 3). Moreover, we found that SlPlsC1 purified with CYMAL-6 lost its activity when the reaction was carried out in the presence of DDM (Supplementary Fig. S1, lane 4), and SlPlsC1 purified with DDM partially recovered its activity when the reaction was carried out in the presence of CYMAL-6 (Supplementary Fig. S1, lane 5). These data suggest that purified SlPlsC1 is reversibly inactivated by DDM. Purification of the active form of AGPAT/PlsC family enzyme has been a long-standing problem. It has been difficult to solubilize this membrane protein without causing its inactivation. Recently, Robertson et al. reported successful purification and crystallographic analysis of PlsC from a thermophilic bacterium, Thermotoga maritima (9). They used DDM as the detergent to purify the enzyme. In contrast to their case, DDM was not suitable for purification of SlPlsC1 as described earlier. Instead, we found that CYMAL-6 is useful as the detergent to solubilize and purify SlPlsC1 in its active form. The reason for the different actions of DDM and CYMAL-6 on SlPlsC1 is not clear at present. However, it may be possible that a linear alkyl chain of DDM (the n-dodecyl group) occludes the enzyme cavity that accommodates the fatty acyl chains of the substrate, whereas CYMAL-6, which has a bulky hydrocarbon moiety (the 6-cyclohexylhexyl group), does not exert this inhibitory effect. Although we have not verified this hypothesis, CYMAL-6 and related detergents may be useful for purification of various AGPAT/PlsC family enzymes. Indeed, we found that the purification method using CYMAL-6 was applicable to other bacterial PlsC homologs (to be reported elsewhere). Enzymatic characterization of SlPlsC1 The enzymatic properties of SlPlsC1 were characterized using commercially available palmitoleoyl-CoA and oleoyl-LPA. After the reaction was terminated, a free CoA released by the reaction was quantified spectrophotometrically using DTNB (24). Thiol production was proportional to the enzyme concentration (0–0.70 µM) and the reaction time (0–10 min) under the standard assay conditions (Supplementary Fig. S2). Using this method, the specific activity of purified SlPlsC1 was calculated to be 5.0 ± 0.3 µmol/min/mg protein. To our knowledge, this is the first example of application of a DTNB assay to quantitative measurement of the activity of AGPAT/PlsC. This method is easy, fast and inexpensive although it is not suitable for characterization of crude AGPAT/PlsC preparations that contain high concentration of thiol compounds such as Cys-containing proteins. Next, the reaction conditions were varied to characterize the enzymatic properties of SlPlsC1. The optimum pH and reaction temperature were found to be 8–9 and 20–25°C, respectively (Fig. 4A and B). Consistent with the psychrotrophic nature of S. livingstonensis Ac10, the activity reduced to 13.3% upon pretreatment at 40°C for 30 min (Fig. 4C). AGPAT activity increased upon the addition of Mg2+, Ca2+ and Mn2+ (Fig. 4D). The kinetic parameters could not be determined because the Km value appeared to be lower than the detection limit of the assay system (∼5 µM). Fig. 4 View largeDownload slide Characterization of enzymatic properties of SlPlsC1. (A) The effect of pH. Circle, Tris-HCl; square, borate-NaOH; triangle, CAPS-NaOH. The activity in Tris-HCl (pH 9.0) is defined as 100%. (B) The effect of reaction temperature. The activity at 20°C is defined as 100%. (C) Thermal stability. SlPlsC1 was preincubated at the indicated temperatures for 30 min prior to the assay. The activity after preincubation at 0°C is defined as 100%. (D) The effect of divalent cations. The activity in the presence of MgCl2 is defined as 100%. Error bars represent standard deviation from three independent experiments. Fig. 4 View largeDownload slide Characterization of enzymatic properties of SlPlsC1. (A) The effect of pH. Circle, Tris-HCl; square, borate-NaOH; triangle, CAPS-NaOH. The activity in Tris-HCl (pH 9.0) is defined as 100%. (B) The effect of reaction temperature. The activity at 20°C is defined as 100%. (C) Thermal stability. SlPlsC1 was preincubated at the indicated temperatures for 30 min prior to the assay. The activity after preincubation at 0°C is defined as 100%. (D) The effect of divalent cations. The activity in the presence of MgCl2 is defined as 100%. Error bars represent standard deviation from three independent experiments. Substrate specificity of SlPlsC1 towards fatty acyl-CoAs and lysophospholipids is shown in Fig. 5. SlPlsC1 showed high specificity towards the unsaturated fatty acyl-CoAs, among which those having multiple cis-double bonds (linoleoyl-, arachidonoyl-, and eicosapentaenoyl-CoAs) were more preferred substrates (Fig. 5A). On the other hand, the saturated fatty acyl-CoAs, especially those with a carbon chain length of 14 or longer, served as poor fatty acyl donors. SlPlsC1 showed almost no activity towards palmitoyl-CoA, which serves as the substrate of PlsC from T. maritima (9). Thus the present study clearly indicates the diverse substrate specificities of the PlsC family enzymes. It should be noted that SlPlsC1 could differentiate between the presence and absence of even a single double bond (e.g. palmitoyl and palmitoleoyl groups). Although the structural basis for this specificity is currently unknown, it is possible that SlPlsC1 has a curved cavity for fatty acyl donors, where a cis-unsaturated acyl chain can be easily accommodated, whereas a saturated long acyl chain would need to adopt