- Email: [email protected]
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is not bound significantly to the adsorbent 6, but is inhibited very strongly by the soluble ligand 8a with a Ki of as low as 8 nM.44 Several factors affect the adsorptive characteristics of the affinity gel such as the unfavorable steric interaction between the protein and the matrix.46 The adsorptive characteristics of the affinity gel 6 might be optimized by taking several factors into consideration, such as the structure of the aglycon moiety of the ligand and the nature of the counteranion of the amidinium group, as well as the physicochemical properties of the matrix and spacer. Finally, the affinity adsorbent with a glycosylamidine as a ligand can be used not only for the purification of a target enzyme, but also for elimination of an undesirable activity from a crude enzyme preparation. This mode of application also holds for the soluble -glycosylamidines as inhibitors to suppress an undesirable activity selectively in a crude enzyme sample. The glycosylamidines thus have potential as a versatile tool for glycosidase studies. Acknowledgment This work was supported in partly by a Grant-in-Aid for Scientific Research [(B)(2) 13480187 for J.H.] from Japan Society for the Promotion of Science.
46
P. Cuatrecasas and C. B. Anfinsen, Annu. Rev. Biochem. 40, 259 (1971).
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Diglycoside-Specific Glycosidases
By Kanzo Sakata, Masaharu Mizutani, Seung-Jin MA, and Jun Hiratake Introduction
A -primeverosidase was reported for the first time as a glycosidase that hydrolyzes a -primeveroside (6-O- -d-xylopyranosyl- -d-glucopyranoside) to a disaccharide (primeverose) and an aglycon (anthraquinone) by Bridel et al. in 1925.1 Although it was suggested that this enzyme occurs widely in plants and microorganisms,2 no -primeverosidase had been purified until Sakata and colleagues isolated it from juvenile tea leaves as a key 1
W. Ho¨sel, ‘‘Glycosidation and Glycosidases,’’ Vol. 7, p. 725. Academic Press, New York, 1981. 2 V. Plouvier, Comp. R. Hebd. Seanaces Acad. Sci. Ser. D 286, 1071 (1980).
METHODS IN ENZYMOLOGY, VOL. 363
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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glycosidase deeply involved in floral tea aroma formation in oolong tea and black tea.3,4 Gene cloning of the tea leaf -primeverosidase and its overexpression have now been accomplished.5,6 Floral tea aroma such as geraniol, linalool, and 2-phenylethanol of oolong tea and black tea were shown to be contained mainly as aroma precursors of diglycosides such as -primeverosides.7,8 A -primeverosidase responsible for the formation of aroma from the glycosidic aroma precursors was purified from the crude enzyme extract prepared from fresh juvenile tea leaves (Camellia sinensis).3,9,10 The purified enzyme shows high substrate specificity toward -primeverosides to yield aglycons and primeverose.11 Since then, many types of -primeverosides having various kinds of aglycons have been isolated from many varieties of plants not only as aroma precursors in flowers and fruits,11–13 but also as cyanogenic glycosides14–16 and as polar constituents.17–23 This fact strongly suggests that -primeverosidases are widely distributed among the plant kingdom. Research to confirm the wide occurrence of -primeverosidases and their physiological roles in plants is now in progress. 3
