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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 563-570 |
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doi:10.1111/j.1745-7270.2006.00196.x |
b-Glucosidase Catalyzing Specific
Hydrolysis of an Iridoid b-Glucoside from Plumeria
obtusa
Doungkamol BOONCLARM1,3,
Thakorn SORNWATANA1,3, Dumrongkiet ARTHAN4,
Palangpon KONGSAEREE2,3, and Jisnuson SVASTI1,3*
1 Department of Biochemistry,
2 Department of Chemistry,
3
Center for Excellence in Protein Structure and Function, Faculty of Science,
Mahidol University, Bangkok 10400, Thailand;
4
Department of Tropical Nutrition and Food Science, Faculty of Tropical
Medicine, Mahidol University, Bangkok 10400, Thailand
Received: March 23,
2006�������
Accepted: April 25,
2006
*Corresponding
author: Tel, 662-201-5845; Fax, 662-201-5843; E-mail, [email protected]
Abstract������� An iridoid b-glucoside, namely plumieride coumarate
glucoside, was isolated from the Plumeria obtusa (white frangipani)
flower. A b-glucosidase, purified to
homogeneity from P. obtusa, could hydrolyze plumieride coumarate
glucoside to its corresponding 13-O-coumarylplumieride. Plumeria b-glucosidase is a monomeric glycoprotein
with a molecular weight of 60.6 kDa and an isoelectric point of 4.90. The
purified b-glucosidase had an optimum pH
of 5.5 for p-nitrophenol (pNP)-b-D-glucoside and for
its natural substrate. The Km values for pNP-b-D-glucoside and Plumeria b-glucoside were 5.04�0.36 mM and 1.02�0.06
mM, respectively. The enzyme had higher hydrolytic activity towards pNP-b-D-fucoside than pNP-b-D-glucoside. No activity was
found for other pNP-glycosides. Interestingly, the enzyme showed a high specificity
for the glucosyl group attached to the C-7 position of the coumaryl moiety of
plumieride coumarate glucoside. The enzyme showed poor hydrolysis of
4-methylumbelliferyl-b-glucoside and esculin, and
did not hydrolyze alkyl-b-glucosides, glucobioses,
cyanogenic-b-glucosides, steroid b-glucosides, nor other iridoid b-glucosides. In conclusion, the Plumeria
b-glucosidase shows high specificity for
its natural substrate, plumieride coumarate glucoside.
Key words������� b-glucosidase; Plumeria
obtusa; iridoid b-glucoside; plumieride
coumarate glucoside; 13-O-coumarylplumieride
b-Glucosidases (EC 3.2.1.21) form a group of glycosidases that catalyze the hydrolysis of b-glucosidic linkage formed between D-glucose and the hydroxyl group of the aglycone. Plant b-glucosidases are involved in a variety of functions such as the release of physiologically active hormones, lignin synthesis, defense mechanisms and cell wall degradation [1]. Various b-glucosidases have been shown to specifically hydrolyze their natural b-glucoside substrates, such as cassava linamarase and linamarin [2], Thai rosewood b-glucosidase and dalcochinin b-glucoside [3], maize b-glucosidase and DIMBO-b-glucoside [4], Polygonum tinctorium b-glucosidase and indoxyl-b-glucoside [5], rice b-glucosidase and oligo-b-glucosides [6], walnut b-glucosidase and hydrojuglone-b-glucoside [7], Solanum b-glucosidase and furostanol glycoside-26-O-b-glucoside [8].
Due to our interest in the specificity of b-glucosidases for their natural substrates, we have screened many species of Thai plants for b-glucosidases and their b-glucoside substrates. An ethanol crude extract of the Plumeria obtusa (Apocynaceae) flower was found to contain a large amount of b-glucoside. An iridoid b-glucoside with two glucosyl groups attached, namely plumieride coumarate glucoside, was subsequently isolated from P. obtusa. This compound was previously found in Allamanda cathartica roots [9], and in the heartwood [10] and bark [11] of P. rubra. Iridoids form a large group of cyclopentane-(c)-pyran monoterpenoids, which have been reported in many varieties of plant species [12-14] and are of pharmaceutical interest. Iridoid glycosides in the Apocynaceae family have been shown to have diverse biological activities, such as algicidal [9], molluscicidal, cytotoxic, antimicrobial [10], plant growth inhibition [15], anti-fungal [16], and anti-fertility [17] activities.