an energetically unfavorable conformation to fit into the curvature. Unlike the donor substrates, SlPlsC1 accepted LPAs having either unsaturated or saturated acyl chains. It showed little activity towards G3P and lysophospholipids with head groups (Fig. 5B), thereby suggesting that SlPlsC1 is dedicated to the production of PA and is not involved in the acyl chain remodeling of phospholipids with a head group. Fig. 5 View largeDownload slide Substrate specificity of SlPlsC1 towards fatty acyl donors (A) and acceptors (B). The acyl moieties are abbreviated as follows: 12:0, lauroyl; 14:0, myristoyl; 16:0, palmitoyl; 16:1, palmitoleoyl; 18:0, stearoyl; 18:1, oleoyl; 18:2, linoleoyl; 20:0, arachidoyl; 20:4, arachidonoyl; 20:5, eicosapentaenoyl. The activity towards palmitoleoyl-CoA and oleoyl-LPA is defined as 100%. Error bars represent standard deviation from three independent experiments. Fig. 5 View largeDownload slide Substrate specificity of SlPlsC1 towards fatty acyl donors (A) and acceptors (B). The acyl moieties are abbreviated as follows: 12:0, lauroyl; 14:0, myristoyl; 16:0, palmitoyl; 16:1, palmitoleoyl; 18:0, stearoyl; 18:1, oleoyl; 18:2, linoleoyl; 20:0, arachidoyl; 20:4, arachidonoyl; 20:5, eicosapentaenoyl. The activity towards palmitoleoyl-CoA and oleoyl-LPA is defined as 100%. Error bars represent standard deviation from three independent experiments. We previously demonstrated that the amounts of EPA-containing phospholipids, but not the others, was notably reduced by disruption of the SlPlsC1 gene (16), implying that this enzyme was dedicated to the incorporation of EPA into phospholipids in vivo. Thus, it was rather unexpected that the purified SlPlsC1 could utilize a broad range of unsaturated fatty acyl-CoAs in vitro (Fig. 5A). A possible explanation for this seeming discrepancy may be that four other PlsC homologs (SlPlsC2, SlPlsC3, SlPlsC4 and SlPlsC5) present in S. livingstonensis Ac10 can compensate for the lack of SlPlsC1 except for the acylation with EPA in vivo. Another possibility is that SlPlsC1 associates with the protein complex for EPA biosynthesis in vivo, which directly couples the EPA production with the incorporation of the nascent eicosapentaenoyl group into LPA. In conclusion, the present study reported a new method to purify PlsC in its active form and demonstrated the occurrence of a new PlsC with unique substrate specificity. Despite many unanswered questions, the successful purification of active SlPlsC1 shall enable us to perform its detailed biochemical and structural analysis, which would result in a deeper understanding of the mechanism underlying the catalytic action of the PlsC family enzymes. Supplementary Data Supplementary Data are available at JB Online. Funding This work was supported in part by the Japan Society for the Promotion of Science KAKENHI (15H06328 to T.O., 15H04474 and 17H04598 to T.K.). Conflict of Interest None declared. References 1 Kennedy E.P. , Weiss S.B. ( 1956 ) The function of cytidine coenzymes in the biosynthesis of phospholipides . J. Biol. Chem. 222 , 193 – 214 Google Scholar PubMed 2 Carter J.R. ( 1968 ) Cytidine triphosphate: phosphatidic acid cytidyltransferase in Escherichia coli . J. Lipid Res. 9 , 748 – 754 Google Scholar PubMed 3 Kanfer J. , Kennedy E.P. ( 1964 ) Metabolism and function of bacterial lipids II . J. Biol. Chem. 239 , 1720 – 1726 Google Scholar PubMed 4 Yao J. , Rock C.O. ( 2013 ) Phosphatidic acid synthesis in bacteria . Biochim. Biophys. Acta. 1831 , 495 – 502 Google Scholar CrossRef Search ADS PubMed 5 Okuyama H. , Wakil S.J. ( 1973 ) Positional specificities of acyl coenzyme A: glycerophosphate and acyl coenzyme A: monoacylglycerophosphate acyltransferases in Escherichia coli . J. 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( 2010 ) PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes . Bioinformatics 26 , 1608 – 1615 Google Scholar CrossRef Search ADS PubMed 20 Bligh E.G. , Dyer W.J. ( 1959 ) A rapid method of total lipid extraction and purification . Can. J. Biochem. Physiol. 37 , 911 – 917 Google Scholar CrossRef Search ADS PubMed 21 Goldman P. , Vagelos P.R. ( 1961 ) The specificity of triglyceride synthesis from diglycerides in chicken adipose tissue . J. Biol. Chem . 236 , 2620 – 2623 Google Scholar PubMed 22 Miroux B. , Walker J.E. ( 1996 ) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels . J. Mol. Biol . 260 , 289 – 298 Google Scholar CrossRef Search ADS PubMed 23 Hirel P.H. , Schmitter M.J. , Dessen P. , Fayat G. , Blanquet S. ( 1989 ) Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid . Proc. Natl. Acad. Sci. USA 86 , 8247 – 8251 Google Scholar CrossRef Search ADS 24 Ellman G.L. ( 1959 ) Tissue sulfhydryl groups . Arch. Biochem. Biophys. 82 , 70 – 77 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AGPAT 1-acyl-sn-glycerol-3-phosphate acyltransferase CAPS N-cyclohexyl-3-aminopropanesulfonic acid CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate CYMAL-6 6-cyclohexyl-1-hexyl-β-d-maltoside DDM n-dodecyl-β-d-maltoside DTNB 5, 5′-dithiobis(2-nitrobenzoic acid) EPA eicosapentaenoic acid GPAT sn-glycerol-3-phosphate acyltransferase G3P sn-glycerol 3-phosphate LPA lysophosphatidic acid LPE lysophosphatidylethanolamine LPG lysophosphatidylglycerol OG n-octyl- β-d-glucoside; PA, phosphatidic acid TLC thin-layer chromatography © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Purification and characterization of 1-acyl-sn-glycerol-3-phosphate acyltransferase with a substrate preference for polyunsaturated fatty acyl donors from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy025