W. Guo, K. Yamauchi, N. Watanabe, T. Usui, S. Luo, and K. Sakata, Biosci. Biotech. Biochem. 59, 962 (1995). 4 K. Sakata, in ‘‘Caffeinated Beverages, Health Benefits, Physiological Effects, and Chemistry’’ (T. H. Parliament, C. Ho, and P. Schieberie, eds.), ACS Symposium Series 754, p. 327. American Chemical Society, Washington, DC, 2000. 5 M. Mizutani, H. Nakanishi, and K. Sakata. in ‘‘2001 International Conference on O-cha (Tea) Culture and Science,’’ Vol. Session II, p. 15. The Organizing Committee of the Conference, Shizuoka, Japan, 2001. 6 M. Mizutani, H. Nakanishi, J. Ema, S.-J. Ma, E. Noguchi, M. Fukuchi-Mizutani, K. Ochiai, Y. Tanaka, and K. Sakata, Plant Physiol. 130, 2164 (2002). 7 K. Sakata, N. Watanabe, and T. Usui, in ‘‘Food for Health in the Pacific Rim, Proceedings of the 3rd International Confrence of Food Science and Technology’’ (J. R. Whitaker et al. eds.), p. 93. Foods Nutrition Press, Inc., Trumbull, CT, 1999. 8 D. Wang, E. Kurasawa, T. Yoshimura, Y. Yamaguchi, K. Kubota, and A. Kobayashi, J. Agric. Food Chem. 49, 1900 (2001). 9 K. Ogawa, Y. Ijima, W. Guo, N. Watanabe, T. Usui, S. Dong, Q. Tong, and K. Sakata, J. Agric. Food Chem. 45, 877 (1997). 10 Y. Ijima, K. Ogawa, N. Watanabe, T. Usui, M. Ohnishi-Kameyama, T. Nagata, and K. Sakata, J. Agric. Food Chem. 46, 1712 (1998). 11 References on glycosidic aroma precursors are cited herein: S. J. Ma, M. Mizutani, J. Hiratake, K. Hayashi, K. Yagi, N. Watanabe, and K. Sakata, Biosci. Biotech. Biochem. 65, 2719 (2001). 12 M. Straubinger, H. Knapp, N. Watanabe, N. Oka, H. Washio, and P. Winterhalter, Natl, Prod. Lett. 13, 5 (1999). 13 H. Mayorga, C. Duque, H. Knapp, and P. Winterhalter, Phytochemistry 59, 439 (2002). 14 D. S. Seigler, Phytochemistry 14, 9 (1975). 15 D. Selmar, R. Lieberei, N. Junqueira, and B. Biehl, Phytochemistry 30, 2135 (1991). 16 J. Rockenbach, A. Nahrstedt, and V. Wray, Phytochemistry 31, 567 (1992).
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-Primeverosidase (EC 3.2.1.149)
Enzyme Assays -Primeverosidase activity is examined by assay using p-nitrophenyl (pNP) -primeveroside or 2-phenylethyl -primeveroside as a substrate. The activity is determined by measuring the liberation of p-nitrophenol or 2-phenylethanol from each glycoside. pNP -Primeveroside is synthesized enzymatically by transglycosylation between xylobiose and pNP -d-glucopyranoside with a -xylosidase from Aspergillus pulverulentus.24 2-Phenylethyl -primeveroside is synthesized chemically by glycosidation of 2-phenylethanol with 2,3,4,20 ,30 ,40 -hexa-O-benzoylprimeverosyl trichloroacetimidate.11 Stock solutions of each substrate (50 mM) are prepared by dissolving the glycosides in deionized water. The enzyme is dissolved in 20 mM citrate buffer (pH 6.0) containing 0.1% bovine serum albumin (BSA). One unit of the -primeverosidase activity is defined as the amount of enzyme liberating 1 mol of p-nitrophenol from pNP -primeveroside per minute in 20 mM citrate buffer (pH 6.0) at 37 . Each reaction mixture (150 l) contains 10 mM substrate, 20 mM citrate buffer (pH 6.0), 0.1% BSA, and 60 l of the enzyme solution. A mix ture without the enzyme is preincubated at 37 , and the reaction is initiated by adding the enzyme. An aliquot (20 l) of each enzyme reaction mixture is withdrawn after various time intervals during the incubation for analyses as follows. Assay with p-Nitrophenyl b-Primeveroside. An aliquot (20 l) of a reaction mixture is added to 80 l of 0.2 M Na2CO3 solution (pH 12), and the liberated p-nitrophenol is determined by measuring the absorbance at 405 nm in a spectrophotometer (UV-3101PC UV-VIS-NIR scanning spectrophotometer, Shimadzu, Japan).