Various natural diglucosides have been found in plants that might be hydrolyzed by b-glucosidase(s) from the same plant. Thus, in the black cherry (Prunus serotina Ehrh.), the b-(1,6)-glucosidic linkage of the cyanogenic diglucoside, amygdalin, is specifically cleaved by the enzyme amygdalin hydrolase into the corresponding cyanogenic monoglucoside, prunasin, which in turn might be cleaved by the enzyme prunasin hydrolase into mandelonitrile and glucose [18]. In addition, a b-glucosidase isolated from ginseng root was recently shown to hydrolyze the steroidal b-(1,2)-diglucoside ginsenoside Rg3 to yield the monoglucoside ginsenoside Rh3 and glucose [19]. Here, we study the cleavage of our natural diglucoside substrate (plumieride coumarate glucoside) from P. obtusa flowers by its natural enzyme isolated from the same source.
Although b-glucosidases and their natural substrates have been studied, b-glucosidases specifically hydrolyzing natural iridoid b-glucosides have not yet been purified or characterized. In this paper, we report the biochemical properties of purified Plumeria b-glucosidase, which specifically hydrolyzes its iridoid-b-diglucoside substrate, namely plumieride coumarate glucoside.
Materials and Methods
Plant materials
Flowers of P. obtusa were collected from Mahidol University (Bangkok, Thailand) in April 2005.
General experimental procedure
The 1H, 13C, and 2-D nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX300 spectrometer (Bruker, Faellanden, Switzerland), operating at 300 MHz for proton and 75 MHz for carbon. The Electrospray ionization-Time of Fight (ESITOF) mass spectra were obtained from a Micromass LCT mass spectrometer (Bruker, Breman, Germany). Isolation of dalcochinin-8'-O-b-D-glucoside [3] and torvoside A [20] from Thai rosewood and Solanum torvum have been described previously; gonocaryoside A and kingiside [21] prepared from Gonocaryum subrostratum were kindly provided by Dr. Chutima LIMMATVATIRAT (Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University, Nakorn-Pathom, Thailand).
Isolation and identification of
the natural substrate and its aglycone
Fresh flowers (2.5 kg) of P. obtusa (white frangipani) were immersed in liquid N2, followed by homogenization and maceration in ethanol overnight. The crude extract (10 g) was evaporated to dryness, and chromatographed on a Sephadex LH-20 column, eluted with MeOH. The b-glucoside containing fractions were pooled, evaporated, then purified by preparative Thin-Layer Chromatography (TLC; silica gel 60G F254) using chloroform:methanol (3:1) as a mobile phase. The b-glucoside band (the most intense band under ultraviolet light) was scraped from the TLC plate. MeOH was added to dissolve the substrate and silica gel was removed by filtration, to yield 200 mg of purified plumieride coumarate glucoside as a yellow powder; 1H-NMR (300 MHz, D2O) d: 5.18 (1H, d, J=6.37 Hz, H-1), 7.31 (1H, s, H-3), 3.69 (1H, s, H-5), 6.23 (1H, dd, J=5.46, 2.25 Hz, H-6), 5.25 (1H, d, J=4.76 Hz, H-7), 2.98 (1H, dd, J=3.14, 3.14 Hz, H-9), 7.20 (1H, s, H-10), 5.5 (1H, d, J=6.48 Hz, H-13), 1.41 (3H, d, J=6.51 Hz, H-14), 3.58 (3H, s, H-16), 6.15 (1H, d, J=15.99 Hz, H-2''), 7.42 (1H, d, J=15.85 Hz, H-3''), 7.28 (1H, d, J=8.53 Hz, H-5''), 6.93 (1H, d, J=8.62 Hz, H-6''), 6.93 (1H, d, J=8.62 Hz, H-8''), 7.28 (1H, d, J=8.53 Hz, H-9''), 4.6 (1H, d, J=7.92 Hz, H-1'), 3.15 (1H, dd, J=8.41, 8.67 Hz, H-2'), 3.47 (1H, dd, J=9.72, 10.56 Hz, H-3'), 3.48 (1H, dd, J=9.72, 10.56 Hz, H-4'), 3.47 (1H, dd, J=9.72, 10.56 Hz, H-5'), 3.75 (2H, dd, J=11.67, 12.59 Hz, H-6'), 4.55 (1H, d, J=9.4 Hz, H-1'''), 3.1 (1H, dd, J=8.41, 8.67 Hz, H-2'''), 3.42 (1H, dd, J=8.96, 9.06 Hz, H-3'''), 3.42 (1H, dd, J=8.96, 9.06 Hz, H-4'''), 3.42 (1H, dd, J=8.96, 9.06 Hz, H-5'''), and 3.7 (2H, dd, J=11.67, 12.59 Hz, H-6'''); 13C-NMR (75 MHz, D2O) d: 93.96, 151.8, 110.6, 38.4, 140.7, 128.4, 97.8, 49.2, 151.6, 133.6, 172.2, 65.8, 19.0, 168.8, 52.4, 167.7, 115.8, 146.1, 128, 130.6, 117.3, 159.2, 117.3, 130.6, 98.6, 73.1, 76.0, 69.8, 76.0, 61.0, 98.6, 73.3, 76.5, 70.0, 76.5, 61.2. Mass Spectrometry [MS; Electrospray ionization (ESI) positive]: m/z 801 [M+Na]+, calculated for C36H42O19.