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Abstract

Abstract 1-Acyl-sn-glycerol-3-phosphate acyltransferase (designated as PlsC in bacteria) catalyzes the acylation of lysophosphatidic acid and is responsible for the de novo production of phosphatidic acid, a precursor for the synthesis of various membrane glycerophospholipids. Because PlsC is an integral membrane protein, it is generally difficult to solubilize it without causing its inactivation, which has been hampering its biochemical characterization despite its ubiquitous presence and physiological importance. Most biochemical studies of PlsC have been carried out using crude membrane preparations or intact cells. In this study, we succeeded in solubilization and purification of a recombinant PlsC in its active form from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10 using 6-cyclohexyl-1-hexyl-β-d-maltoside as the detergent. We characterized the purified enzyme and found that it has a substrate preference for the acyl donors with a polyunsaturated fatty acyl group, such as eicosapentaenoyl group. These results provide a new method for purification of the PlsC family enzyme and demonstrate the occurrence of a new PlsC with unique substrate specificity. 1-acyl-sn-glycerol-3-phosphate acyltransferase, lipid metabolism, membrane protein, phospholipid, PlsC 1,2-Diacyl-sn-glycerol 3-phosphate, or phosphatidic acid (PA), is a precursor for the synthesis of various glycerophospholipids, in both eukaryotes and bacteria, which constitute biological membranes (1–3). The de novo synthesis of PA involves two distinct acyltransferases that catalyze the sequential acylation of sn-glycerol 3-phosphate (G3P) (4) (Fig. 1). The first step is the acylation of hydroxyl group at the sn-1 position of G3P to produce 1-acyl-sn-glycerol 3-phosphate, or lysophosphatidic acid (LPA), by the action of G3P acyltransferase (GPAT). There are two types of GPATs—PlsB and PlsY—which show no homology in their amino acid sequences. The second acylation step, where a hydroxyl group at the sn-2 position of LPA is acylated to yield PA, is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT; EC number: 2.3.1.51) (5). This enzyme, designated as PlsC in bacteria, is a membrane protein that has four acyltransferase motifs (6). An AGPAT protein homologous to PlsC also occurs in eukaryotes (including yeast, fungi, plants and mammals), and its role in PA production in these organisms is conserved (7, 8). Fig. 1 View largeDownload slide Schematic representation of the de novo glycerophospholipid biosynthesis pathway. R and R′ represent hydrocarbon chains, and X represents a polar head group. ACP stands for acyl carrier protein. Fig. 1 View largeDownload slide Schematic representation of the de novo glycerophospholipid biosynthesis pathway. R and R′ represent hydrocarbon chains, and X represents a polar head group. ACP stands for acyl carrier protein. Despite such ubiquitous presence and an important role in lipid metabolism, purification of AGPATs/PlsCs in their active forms has not been reported until recently (9). The difficulty in purification of these enzymes is supposed to be due to their membrane association: AGPATs/PlsCs tend to be inactivated following solubilization from their native membranous milieu into the detergent micelles. Therefore, biochemical insights into the enzymatic characteristics of AGPATs/PlsCs have been limited. Because AGPATs/PlsCs play a major role in determining the acyl chain composition of membrane phospholipids, the substrate specificity of these enzymes can significantly affect the physicochemical and biochemical properties of biological membrane. Various bacteria possess more than one plsC gene (10–14), and the occurrence of multiple PlsCs, which possibly have different substrate specificities, in a single bacterium could be a considerable advantage in modifying the fatty acyl composition of cell membrane in response to the changes in extracellular environment. So far, although mutagenesis experiments using intact cells or crude extracts provided a clue to estimate their properties (10–13), potential functional redundancy between different PlsC homologs in vivo and lack of in vitro characterization of purified PlsCs have been obscuring their precise enzymatic characteristics, including their fatty acyl chain selectivity. A cold-adapted bacterium Shewanella livingstonensis Ac10, which was originally isolated from the Antarctic seawater, grows in the temperature range of 4–25°C (optimum temperature, 18°C). In response to the low temperature, the bacterium produces glycerophospholipids that are esterified at the sn-2 position with a polyunsaturated fatty acid, namely eicosapentaenoic acid (EPA) (15). The EPA-containing phospholipids play an important role in survival of the bacterium in a cold environment (15). There are five putative plsC genes in the genome of S. livingstonensis Ac10 (the gene products are designated as SlPlsC1, SlPlsC2, SlPlsC3, SlPlsC4 and SlPlsC5). We previously revealed that the deletion of the SlPlsC1 gene caused a marked decrease in the amounts of EPA-containing phospholipids, without significantly altering the composition of other phospholipids (16). Thus, SlPlsC1 appeared to be dedicated for the in vivo acylation of LPA with EPA. To determine its precise biochemical characteristics, we established a method to purify SlPlsC1 in its active