17
L. H. W. van der Plas, M. J. M. Hargendoorn, and D. C. Jamar, J. Plant Physiol. 152, 235 (1998). 18 G. C. H. Derksen, T. A. van Beek, A. Groot, and A. Capelle, J. Chromatogr. A 816, 277 (1998). 19 Y. Lu, P.-J. Xu, Z.-N. Chen, and G.-M. Liu, Phytochemistry 49, 1135 (1998). 20 S. D. Petrovic, M. S. Gorunovic, V. Wray, and I. Merfort, Phytochemistry 50, 293 (1999). 21 A. Tamaki, H. Otsuka, and T. Ide, J. Nat. Prod. 62, 1074 (1999). 22 S. Yamamura, K. Osawa, K. Ohtani, R. Kasai, and K. Yamasaki, Phytochemistry 48, 131 (1998). 23 J. W. Jaroszewski, A. B. Rasmussen, H. B. Rasmussen, C. E. Olsen, and L. B. Jfrgensen, Phytochemistry 42, 649 (1996). 24 T. Murata, M. Shimada, N. Watanabe, K. Sakata, and T. Usui, J. Appl. Glycosci. 46, 431 (1999).
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Assay with 2-Phenylethyl b-Primeveroside. For the reaction with 2phenylethyl glycosides, an aliquot (20 l) of a reaction mixture is added to 2 l of 1 N NaOH, and then 8 l of an MeCN solution containing 3 g of benzyl alcohol is added as an internal standard. A sample (20 l) from each mixture (30 l) is injected to a HPLC. Analysis of liberated 2-phenylethanol is performed under the following HPLC conditions: column, YMC-pack ODS-AQ (4.6 250 mm) (YMC Co., Japan); detection, 210 nm with a Waters 996 photodiode array detector; column temperature, 40 ; mobile phase, 33% MeCN; and flow rate, 1.0 ml/min. Benzyl alcohol and 2-phenylethanol are detected at tR 6.7 and 8.7 min, respectively. The hydrolysis rate is determined from the linear portion (within 10% hydrolysis of the substrate) of each progress curve. Standard curves can be prepared by measuring the value of various concentrations of p-nitrophenol or 2-phenylethanol. Protein concentration is determined by the Bradford method25 using the Coomassie protein assay reagent (Pierce, Rockford, IL) with BSA as a standard. Purification of A b-Primeverosidase from a Tea Plant (Camellia sinensis var. sinensis cv. Yabukita) Fresh tea leaves are finely chopped, crushed in dry ice-acetone by a homogenizer, and filtered in vacuo. The residue is washed with chilled acet one (20 ) until the filtrate becomes nearly colorless. The residue is spread on filter paper and is then placed in vacuo to remove acetone. The residual acetone powder is stored at 20 before use. The acetone powder (100 g, equivalent to 600 g of the fresh leaves) is suspended in 0.1 M citrate buffer (pH 6.0, 2 liters), stirred for 4 h at 4 , and centrifuged at 14,000 g for 20 min. To the supernatant, an equal volume of chilled acetone (20 ) is added gradually with stirring, and the mixture is left overnight at 4 . The precipitate obtained by centrifugation at 14,000 g for 20 min is dissolved in 0.1 M citrate buffer (pH 6.0, 500 ml). Ammonium sulfate is added up to 40% saturation, and the mixture is centrifuged at 14,000 g for 20 min. The supernatant is subjected to a butyl-Toyopearl 650M column (Tosoh Corp., Tokyo, Japan) equilibrated with 20 mM citrate buffer (pH 6.0) containing 40% ammonium sulfate. The enzyme fractions are eluted by a linear gradient of ammonium sulfate from 40 to 0% saturation in 1 liter each of 20 mM citrate buffer (pH 6.0) with a flow rate of 5 ml/min. -Primeverosidase fractions are combined and concentrated by ultrafiltration (Amicon PM-10, Grace Japan Co., Ltd., Tokyo, Japan) and are dialyzed against 20 mM citrate buffer (pH 6.0) containing 50 mM NaCl. 25