Plumieride coumarate glucoside (50 mg) was incubated overnight with purified Plumeria b-glucosidase in 0.1 M sodium acetate buffer, pH 5.5, 37 �C. After solvent evaporation, the reaction mixture was separated using preparative TLC. Chloroform:methanol (3:1) was used as a mobile phase. MeOH was used to elute compounds from silica gel, to afford 20.0 mg of 13-O-coumarylplumieride as yellow powder; 1H-NMR (300 MHz, D2O) d: 5.28 (1H, d, J=5.6 Hz, H-1), 7.55 (1H, s, H-3), 3.88 (1H, ddd, H-5), 6.43 (1H, dd, J=2.36, 2.36 Hz, H-6), 5.53 (1H, dd, J=2.24, 2.33 Hz, H-7), 2.84 (1H, dd, J=7.32, 5.76 Hz, H-9), 7.48 (1H, s, H-10), 5.65 (1H, d, J=5.59 Hz, H-13), 1.5 (3H, d, J=6.64 Hz, H-14), 3.67 (3H, s, H-16), 6.34 (1H, d, J=15.89 Hz, H-2''), 7.6 (1H, d, J=15.95 Hz, H-3''), 7.5 (1H, d, J=8.81 Hz, H-5''), 6.85 (1H, d, J=8.55 Hz, H-6''), 6.75 (1H, d, J=8.69 Hz, H-8''), 7.45 (1H, d, J=8.81, H-9''), 4.65 (1H, d, J=7.83 Hz, H-1'), 3.15 (1H, dd, J=8.03, 8.58 Hz, H-2'), 3.35 (1H, dd, H-3'), 3.25 (1H, dd, H-4'), 3.25 (1H, dd, J=8.03, 8.58 Hz, H-5'), 3.8 (2H, dd, J=11.42, 11.42 Hz, H-6'); 13C-NMR (75 MHz, D2O) d: 92.97, 151.77, 109.55, 39.94, 141.21, 129.03, 96.74, 49.81, 151.77, 133.44, 170.17, 64.71, 19.38, 166.79, 51.42, 166.28, 114.11, 146.07, 125.42, 130.77, 116.45, 161.18, 115.47, 130.77, 99.1, 73.8, 77.1, 70.73, 78.0, 61.8. MS (ESI positive): m/z 639 [M+Na]+, calculated for C30H32O14.