form and characterized the purified enzyme, which revealed its broad substrate specificity with a preference for various polyunsaturated fatty acyl donors. Materials and Methods Materials Palmitoleoyl-CoA was purchased from Sigma–Aldrich (St. Louis, MO, USA). LPAs, lysophosphatidylethanolamine (LPE) and lysophosphatidylglycerol (LPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 6-Cyclohexyl-1-hexyl-β-d-maltoside (CYMAL-6) was purchased from Anatrace (Maumee, OH, USA). n-Dodecyl-β-d-maltoside (DDM), n-octyl-β-d-glucoside (OG), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), arachidonic acid and EPA were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals used were of analytical grade. Heterologous expression, solubilization and purification of SlPlsC1 Escherichia coli C43(DE3) was transformed with a pET21a-derived expression vector (pET21a-SlPlsC1) that encoded SlPlsC1 fused with a C-terminal hexahistidine-tag, which we constructed previously (16). The transformant was cultured in M9YG medium (1 L) (17) supplemented with ampicillin (100 mg/L) on a reciprocal shaker (100 min−1) at 37°C. After the optical density at 600 nm reached ∼0.7, the culture was incubated at 18°C for additional 20 h. The cells were collected and stored at −30°C until further use. The following procedure was performed on ice or in a cold room. The collected cells (about 3 g) were disrupted by sonication in buffer A [6 ml; sodium phosphate (20 mM, pH 7.4), NaCl (0.5 M), imidazole (20 mM), dithiothreitol (0.5 mM) and glycerol (10%)]. After centrifugation (10,110 g for 30 min at 4°C), the supernatant was recovered and subjected to ultracentrifugation (150,000 g for 1 h at 4°C). The membrane pellet was resuspended in buffer A (6 ml) containing one of the following detergents present at four times higher concentration than their critical micelle concentrations: CYMAL-6 (2.2 mM), DDM (0.68 mM), OG (100 mM) or CHAPS (32 mM). After incubating with gentle shaking for 2 h, the suspension was subjected to ultracentrifugation (100,000 g for 1 h at 4°C) to collect the detergent-solubilized membrane proteins into the supernatant. The proteins solubilized with either CYMAL-6 or DDM were applied onto the HisTrap FF column (1 ml; GE Healthcare, Buckinghamshire, UK), which was preequilibrated with buffer B [sodium phosphate (20 mM, pH 7.4), NaCl (0.5 M), glycerol (10%), and either CYMAL-6 (1.1 mM) or DDM (0.34 mM)] containing 20 mM imidazole. The column was washed with buffer B containing 60 mM imidazole, and subsequently eluted with buffer B containing 0.5 M imidazole. The eluate was concentrated using the Amicon 10 kDa molecular weight cut-off centrifugal filter (Merck Millipore, Darmstadt, Germany). For long-term storage, glycerol was added to the concentrate at a final concentration of 30%. The fractions collected during purification were analyzed by 12.5% SDS-PAGE. Protein concentration was determined by the Bradford method using the Protein Assay CBB solution (Nacalai Tesque). Western blot analysis The protein samples were separated on a 12.5% SDS-PAGE gel and subsequently transferred onto an Immobilon-P membrane (Merck Millipore). The hexahistidine-tagged SlPlsC1 was specifically labelled with anti 6 × histidine, monoclonal antibody 9F2 (Wako Chemicals, Kyoto, Japan) and was detected using a peroxidase-conjugated anti-rat IgG antibody (Sigma–Aldrich) and Chemi-Lumi One Ultra (Nacalai Tesque). The membrane was scanned with an imaging system C-DiGit (M&S TechnoSystems, Osaka, Japan). N-Terminal amino acid sequence analysis The N-terminal amino acid sequence was determined using the PPSQ-31 A protein sequencer (Shimadzu, Kyoto, Japan). The presence or absence of an N-terminal signal sequence was analyzed using the PSORTb ver3.0.2 (http://www.psort.org/psortb/) and SOSUIsignal (http://harrier.nagahama-i-bio.ac.jp/sosui/sosuisignal/sosuisignal_submit.html) webservers (18, 19). Qualitative AGPAT assay A reaction mixture (120 µl) for the crude membrane fraction contained palmitoleoyl-CoA (0.5 mM), oleoyl-LPA (0.5 mM), Tris-HCl (10 mM, pH 9.0), MgCl2 (1 mM), CYMAL-6 (1.1 mM) and the membrane fraction (ca. 0.8 µg protein). A reaction mixture (80 µl) for the detergent-solubilized fraction contained palmitoleoyl-CoA (0.5 mM), oleoyl-LPA (0.5 mM), Tris-HCl (10 mM, pH 9.0), MgCl2 (1 mM), the detergent-solubilized fraction (ca. 0.4 µg protein), and one of the following detergents: CYMAL-6 (1.1 mM), DDM (0.34 mM), OG (50 mM) or CHAPS (16 mM). The composition of a reaction mixture (80 µl) for purified SlPlsC1 was the same as for the membrane fraction except that the membrane fraction was replaced with purified SlPlsC1 (2.8 µg). These reaction mixtures were incubated at 20°C for 10 min, and the product was extracted using the Bligh and Dyer method (20). The extracted product was analyzed on the silica gel 60 thin-layer chromatography (TLC) plate (Merck Millipore) that was developed using the chloroform–acetone–methanol–acetic acid–water (5:2:1:1:0.5 v/v) solvent system. The TLC plates were stained with molybdenum blue solution. Also, the extracted product was dissolved in the acetonitrile–methanol (2:1 v/v) solvent system containing 0.1% triethylamine and analyzed using the API 3000 mass spectrometer (SCIEX, Ontario, Canada) equipped with an electrospray ionization source under the following conditions: polarity, negative; scan range, m/z 200–900; nebulizer gas, 8; curtain gas, 8; ionspray voltage, −4500 V; declustering potential, −30 V; focusing potential, −200 