M. Bradford, Anal. Biochem. 72, 248 (1976).
448
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Purification step
SINENSIS
cv. Yabukita
Total Total Yield Specific activity Purification protein (mg) activity (unit) (%) (unit/mg protein) fold (-fold)
Buffer extract Acetone ppt 40% (NH4)2SO4 sup Butyl-Toyopearl CM-Toyopearl Mono S
1000 220 110 7.7 0.54 0.30
40 38 32 3.8 0.70 0.51
100 95 80 9.3 1.8 1.3
0.04 0.17 0.29 0.49 1.3 1.7
1.0 4.3 7.3 12 33 43
The fraction is applied to a CM Toyopearl column (22 130 mm, Tosoh), and -primeveroidase fractions are eluted by a linear gradient of NaCl from 50 to 150 mM in 500 ml citrate buffer (pH 6.0). The -primeverosidase fraction is placed on a column (5 50 mm) of Mono S HR (Amersham Pharmacia Biotech Co. Ltd., Tokyo, Japan) equilibrated with 20 mM citrate buffer (pH 6.0). The -primeverosidase is eluted with a linear gradient of NaCl from 0.1 to 0.25 M at a flow rate of 1 ml/min. Because -primeverosidase is nearly coeluted with -glucosidases in each of the column chromatographis, -glucosidases are thoroughly eliminated from the -primeverosidase fractions by tracing both the activities using pNP -d-glucopyranoside and -primeveroside as substrates during the whole purification process. The purification procedures are summarized in Table I. -Primeverosidases from fresh leaves of two other cultivars for black tea (C. sinensis var. assamica) and oolong tea (C. sinensis var. sinensis cv. Shuixian) are also purified in the same way.9,10 Enzymatic Characteristics The purified -primeverosidase from cv. Yabukita gives a single band with an apparent molecular mass of 61 kDa on SDS–PAGE. The molecular weight has been determined to be 60,480 by TOFMS analysis.10 The enzymatic properties of -primeverosidases from three different cultivars for green tea, oolong tea, and black tea are summarized in Table II.9,10 Substrate Specificity In order to clarify the substrate specificity of the -primeverosidase with respect to the glycon moiety, nine kinds of diglycosides (1–9) and a glucoside (10) of 2-phenylethanol were synthesized (Fig. 1).11 These glycosides are designed by considering the structures of the natural aroma precursors isolated from plants along with the ease of synthesis.
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TABLE II Summary of Enzymatic Properties of -Primeverosidase -Primeverosidase Black tea MW pI Optimum temperature ( )a Stable temperatureb Optimum pHc pH stabilityd Specific activity (unit/mg)e
60,310 9.5 45 40 4 4–5 0.99
Oolong tea 60,480 9.5 45 40 4 3–5 0.98
Green tea 60,170 9.4 45 45 4 4–5 0.90
a
The optimum temperature of the enzyme was measured with pNP -primeveroside in 20 mM citrate buffer (pH 6.0) at various temperatures (30–70 ). b The thermal stability of the enzyme was estimated from the residual activities after incubation in 20 mM citrate buffer (pH 6.0) at various temperatures (30–60 ). c The optimum pH of the enzyme was examined in buffers with various pH values (2.0–9.0) at 37 for 1 h. d The pH stability of the enzyme was estimated from residual activities after incubation in the buffers with various pH values (2.0–9.0) at 37 for 1 h. The buffers used were 20 mM Clark and Lubs buffer (pH 2.0), 20 mM citrate buffer (pH 3.0–6.0), and 20 mM Tris–HCl buffer (pH 7.0–9.0). e -Primeverosidase activity was measured with pNP -primeveroside in 20 mM citrate buffer (pH 6.0) at 37 .