Purification of Plumeria
b-glucosidase
In order to remove phenolic compounds, the fresh flowers of P. obtusa were immersed in liquid nitrogen and homogenized immediately with extraction buffer [50 mM sodium acetate buffer, pH 5.5, containing 1 mM phenylmethylsulphonyl fluoride, and 5% (W/V) poly�vinylpoly�rrolidone ]. The plant extract was filtered and centrifuged at 10,000 g for 30 min. Dowex 2x8-400 resin (Sigma, Steinhiem, Germany) was then added to the supernatant and the suspension was subsequently filtered and centrifuged at 10,000 g for 30 min. Ammonium sulfate was added to the supernatant to a final concentration of 80% (W/V). The pellet obtained by centrifugation at 10,000 g for 30 min was resuspended in 10 mM sodium phosphate buffer, pH 7.0, then dialyzed overnight at 4 �C against the same buffer to obtain crude extract. As the enzyme assay using glucose oxidase kit was interfered with by polyphenolic compounds of the crude extract, calculations were made using the activity at the ammonium sulfate fractionation step as 100% activity. The dialyzed enzyme was loaded at 0.5 ml/min onto a diethylaminoethyl (DEAE)-cellulose column (1.8 cm�12 cm; Whatman, Maidstone, England) equilibrated with 10 mM sodium phosphate buffer, pH 7.0. The bound components were eluted by applying a stepwise gradient of 0.1 M NaCl and 0.5 M NaCl in the same buffer. Active fractions from the DEAE-cellulose column eluted at 0.1 M NaCl were pooled, supplemented with ammonium sulfate to 1.0 M final concentration, then loaded onto a butyl-Toyopearl column (1.8 cm�10 cm; TosoHaas, Tokyo, Japan) previously equilibrated with 10 mM sodium phosphate buffer, pH 7.0, containing 1 M ammonium sulfate. The bound proteins were eluted with a linear gradient of 1.0-0 M ammonium sulfate in the same buffer (10+10 column volume). The b-glucosidase-containing fractions were pooled and concentrated using Aquasorb (BM Laboratories, Bangkok, Thailand). The concentrated pool was applied onto a Sephacryl S-200 HR column (2.5 cm�52 cm; Pharmacia, Uppsala, Sweden) equilibrated with 10 mM sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl. After that the active fractions were pooled and loaded onto a Con A-Sepharose column (1.0 cm9 cm; Pharmacia) equilibrated with 10 mM sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl. The bound components were eluted with 0.3 M of a-methyl glucoside in the same buffer. Active fractions were pooled and dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0. The dialyzed fraction was loaded onto a DEAE-cellulose column (0.5 cm�13 cm; Whatman) equilibrated with 10 mM sodium phosphate buffer, pH 7.0. A linear gradient of 0-0.3 M NaCl (20+20 column volume) in the same buffer was then applied to elute the bound protein.
Enzyme assay and protein
determination
Hydrolytic activity of b-glucosidase was determined either by measuring p-nitrophenol (pNP) released from pNP-glycosides or by glucose released by cleavage of pNP or other glycosides. For determination of pNP released, 1 ml of reaction mixture, containing 50 ml of appropriately diluted� enzyme and a final concentration of 2.5 mM p-nitrophenyl-b-D-glucoside (pNP-b-D-Glc) or pNP-glycoside, was incubated� in 0.1 M sodium acetate buffer, pH 5.5, at 37 �C for 30 min. Reactions were then stopped by adding 2 ml of 2 M sodium carbonate. pNP released was measured with a spectrophotometer at 400 nm, and one nkat of b-glucosidase was defined as the amount of enzyme releasing� 1 nmol pNP per second from 2.5 mM pNP-b-D-glucoside or pNP glycoside at 37 �C and pH 5.5.
Detection of glucose released for b-glucosidase assay was carried out by the glucose oxidase procedure (using 4-aminoantipyrene as a chromophore) [22]. Enzyme assay was performed in a reaction mixture (100 ml) containing appropriately diluted enzyme and 2.5 mM of final concentration of the substrate in 0.1 M sodium acetate buffer, pH 5.5, at 37 �C for 30 min. The reaction was stopped by boiling for 5 min, then 1.0 ml of a glucose oxidase reagent kit (BM Laboratories) was added to each reaction. The reaction was further incubated at 37 �C for 15 min. Glucose released was measured with a spectrophotometer at 505 nm. One nkat of b-glucosidase, measured in this manner, was defined as the amount of enzyme releasing 1 nmol glucose per sec from 2.5 mM glycoside at 37 �C and pH 5.5.
Kinetic studies were carried out by enzymatic assay using a glucose oxidase kit and substrate concentrations were varied from 0.2 mM to 8 mM for the natural substrate, and from 0.4 mM to 15 mM for pNP-b-D-Glc. Kinetic constants were determined using the Prism 3 program (GraphPad Software, San Diego, USA). Protein contents were followed in column effluents at 280 nm or otherwise measured by the Bio-Rad protein assay kit (Bio-Rad, Hercules, USA) based on the Bradford method [23], using bovine serum albumin as a standard.