V; and entrance potential, −10 V. Quantitative AGPAT assay A mixture containing palmitoleoyl-CoA (0.5 mM), oleoyl-LPA (0.5 mM), Tris-HCl (10 mM, pH 9.0), MgCl2 (1 mM) and CYMAL-6 (1.1 mM) was prepared and aliquoted to each reaction tube (36 µl). After preincubating the mixture at 20°C for 1 min, purified SlPlsC1 (4 µl) was added to a final concentration of 0.11 µM (3.1 µg/ml). The reaction was performed at 20°C for 2–3 min and stopped by adding guanidine-HCl (5 M, 40 µl) in HEPES-NaOH (0.5 M, pH 8.0). All reactions were stopped before ∼20% of the substrates were consumed. Next, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; 2 mM, 20 µl) in HEPES-NaOH (0.5 M, pH 8.0) was added, and the reaction mixture was allowed to stand at about 25°C for ∼10 min. The mixture (90 µl) was transferred to a 96-well microplate and absorption was measured at 412 nm using the SpectraMax 190 (Molecular Devices, Sunnyvale, CA, USA). A standard curve was plotted with cysteine. To characterize the enzymatic properties of SlPlsC1, the above-mentioned standard assay conditions were modified as follows: (i) the buffer was replaced with Tris-HCl (pH range 7.5–9), borate-NaOH (pH range 8.5–10) and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS)-NaOH (pH range 10–11); (ii) the reaction was performed in the range of 4–40°C; (iii) the enzyme solution was preincubated in the range of 0–50°C for 30 min before the reaction; (iv) MgCl2 (1 mM) was replaced with CaCl2 (1 mM), MnCl2 (1 mM), NiCl2 (1 mM) or EDTA (10 mM); (v) palmitoleoyl-CoA was replaced with lauroyl-, myristoyl-, palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, arachidoyl-, arachidonoyl-, or eicosapentaenoyl-CoA; (vi) oleoyl-LPA was replaced with palmitoyl-LPA, palmitoleoyl-LPA, oleoyl-LPE, oleoyl-LPG or G3P. Synthesis of fatty acyl-CoAs Various acyl-CoAs were synthesized via the mixed anhydride method (21). Briefly, to various fatty acids (25 µmol), tetrahydrofuran (0.5 ml) containing ethyl chloroformate (4.8 µl), triethylamine (3.5 µl) and butylhydroxytoluene (5.5 mg) was added. After vigorous shaking at room temperature for 2 h, the solvent was evaporated. The residue was dissolved in tetrahydrofuran (1 ml), and the solution was briefly centrifuged. The supernatant (0.5 ml) was transferred to a fresh tube, to which CoA (0.25 ml, 20 mM) in sodium bicarbonate solution (0.1 M) was added. After vigorous shaking at room temperature for 2 h, the reaction mixture was washed twice with diethyl ether (0.5 ml). The fatty acyl-CoAs were precipitated by adding 1/10 volume of 10% perchloric acid and following centrifugation. The precipitate was washed sequentially with 0.5 ml each of diethyl ether, acetone and diethyl ether. The synthetic acyl-CoAs were purified using the high-performance liquid chromatography system equipped with the CAPCELL PAK C18 column (4.6 × 250 mm; Shiseido, Tokyo, Japan). The acyl-CoAs were eluted using ammonium formate (0.1 M)–methanol solvent system with a linear gradient of 70–95%(v/v) of methanol at a flow rate of 0.5 ml/min over 20 min. The elution was monitored by measuring the absorption at 254 nm. The purity of synthetic acyl-CoAs was checked by normal-phase TLC with the n-butanol–acetic acid–water (5:2:3 v/v) solvent system. Results and Discussion Expression, solubilization and purification of recombinant SlPlsC1 In most cases, overexpression of a membrane protein is toxic to the host cells. Generally, this results in a significantly lower expression of the protein or the formation of inclusion bodies. To circumvent this problem, we constructed an expression system for SlPlsC1 using E. coli C43(DE3) (22) and employed mild expression conditions, in which the recombinant cells were cultured in M9YG medium at 18°C without the addition of isopropyl-β-d-1-thiogalactoside. The attempts to express SlPlsC1 either in Luria–Bertani broth or using a low-copy vector pSTV28 gave lower expression level (data not shown). The cultured cells were disrupted, and the cell-free extract was recovered and subjected to ultracentrifugation to obtain membrane pellets. The SDS-PAGE analysis showed that a ∼31 kDa protein was present in the 150,000 g pellet from the E. coli cells harboring pET21a-SlPlsC1 (calculated mass of tagged SlPlsC1, 28.1 kDa), whereas the same protein was not present in the corresponding fraction of the E. coli cells harboring the empty vector (Fig. 2A, lanes 4 and 8). The protein band was identified to be recombinant SlPlsC1 by western blotting using the anti-hexahistidine antibody (Fig. 2C and D, lane 1) and also by in vitro assay showing that the membrane fraction from E. coli harboring pET21a-SlPlsC1 was capable of producing PA more efficiently than that from the control E. coli cells (Fig. 3A). Fig. 2 View largeDownload slide SDS-PAGE and western blot analyses of the fractions collected during purification. (A) The supernatants and pellets obtained by centrifugation of the disrupted E. coli cells harboring pET21a (lanes 1 and 2, respectively) and those obtained by subsequent ultracentrifugation (lanes 3 and 4, respectively). The corresponding fractions from E. coli cells harboring pET21a-SlPlsC1 are shown in lanes 5–8, respectively. (B) The supernatants and pellets (odd- and even-numbered lanes, respectively) obtained by ultracentrifugation after solubilization with the indicated detergents. Mock indicates the samples prepared in the absence of detergents. (C) and (D) The membrane fraction (lane 1), the supernatant obtained by ultracentrifugation after