-Primeverosides, -vicianosides, -acuminosides, -gentiobiosides, and 6-O- -l-arabinofuranosyl- -d-glucopyranoside have been found in tea leaves as well as in several kinds of flowers and fruits.11 The aglycon moiety is fixed as 2-phenylethyl group, as 2-phenylethanol is one of the most common aroma compounds and its diglycosides ( -primeveroside, accuminoside, and 6-O- -l-arabinofuranosyl- -d-glucopyranoside) have been isolated from tea leaves as well as from several kinds of flowers and fruits as aroma precursors.11 The hydrolysis rate for the -primeverosidase purified from tea leaves is determined by using 10 mM of each synthetic 2-phenylethyl -glycoside (1–10) and pNP -glycoside (11–15) (Table III). The purified enzyme shows highly selective activity toward the diglycosidie substrates, -primeverosides (1 and 11). Interestingly, all the diglycosides (1–5 and 11) hydrolyzed by the enzyme are naturally occurring and have a (1–6) or an (1–6) glycosidic linkage in the glycon moiety. None of the unnatural diglycosides (6–9) with a (1–4)- or an (1–4)-glycosidic linkage are hydrolyzed. The 2-phenylethyl group seems to be preferred more as an aglycon for the -primeverosidase than pNP, as the hydrolysis rate of 1 was about twice
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Fig. 1. 2-Phenylethyl glycosides designed to study the substrate specificity of the -primeverosidase from tea leaves (cv. Yabukita).
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TABLE III Substrate Specificity of -Primeverosidase Purified from Juvenile Tea Leaves
Substrate 2-Phenylethyl -primeveroside (1) 2-Phenylethyl -vicianoside (2) 2-Phenylethyl -acuminoside (3) 2-Phenylethyl -gentiobioside (4) 2-Phenylethyl 6-O- -l-arabinofuranosyl -d-glucopyranoside (5) 2-Phenylethyl -lactoside (6) 2-Phenylethyl -cellobioside (7) 2-Phenylethyl -maltoside (8) 2-Phenylethyl -melibioside (9) 2-Phenylethyl -d-glucopyranoside (10) pNP -Primeveroside (11) pNP -d-Glucopyranoside (12) pNP -d-Galactopyranoside (13) pNP -d-Xylopyranoside (14) pNP -d-Arabinopyranoside (15) a
Hydrolysis activitya (mol/min/mg protein)
Relative activity (%)
39.1 1.16 0.301 0.098
100 2.97 0.60 0.34
0.027 NDb ND ND ND ND 21.3 0.091 ND ND ND
0.05 ND ND ND ND ND 54.4 0.23 ND ND ND
Hydrolysis activity is expressed by initial velocity (mol/ml/min) per protein concentration (mg/ml). The activity was determined by the liberation of 2-phenyethanol or p-nitrophenol from each glycoside. The liberated 2-phenyethanol was analyzed by HPLC, and p-nitrophenol was determined at 405 nm. Each reaction mixture contained 10 mM of each substrate, tea leaf -primeverosidase (0.22 unit/ml), and 20 mM citrate buffer (pH 6). The mixtures were incubated at 37 . [S.-J. Ma et al., Biosci. Biochem. Biotechnol. 65, 2719 (2001)].
as high as that of 11. This is due in part to the fact that pNP glycoside is not a natural substrate in tea leaves. To define substrate specificity with respect to the glycon moiety more quantitatively, the kinetic parameters (Km and kcat) for 1 and 2 are determined by the reaction of various concentrations of 1 (0.5–4.0 mM) or 2 (5–25 mM) with the enzyme under the respective assay condition as described earlier. The Michaelis constant of 1 (Km ¼ 2.00 mM) is 7.5-fold higher than that of 2 (Km ¼ 14.9 mM), and the specificity constant for 1 (kcat/Km ¼ 21.9 s1 mM) is 56-fold higher than that for 2 (kcat/Km ¼ 0.391 s1mM). These results indicate that the substrate specificity of the tea leaf -primeverosidase is highly specific for -primeverosides, although this enzyme hydrolyzes 2-phenylethyl -vicianoside and -acuminoside as well as -primeveroside.