Determination of optimum pH
The optimum pH of the purified Plumeria b-glucosidase for the synthetic (pNP-b-D-Glc) and natural (plumieride coumarate glucoside) substrates was determined by conducting the enzyme assay in various 0.1 M citrate-phosphate buffers ranging from pH 3.0 to 7.5, and measuring glucose released by the glucose oxidase method.
Native molecular weight
determination
The native molecular weight of the enzyme was determined using a Sephacryl S-200 HR column (2.5 cm�52 cm; Pharmacia) calibrated with b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and ribonuclease (13.7 kDa).
Analytical gel electrophoresis
To determine subunit molecular weight and to check purity, the purified enzyme was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% separating gel and a 4% stacking gel (Hoefer mini-gel system; Hoefer Pharmacia Biotech, San Francisco, USA), according to the procedure of Laemmli [24]. Isoelectric point (pI) of the purified Plumeria b-glucosidase was determined by agarose isoelectric focusing (IEF) carried out in a Bio-Rad mini-gel IEF apparatus. After the electrophoresis was complete, the protein band was detected by staining with Coomassie blue R-250.
Effect of various compounds on
Plumeria -glucosidase activity
The purified b-glucosidase was preincubated for 30 min at 37 �C in the presence of various reagents. The enzyme was then assayed under standard conditions using pNP-b-D-Glc as a substrate.
Results
Incubation of the crude ethanol extract (containing natural substrate) of the flowers of P. obtusa with the aqueous extract (containing b-glucosidase enzyme) from the flowers of P. obtusa yielded an aglycone and a D-glucose (analyzed by TLC, data not shown). The natural substrate of b-glucosidase from the flowers of P. obtusa was then isolated and identified for further studies on the enzyme activity. Structure elucidation of a natural substrate of Plumeria b-glucosidase was carried out by comparison with those of its analogs [9]. Mass spectrometry data of the glycoside of m/z 801 corresponded to [M+Na]+, giving a molecular weight of 778. The 1H and 13C NMR and mass spectroscopic data showed signals corresponding to an iridoid b-glucoside, containing two glucosyl groups. Products obtained after the hydrolysis of plumieride coumarate glucoside by Plumeria b-glucosidase were characterized, and compared with published data [9]. Mass spectrometry data of the aglycone after hydrolysis with Plumeria b-glucosidase showed a signal at m/z 639 corresponding to [M+Na]+, giving a molecular weight of 616. The 1H, 13C and 2-D NMR spectra of aglycone were less complex than those of the b-glucoside substrate, particularly in the sugar resonance. These mass and NMR spectral data suggested that one unit of glucose was removed after digestion with purified Plumeria b-glucosidase. Analysis of NMR spectral data revealed that the hydrolysis occurred specifically at the glucosyl group attached to the phenolic ring at the C-7 position, whereas the second glucose attached to the C-1 position of plumieride coumarate glucoside was not cleaved by Plumeria b-glucosidase. The 1H, 13C, 2-D NMR and mass spectroscopic data showed signals corresponding to an iridoid b-glucoside, namely 13-O-coumarylplumieride or plumieride coumarate glucoside, previously found in the A. cathartica roots [9] and P. rubra bark [11]. Scheme 1 shows the hydrolysis of plumieride coumarate glucoside by b-glucosidase from the flowers of P. obtusa to form 13-O-coumarylplumieride.
A b-glucosidase specifically hydrolyzing plumieride coumarate glucoside
was subsequently purified to homogeneity from P. obtusa flowers by five
chromatographic steps on DEAE-cellulose, butyl-Toyopearl, Sephacryl S-200 HR,
Con A-Sepharose and DEAE-cellulose columns. The final yield of the purification
was 11% with 217-fold purification (Table 1). Plumeria b-glucosidase was
purified to homogeneity as revealed by SDS-PAGE and agarose IEF. Agarose IEF gel
and protein staining showed a single band protein corresponding to a pI of
approximately 4.90 [Fig. 1(A), lane 2]. In addition, a single protein
band was found at 60.6 kDa on SDS-PAGE [Fig. 1(B), lane 2], while the
molecular weight of the native enzyme was estimated by Sephacryl S-200 to be 54
kDa. As this enzyme could bind to Con A-Sepharose, Plumeria b-glucosidase is
probably a glycoprotein. The optimum pH for b-glucosidase activity using
pNP-b-D-Glc or natural b-glucoside substrate was found to be 5.5 (data
not shown). The enzyme retained 75% relative activity in the pH range 4.5-6.5.