solubilization with CYMAL-6 (lane 2), and the eluate obtained after the affinity chromatography (lane 3) were visualized by CBB staining (C) and chemiluminescence (D). The arrowheads indicate the position of the SlPlsC1 band. Fig. 2 View largeDownload slide SDS-PAGE and western blot analyses of the fractions collected during purification. (A) The supernatants and pellets obtained by centrifugation of the disrupted E. coli cells harboring pET21a (lanes 1 and 2, respectively) and those obtained by subsequent ultracentrifugation (lanes 3 and 4, respectively). The corresponding fractions from E. coli cells harboring pET21a-SlPlsC1 are shown in lanes 5–8, respectively. (B) The supernatants and pellets (odd- and even-numbered lanes, respectively) obtained by ultracentrifugation after solubilization with the indicated detergents. Mock indicates the samples prepared in the absence of detergents. (C) and (D) The membrane fraction (lane 1), the supernatant obtained by ultracentrifugation after solubilization with CYMAL-6 (lane 2), and the eluate obtained after the affinity chromatography (lane 3) were visualized by CBB staining (C) and chemiluminescence (D). The arrowheads indicate the position of the SlPlsC1 band. Fig. 3 View largeDownload slide Analysis of the products of the in vitro AGPAT assay. (A) AGPAT assay using the membrane fraction from the E. coli cells harboring pET21a (lane 2) and that from the E. coli cells harboring pET21a-SlPlsC1 (lane 3). Lane 1 shows authentic PA. (B) AGPAT assay using the soluble fractions collected after incubation without detergents (lane 2) or with CYMAL-6 (lane 3), DDM (lane 4), OG (lane 5), or CHAPS (lane 6). Lane 1 shows authentic PA. (C) AGPAT assay of the enzyme purified with CYMAL-6. Lane 1, authentic PA; Lane 2, complete reaction; Lane 3, reaction using heat-denatured SlPlsC1; Lane 4, reaction without oleoyl-LPA; Lane 5, reaction without palmitoleoyl-CoA (ori., origin; s.f., solvent front). (D) Mass spectrometric analysis of PA produced by purified SlPlsC1. Front graph, complete reaction; rear graph, reaction using heat-denatured SlPlsC1. Fig. 3 View largeDownload slide Analysis of the products of the in vitro AGPAT assay. (A) AGPAT assay using the membrane fraction from the E. coli cells harboring pET21a (lane 2) and that from the E. coli cells harboring pET21a-SlPlsC1 (lane 3). Lane 1 shows authentic PA. (B) AGPAT assay using the soluble fractions collected after incubation without detergents (lane 2) or with CYMAL-6 (lane 3), DDM (lane 4), OG (lane 5), or CHAPS (lane 6). Lane 1 shows authentic PA. (C) AGPAT assay of the enzyme purified with CYMAL-6. Lane 1, authentic PA; Lane 2, complete reaction; Lane 3, reaction using heat-denatured SlPlsC1; Lane 4, reaction without oleoyl-LPA; Lane 5, reaction without palmitoleoyl-CoA (ori., origin; s.f., solvent front). (D) Mass spectrometric analysis of PA produced by purified SlPlsC1. Front graph, complete reaction; rear graph, reaction using heat-denatured SlPlsC1. Next, a detergent suitable for solubilizing SlPlsC1 was screened by testing the solubilization efficiency and enzymatic activity of the solubilized protein. The detergents used were CYMAL-6, DDM, OG and CHAPS, which are considered to have low impact on the structural integrity of membrane proteins. The membrane fraction was incubated in a buffer containing one of these detergents. After ultracentrifugation, SlPlsC1 was recovered in the supernatant with either of these detergents (Fig. 2B). However, we noticed that only the recombinant protein that was solubilized with either CYMAL-6 or DDM retained the PA-producing activity (Fig. 3B, lanes 3 and 4). Notably, the sample prepared in the absence of detergents also produced PA (Fig. 3B, lane 2). This was probably because a small amount of SlPlsC1, which was not extracted from the membrane, remained in the supernatant (Fig. 2B, lane 1). Thus, the supernatants obtained from the CYMAL-6- or DDM-treated samples were subjected to Ni–NTA affinity chromatography. As shown in the lane 3 of Fig. 2C and D, SlPlsC1 was purified to homogeneity using CYMAL-6, and the protein yield was approximately ∼0.2 mg/g of wet cells. A comparable result was obtained by purification using DDM (data not shown). The N-terminal residues of the purified protein were identified as 1-MLLIARS-7, which corresponded to the estimated N-terminal sequence of the nascent SlPlsC1. This is consistent with the previous report that the N-terminal Met residue is not removed when the second residue has a bulky side chain (23) and with the computational estimation that a conventional cleavable signal peptide is absent in SlPlsC1. We examined AGPAT activity of the purified recombinant proteins, with palmitoleoyl-CoA and oleoyl-LPA as the acyl donor and acceptor substrates, respectively. Product analysis using normal-phase TLC showed that the CYMAL-6-solubilized SlPlsC1 did remain active (Fig. 3C, lane 2; Supplementary Fig. S1, lane 2). PA production was dependent on the presence of the native enzyme and both substrates. The product was further analyzed using mass spectrometry, which showed product ions of m/z 671.8 (Fig. 3D). This m/z value was similar to the theoretical mass of PA with palmitoleoyl and oleoyl groups (calculated mass of [M–H]− ion, 671.5). These results demonstrated that SlPlsC1 retained AGPAT activity after solubilization and purification when CYMAL-6 was used as the detergent. In contrast to the CYMAL-6-solubilized enzyme, the DDM-solubilized enzyme was inactive