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Cloning of Tea Leaf b-Primeverosidase The purified -primeverosidase described previously is applied to reversed-phase HPLC using a CAPCELL PAK C18 SG300 column (4.6 150 mm, Shiseido, Tokyo, Japan), and a single peak fraction is collected and directly analyzed to determine the N-terminal amino acid sequence. The fraction is further digested with a lysyl endopeptidase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) or trypsin (Wako), and the resulting peptide fragments are separated by reversed-phase HPLC using a CAPCELL PAK C18 SG120 column (4.6 250 mm, Shiseido). Amino acid sequences are analyzed by automated Edman degradation using an ABI 492 protein sequencer. The N-terminal and internal amino acid sequences thus determined are shown with an underline in Fig. 2. With the aid of the amino acid sequences determined from the purified primeverosidase, the cDNA encoding tea -primeveosidase is isolated from cv. Yabukita.5 Total RNA is isolated from fresh leaves of cv. Yabukita, and the mRNA is purified with an oligo(dT) cellulose column type 7 (Faversham Biosciences, Piscataway, New Jersey). The cDNA library is constructed from the mRNA with a double-stranded Uni-ZAP XR vector (Stratagene, San Diego, CA) following the manufacturer’s instructions. The oligonucleotide primer, BGLU1:50 -GGIGA(T/C)GTIGCIGA(T/C)GA(T/C)TT (T/C)TA (T/C)CA-30 is generated from the internal amino acid sequence, GDVADDFYH, determined from the purified -primeverosidase. By using a set of BGLU1 and an oligo(dT)16 primer, polymerase chain reaction (PCR) is performed through 30 cycles of 60 s at 94 , 90 s at 45 , and 60 s at 72 with the mass-excised plasmid library as templates. PCR products are separated by agarose gel (2%) electrophoresis, and the major band is cloned into the pGEM-T vector (Promega, Madison, WI). The cDNA fragment is labeled with [32P]dCTP by a random priming method, and about 500,000 plaques from the tea cDNA library are screened with the labeled fragment as a probe. The isolated cDNA consists of a 1524-bp open reading frame encoding a polypeptide of 507 amino acid residues, 8-bp 50 -untranslated region, a 179-bp 30 -noncoding region, and a poly(A) tail (Fig. 2). The deduced amino acid sequence of the -primeverosidase has high homology with -glucosidases from various plants such as amygdalin hydrolase (58%) from black cherry,26 -glucosidase for indoxyl -d-glucoside (55%) from the indigo,27 and linamarase from cassava (50%).28 Many 26
L. Zheng and J. E. Poulton, Plant Physiol. 109, 31 (1995). Y. Minami, Y. Shigeta, U. Tokumoto, Y. Tanaka, K. Yonekura-Sakakibara, H. Oh-oka, and Matsubara, Plant Sci. 142, 219 (1999). 28 M. A. Hughes, K. Brown, A. Pancoro, S. Murray, E. Oxtoby, and J. Hughes, Arch. Biochem. Biophys. 295, 273 (1992). 27
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Fig. 2. Nucleotide and predicted amino acid sequences of -primeverosidase cDNA from cv. Yabukita. Peptide sequences determined from the purified protein are underlined. The arrow indicates the position of the N-terminal amino acid sequence of the purified protein and also the possible cleavage site predicted by Psort analysis. Possible N-glycosylation sites are boxed. Catalytic residues in conserved sequence motifs in family 1 glycosyl hydrolases are double underlined.