The specificity of enzyme for the glycone moiety was tested by incubating purified b-glucosidase from the flowers of P. obtusa with 2.5 mM pNP glycosides, and following release of pNP. The results showed that, relative to the activity with pNP-b-D-Glc (100%), the enzyme showed higher activity towards pNP-b-D-Fuc (150%) and lower activity towards pNP-b-D-Gal (34%), but was unable to hydrolyze other pNP-glycosides (pNP-b-D-Man, pNP-a-D-Gal, pNP-a-D-Glc, and pNP-a-L-Fuc).
Plumeria b-glucosidase was found to show high specificity towards the diglucoside, plumieride coumarate glucoside, but was unable to hydrolyze the monoglucoside, 13-O-coumarylplumieride (the product from the hydrolysis of plumieride coumarate glucoside). This indicates that only the glucose unit attached to the phenolic ring at the C-7'' position can be cleaved by Plumeria b-glucosidase. Interestingly, this enzyme did not hydrolyze other iridoid-b-glucosides, including gonocaryoside A, or its derivative kingiside, indicating that this enzyme is specific for the aglycone moiety of iridoid-b-glucoside. Plumeria b-glucosidase was also able to hydrolyze pNP-b-D-Glc and 4-MU-b-glucoside (Table 2). In addition, it showed little hydrolysis of dalcochinin-8'-O-b-glucoside, which is the natural substrate of Thai rosewood [3], and esculin, but was not able to hydrolyze cyanogenic glucoside (linamarin and amygdalin), torvoside A (a steroid-b-glucoside isolated� from S. torvum) [23], b-linked glucobioses (gentiobiose and cellubiose), aromatic glucosides (arbutin and salicin), or alkyl-glucosides (methyl-b-glucoside and hexyl-b-glucoside).
Kinetic parameters of b-glucosidase were determined for its natural substrate, plumieride coumarate glucoside, and compared to a synthetic substrate (pNP-b-D-Glc). The Km value for plumieride coumarate glucoside (1.02�0.06) mM was lower than that for pNP-b-D-Glc (5.04�0.36) mM (Table 3), indicating that Plumeria b-glucosidase has a higher affinity for its natural substrate. Although the kcat for plumieride coumarate glucoside (13.9�0.3) s-1, was lower than for pNP-b-D-Glc (30.7�0.9) s-1, the catalytic efficiency (kcat/Km) of the plumieride coumarate glucoside is higher than pNP-b-D-Glc (Table 3), indicating that this enzyme shows a more efficient hydrolysis of its natural substrate than of the synthetic substrate.
D-glucono-1,5-lactone, a specific b-glucosidase inhibitor, at 1 mM, caused 44% inhibition of Plumeria b-glucosidase activity, divalent ions and EDTA showed little inhibitory effect on b-glucosidase activity. Additionally, Hg2+ and p-chloromercurybenzoate had little effect on the Plumeria b-glucosidase activity, indicating that no sulfhydryl� groups are involved in the catalytic activity (Table 4).