after purification (Supplementary Fig. S1, lane 3). Moreover, we found that SlPlsC1 purified with CYMAL-6 lost its activity when the reaction was carried out in the presence of DDM (Supplementary Fig. S1, lane 4), and SlPlsC1 purified with DDM partially recovered its activity when the reaction was carried out in the presence of CYMAL-6 (Supplementary Fig. S1, lane 5). These data suggest that purified SlPlsC1 is reversibly inactivated by DDM. Purification of the active form of AGPAT/PlsC family enzyme has been a long-standing problem. It has been difficult to solubilize this membrane protein without causing its inactivation. Recently, Robertson et al. reported successful purification and crystallographic analysis of PlsC from a thermophilic bacterium, Thermotoga maritima (9). They used DDM as the detergent to purify the enzyme. In contrast to their case, DDM was not suitable for purification of SlPlsC1 as described earlier. Instead, we found that CYMAL-6 is useful as the detergent to solubilize and purify SlPlsC1 in its active form. The reason for the different actions of DDM and CYMAL-6 on SlPlsC1 is not clear at present. However, it may be possible that a linear alkyl chain of DDM (the n-dodecyl group) occludes the enzyme cavity that accommodates the fatty acyl chains of the substrate, whereas CYMAL-6, which has a bulky hydrocarbon moiety (the 6-cyclohexylhexyl group), does not exert this inhibitory effect. Although we have not verified this hypothesis, CYMAL-6 and related detergents may be useful for purification of various AGPAT/PlsC family enzymes. Indeed, we found that the purification method using CYMAL-6 was applicable to other bacterial PlsC homologs (to be reported elsewhere). Enzymatic characterization of SlPlsC1 The enzymatic properties of SlPlsC1 were characterized using commercially available palmitoleoyl-CoA and oleoyl-LPA. After the reaction was terminated, a free CoA released by the reaction was quantified spectrophotometrically using DTNB (24). Thiol production was proportional to the enzyme concentration (0–0.70 µM) and the reaction time (0–10 min) under the standard assay conditions (Supplementary Fig. S2). Using this method, the specific activity of purified SlPlsC1 was calculated to be 5.0 ± 0.3 µmol/min/mg protein. To our knowledge, this is the first example of application of a DTNB assay to quantitative measurement of the activity of AGPAT/PlsC. This method is easy, fast and inexpensive although it is not suitable for characterization of crude AGPAT/PlsC preparations that contain high concentration of thiol compounds such as Cys-containing proteins. Next, the reaction conditions were varied to characterize the enzymatic properties of SlPlsC1. The optimum pH and reaction temperature were found to be 8–9 and 20–25°C, respectively (Fig. 4A and B). Consistent with the psychrotrophic nature of S. livingstonensis Ac10, the activity reduced to 13.3% upon pretreatment at 40°C for 30 min (Fig. 4C). AGPAT activity increased upon the addition of Mg2+, Ca2+ and Mn2+ (Fig. 4D). The kinetic parameters could not be determined because the Km value appeared to be lower than the detection limit of the assay system (∼5 µM). Fig. 4 View largeDownload slide Characterization of enzymatic properties of SlPlsC1. (A) The effect of pH. Circle, Tris-HCl; square, borate-NaOH; triangle, CAPS-NaOH. The activity in Tris-HCl (pH 9.0) is defined as 100%. (B) The effect of reaction temperature. The activity at 20°C is defined as 100%. (C) Thermal stability. SlPlsC1 was preincubated at the indicated temperatures for 30 min prior to the assay. The activity after preincubation at 0°C is defined as 100%. (D) The effect of divalent cations. The activity in the presence of MgCl2 is defined as 100%. Error bars represent standard deviation from three independent experiments. Fig. 4 View largeDownload slide Characterization of enzymatic properties of SlPlsC1. (A) The effect of pH. Circle, Tris-HCl; square, borate-NaOH; triangle, CAPS-NaOH. The activity in Tris-HCl (pH 9.0) is defined as 100%. (B) The effect of reaction temperature. The activity at 20°C is defined as 100%. (C) Thermal stability. SlPlsC1 was preincubated at the indicated temperatures for 30 min prior to the assay. The activity after preincubation at 0°C is defined as 100%. (D) The effect of divalent cations. The activity in the presence of MgCl2 is defined as 100%. Error bars represent standard deviation from three independent experiments. Substrate specificity of SlPlsC1 towards fatty acyl-CoAs and lysophospholipids is shown in Fig. 5. SlPlsC1 showed high specificity towards the unsaturated fatty acyl-CoAs, among which those having multiple cis-double bonds (linoleoyl-, arachidonoyl-, and eicosapentaenoyl-CoAs) were more preferred substrates (Fig. 5A). On the other hand, the saturated fatty acyl-CoAs, especially those with a carbon chain length of 14 or longer, served as poor fatty acyl donors. SlPlsC1 showed almost no activity towards palmitoyl-CoA, which serves as the substrate of PlsC from T. maritima (9). Thus the present study clearly indicates the diverse substrate specificities of the PlsC family enzymes. It should be noted that SlPlsC1 could differentiate between the presence and absence of even a single double bond (e.g. palmitoyl and palmitoleoyl groups). Although the structural basis for this specificity is currently unknown, it is possible that SlPlsC1 has a curved cavity for fatty acyl donors, where a cis-unsaturated acyl chain can be easily accommodated, whereas a