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glycoside hydrolases from bacteria to mammals have been classified into 83 families according to amino acid sequence similarity (http:// afmb.cnrs-mrs.fr/pedro/CAZY/ghf.html), and tea -primeverosidase is classified in a family 1 glycosyl hydrolase because -primeverosidase shows high similarities to the family 1 glycoside hydrolases from various plants. Expression in Escherichia coli The cDNA encoding the mature -primeverosidase is amplified by PCR with a set of primers (50 -TCTAGAGCTCAAATCTCCTCCTTCAAC and 50 -GTCGACCTACTTGAGGAGGAATTTCTT) and is ligated into the pMALc2 vector (New England Biolabs, Inc., Beverly, MA) to generate pMALc2-Pri. Escherichia coli (JM109) is transformed with pMALc2-Pri, and the expression of -primeverosidase fused with a maltose-binding protein is induced by the addition of 0.1 mM isopropyl -d-thiogalactoside (IPTG). The cells are further grown at 20 , 150 rpm, for 24 h. The cells are collected by centrifugation at 5000 g for 10 min and are resuspended in 50 mM citrate buffer, pH 6.0, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The suspension is sonicated 15 times for 30 s at 30-s intervals and is centrifuged at 28,000 g for 30 min. The cell extract is applied to the amylose resin column, and the fusion protein is purified according to the manufacturer’s instructions. When the recombinant fusion protein between a maltose-binding pro tein and the mature primeverosidase is expressed at 37 , all the recombinant proteins are found as inclusion bodies, and -primeverosidase activity is not detected in the cell lysates. However, when the transformed cells are grown at 20 in the presence of 0.1 mM IPTG, a part of the recombinant proteins is detected in the soluble fractions. The recombinant fusion protein purified by amylose resin affinity chromatography is able to hydrolyze either pNP -primeveroside or 2-phenylethyl -primeveroside. Mode of Hydrolysis by b-Primeverosidase pNP -Primeveroside is incubated with either the -primeverosidase purified from tea leaves or the recombinant protein produced in E. coli, and the hydrolysates are analyzed by TLC. TLC is carried out on silica gel 60 F254 plates using a solvent system of butanol–pyridine–water–acetic acid (6:4:3:1, v/v). Glycosides and sugars are detected by heating the plate at 120 after spraying with 0.2% naphthoresorcinol in H2SO4–EtOH (1:19, v/v). The spot corresponding to primeverose (Rf 0.39) is clearly observed in each of the hydrolysates, but no spots for glucose (Rf 0.48) and xylose (Rf 0.60) are detected (Fig. 3). This confirms that -primeverosidase is a unique diglycosidase specifically hydrolyzing the -glycosidic bond between an
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Fig. 3. Thin-layer chromatogram of hydrolysis of pNP-primeveroside. pNP-Primeverosides at a final concentration of 5 mM were incubated in citrate buffer (pH 6.0) with either the tea -primeverosidase or the recombinant -primeverosidase expressed in E. coli. Products of the reaction were analyzed by TLC. Lanes 1, 2, 3, 4, and 7 were standard. Lane 1, pNPprimeveroside; lane 2, glucose; lane 3, xylose; lane 4, primeverose; lane 5, hydrolysis product by the tea -primeverosidase; lane 6, hydrolysis product by the recombinant enzyme; and lane 7, standard mixture (glucose, xylose, primeverose).
aglycon and a primeverose without cleaving the interglycosidic bond between the sugars. The stereochemistry for the enzymatic hydrolysis by -primeverosidase is analyzed by 1H-NMR (Fig. 4). The method is essentially the same as described by Wang et al.29 2-Phenylethyl -primeveroside (10 mg) is dissolved in 0.7 ml of D2O. -Primeverosidase (0.2 unit) purified from tea leaves is lypophilized and redissolved in 40 l of D2O. The enzyme (20 l) is added to an NMR tube containing the substrate, and the tube is incubated at 37 . The 1H-NMR measurement at the time intervals is per formed at 25 because the axial proton of -anomer of the released primeverose is overlapped with the HDO signal at 37 . NMR spectra are recorded on a JEOL JNM-AL 400 spectrometer. 1H-NMR spectra of the reaction mixture containing 2-phenylethyl -primeveroside and -primeverosidase reveal that the -anomer (Ha, 4.43, J ¼ 8.1 Hz) of primeverose is formed first. The -anomer (He, 5.04, J ¼ 3.8 Hz) of primeverose appears later as a 29
A. W. Wang, S. He, J. H. Grubb, W. S. Sly, and S. G. Withers, J. Biol. Chem. 273, 34057 (1998).
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Fig. 4. Time course of hydrolysis of 2-phenylethyl primeveroside catalyzed by the tea -primeverosidase, followed by 1H-NMR. (A) Postulated retaining hydrolysis of 2-phenylethyl primeveroside by the tea enzyme. (B) Spectra recorded 0, 5, 10, 20, 40, 90, and 180 min after addition of the enzyme. Ha and He indicate the H-1 resonances of - and -primeverose, respectively.