Discussion
In this study, we have purified an iridoid b-glucoside, plumieride coumarate glucoside, and its specific b-glucosidase from P. obtusa. The pI of this enzyme is similar to that of b-glucosidases purified from Polygonum tinctorium [5], Thai rosewood [25], and ripe sweet cherry [26].� The similarity in the molecular weights determined by denaturing SDS-PAGE and native gel filtration suggest that Plumeria b-glucosidase is likely to be monomeric, as found in Polygonum tinctorium [5], rice [6], and S. torvum [8] b-glucosidases. In addition, the enzyme is likely to be a glycoprotein, as is the case for many b-glucosidases for example cassava [2], rice [6], S. torvum [8], Thai rosewood [27], and oat [29]. In conclusion, the purified b-glucosidase is a monomeric glycoprotein. In terms of hydrolytic activity, the enzyme has optimum pH of 5.5, similar to many b-glucosidases. The Plumeria enzyme shows specificity towards b-linked glucose, fucose, and galactose moieties, similar to the glycone specificity of b-glucosidases from widely different sources such as walnut [7], Thai rosewood [25], ripe sweet cherry [26] and oat [28,29]. As for aglycone specificity, the results suggest that Plumeria b-glucosidase shows narrow substrate specificity for its iridoid-b-glucoside substrate and shows little hydrolysis of other b-glucosides having aromatic aglycones. Is this similar to Podophyllum peltatum? b-Glucosidase, which is very specific for its lignin natural substrate podophyllotoxin-4-b-D-glucoside, cannot hydrolyze pNP-b-D-glycosides, and has low activity towards b-linked oligosaccharides [30]. The enzyme also has higher catalytic efficiency (kcat/Km) for plumieride coumarate glucoside than pNP-b-D-Glc, indicating that it shows a more efficient hydrolysis of its natural substrate than of the synthetic substrate.� Like other plant b-glucosidases, for example, rice [6], Thai rosewood [25], sweet cherry [26] and oat [28,29], the P. obtusa enzyme is inhibited by D-glucono-1,5-lactone.
Plumieride coumarate glucoside, the diglucoside substrate, consists of two glucoses attached to different positions of the aglycone. This contrasts with other diglucosides, such as amygdalin, which contains b(1-6) glucobiose or ginsenoside, and Rg3, which contains b(1-2) glucobiose linked to the aglycone. The corresponding b-glucosidase enzymes from black cherry and ginseng cleave their respective natural substrates at the b-glucosidic linkage within the glucobiose moiety to yield the various corresponding monoglucosides and glucose [18-19]. However, unlike the other diglucosides mentioned above which have the two glucose residues covalently linked to each other, the plumieride coumarate glucoside substrate isolated here from P. obtusa contains two glucosyl groups attached to the aglycone at different positions, at the C-1 position of the iridoid moiety and at the C-7'' position of the coumaryl moiety. In conclusion, Plumeria b-glucosidase shows specific cleavage at the glucosyl group linked to the C-7'' position of the coumaryl moiety.
Previously, Ligustrum obtusifolium b-glucosidase was reported to be able to hydrolyze an iridoid b-glucoside, namely oleuropein, to give the activated products, which could form covalent adducts with the proteins of predators, presumably as a defense mechanism [31]. To our knowledge, however, the present work is the first report on the biochemical properties of a purified b-glucosidase having a high specificity for iridoid-b-glucoside.
Plumieride coumarate glucoside can be detected not only in flowers but also in other tissues of P. obtusa, such as leaf and stem, but in lower levels (unpublished data). In addition, it is clear that the b-glucoside and its specific b-glucosidase from the flowers of P. obtusa come in contact, as they occur in the same tissue, permitting the hydrolysis of the natural substrate. Other b-glucoside and specific b-glucosidase enzyme combinations were shown to play various roles in lignin synthesis, phytohormone activation and defense mechanisms [1]. However, there is no information available on the physiological functions of Plumeria b-glucosidase and its natural substrate. Iridoid b-glucosides isolated from the Apocynaceae family is known to have a variety of biological effects. Previously, plumieride was shown to have cytotoxic [11], plant growth inhibiting [15], and anti-fungal [16] activities. In addition, plumieride has been reported to arrest spermatogenesis when given to male rats, resulting in significant reduction of sperm mobility and sperm density [17]. Phytotoxic iridoids containing a spiro-lactone ring, isoplumericin and plumericin, are known to have algicidal [9], molluscicidal, antibacterial, and cytotoxic [10] activities. As the skeleton structures of these toxic compounds are similar to those of plumieride coumarate glucoside and 13-O-coumarylplumieride, it is possible that they might be metabolic products of plumieride coumarate glucoside and 13-O-coumarylplumieride, which might be considered to be the precursors of the toxic compounds. Therefore, it is possible that the storage iridoid b-glucoside compounds in the P. obtusa flowers are deglucosylated by intrinsic or extrinsic b-glucosidases, after which they are metabolized into toxic compounds used for self-defense against herbivores, insects, pests and microbes.
Acknowledgements
Jisnuson SVASTI is a Senior Research Scholar of the Thailand Research Fund. We thank Dr. Chutima LIMMATVATIRAT (Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University, Nakorn-Pathom, Thailand) for providing gonocaryoside A.
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