saturated long acyl chain would need to adopt an energetically unfavorable conformation to fit into the curvature. Unlike the donor substrates, SlPlsC1 accepted LPAs having either unsaturated or saturated acyl chains. It showed little activity towards G3P and lysophospholipids with head groups (Fig. 5B), thereby suggesting that SlPlsC1 is dedicated to the production of PA and is not involved in the acyl chain remodeling of phospholipids with a head group. Fig. 5 View largeDownload slide Substrate specificity of SlPlsC1 towards fatty acyl donors (A) and acceptors (B). The acyl moieties are abbreviated as follows: 12:0, lauroyl; 14:0, myristoyl; 16:0, palmitoyl; 16:1, palmitoleoyl; 18:0, stearoyl; 18:1, oleoyl; 18:2, linoleoyl; 20:0, arachidoyl; 20:4, arachidonoyl; 20:5, eicosapentaenoyl. The activity towards palmitoleoyl-CoA and oleoyl-LPA is defined as 100%. Error bars represent standard deviation from three independent experiments. Fig. 5 View largeDownload slide Substrate specificity of SlPlsC1 towards fatty acyl donors (A) and acceptors (B). The acyl moieties are abbreviated as follows: 12:0, lauroyl; 14:0, myristoyl; 16:0, palmitoyl; 16:1, palmitoleoyl; 18:0, stearoyl; 18:1, oleoyl; 18:2, linoleoyl; 20:0, arachidoyl; 20:4, arachidonoyl; 20:5, eicosapentaenoyl. The activity towards palmitoleoyl-CoA and oleoyl-LPA is defined as 100%. Error bars represent standard deviation from three independent experiments. We previously demonstrated that the amounts of EPA-containing phospholipids, but not the others, was notably reduced by disruption of the SlPlsC1 gene (16), implying that this enzyme was dedicated to the incorporation of EPA into phospholipids in vivo. Thus, it was rather unexpected that the purified SlPlsC1 could utilize a broad range of unsaturated fatty acyl-CoAs in vitro (Fig. 5A). A possible explanation for this seeming discrepancy may be that four other PlsC homologs (SlPlsC2, SlPlsC3, SlPlsC4 and SlPlsC5) present in S. livingstonensis Ac10 can compensate for the lack of SlPlsC1 except for the acylation with EPA in vivo. Another possibility is that SlPlsC1 associates with the protein complex for EPA biosynthesis in vivo, which directly couples the EPA production with the incorporation of the nascent eicosapentaenoyl group into LPA. In conclusion, the present study reported a new method to purify PlsC in its active form and demonstrated the occurrence of a new PlsC with unique substrate specificity. Despite many unanswered questions, the successful purification of active SlPlsC1 shall enable us to perform its detailed biochemical and structural analysis, which would result in a deeper understanding of the mechanism underlying the catalytic action of the PlsC family enzymes. Supplementary Data Supplementary Data are available at JB Online. Funding This work was supported in part by the Japan Society for the Promotion of Science KAKENHI (15H06328 to T.O., 15H04474 and 17H04598 to T.K.). Conflict of Interest None declared. References 1 Kennedy E.P. , Weiss S.B. ( 1956 ) The function of cytidine coenzymes in the biosynthesis of phospholipides . J. Biol. Chem. 222 , 193 – 214 Google Scholar PubMed 2 Carter J.R. ( 1968 ) Cytidine triphosphate: phosphatidic acid cytidyltransferase in Escherichia coli . J. Lipid Res. 9 , 748 – 754 Google Scholar PubMed 3 Kanfer J. , Kennedy E.P. ( 1964 ) Metabolism and function of bacterial lipids II . J. Biol. Chem. 239 , 1720 – 1726 Google Scholar PubMed 4 Yao J. , Rock C.O. ( 2013 ) Phosphatidic acid synthesis in bacteria . Biochim. Biophys. Acta. 1831 , 495 – 502 Google Scholar CrossRef Search ADS PubMed 5 Okuyama H. , Wakil S.J. ( 1973 ) Positional specificities of acyl coenzyme A: glycerophosphate and acyl coenzyme A: monoacylglycerophosphate acyltransferases in Escherichia coli . J. 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( 2010 ) PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes . Bioinformatics 26 , 1608 – 1615 Google Scholar CrossRef Search ADS PubMed 20 Bligh E.G. , Dyer W.J. ( 1959 ) A rapid method of total lipid extraction and purification . Can. J. Biochem. Physiol. 37 , 911 – 917 Google Scholar CrossRef Search ADS PubMed 21 Goldman P. , Vagelos P.R. ( 1961 ) The specificity of triglyceride synthesis from diglycerides in chicken adipose tissue . J. Biol. Chem . 236 , 2620 – 2623 Google Scholar PubMed 22 Miroux B. , Walker J.E. ( 1996 ) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels . J. Mol. Biol . 260 , 289 – 298 Google Scholar CrossRef Search ADS PubMed 23 Hirel P.H. , Schmitter M.J. , Dessen P. , Fayat G. , Blanquet S. ( 1989 ) Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid . Proc. Natl. Acad. Sci. USA 86 , 8247 – 8251 Google Scholar CrossRef Search ADS 24 Ellman G.L. ( 1959 ) Tissue sulfhydryl groups . Arch. Biochem. Biophys. 82 , 70 – 77 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AGPAT 1-acyl-sn-glycerol-3-phosphate acyltransferase CAPS N-cyclohexyl-3-aminopropanesulfonic acid CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate CYMAL-6 6-cyclohexyl-1-hexyl-β-d-maltoside DDM n-dodecyl-β-d-maltoside DTNB 5, 5′-dithiobis(2-nitrobenzoic acid) EPA eicosapentaenoic acid GPAT sn-glycerol-3-phosphate acyltransferase G3P sn-glycerol 3-phosphate LPA lysophosphatidic acid LPE lysophosphatidylethanolamine LPG lysophosphatidylglycerol OG n-octyl- β-d-glucoside; PA, phosphatidic acid TLC thin-layer chromatography © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Feb 5, 2018

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