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consequence of mutarotation. Thus tea -primeverosidase is a retaining glycosidase as has been observed for other family 1 -glucosidases (Fig. 4). Diglycosidases from Other Plants and Microrganisms
A -primeverosidase has been found to be a real diglycoside-specific glycosidase, e.g., diglycosidase, which shows very high substrate specificity toward -primeverosides. In the early 1920s a few kinds of diglycosidases, which hydrolyzes diglycosides into aglycons and disaccharides, such as 6-O- -l-rhamnopyranosyl-d-glucose (rutinose), 6-O- -l-arabinopyranosyl-d-glucose (vicianose), and primeverose, were reported. However, most reports dealt with the crude enzyme, and only four kinds of diglycosidases have been purified and discussed in terms of their enzymic characteristics as shown in this section. Vicianin Hydrolase The occurrence of cyanogenic diglycosidases in Vicia angustifolla1 and Geumurbanaum30 has been reported. A vicianin hydrolase, which hydrolyzes vicianin to liberate vicianose and (R)-mandelonitrile, was purified from squirrel’s foot fern (Davallia tricomanoides).31 The enzyme was shown to have molecular weight of 340,000, a Km value of 4.9 mM for vicianin, and an optimum pH 5.5. SDS–PAGE analysis yielded three polypeptide bands at 56, 49, and 32.5 kDa. Rutinosidase (Rutinase) Occurrences of this enzyme in many kinds of plants have been reported for a long time. Two kinds of rutin hydrolases have been purified from the seeds of Fagopyrum tartaricum.32 They are both monomeric proteins of 68 kDa and show an optimum pH at 5.0. Km values for rutin are 130 and 120 mM, respectively. Quite recently, a rutinosidase has been purified from Penicillium rugulosum, which was cultured in a medium containing 2% rutin as a sole carbon sauce.33 This enzyme was shown to be a homotetramer with a molecular weight of 245,000 and an optimum pH at 2.2. An Endoglycosidase from Grape A diglycosidase (molecular weight 58,000; optimum pH, 4–5) was purified from Muscatt grape peel guided by rutinosidase activity measured with 30
M. Psenak, A. Jindra, P. Kovacs, and Dulovocova, Planta Med. 19, 154 (1970). P. A. Lizotte and J. E. Poulton, Plant Physiol. 86, 322 (1988). 32 T. Yasuda and H. Nakagawa, Phytochemistry 37, 133 (1994). 33 T. Narikawa, H. Shinoyama, and T. Fujii, Biosci. Biotechnol. Biochem. 64, 1317 (2000). 31
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enzymes and cells
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pNP -rutinoside.34 The glycosidase showed diglycosidase activity by hydrolyzing -vicianosides and -acuminosides with 2-phenylethanol as their aglycons to liberate the corresponding disaccharides and aglycons. The Km value of this enzyme for pNP -rutinoside is 1.69 mM. Furcatin Hydrolase A partially purified enzyme from Viburnum furcatum was confirmed to hydrolyze furcatin (p-allylphenyl -d-apiofuranosyl- -d-glucopyranoside) into acuminose and an aglycon.35 Quite recently the partial cDNA of the diglycosidase was isolated by the RT-PCR method using primers designed for the conserved regions of the glycosyl hydrolase family 1. A full-length cDNA clone encoding putative furcatin hydrolase was isolated by screening of a cDNA library constructed from the leaves of V. furcatum and 50 -race PCR (unpublished data). The amino acid sequence of furcatin hydrolase showed 64% sequence identity with that of the tea leaf -primeverosidase. This is the second example of cDNA cloning of a diglycoside-specific glycosidase. A b-Primeverosidase-like Diglycosidase A -diglycosidase was purified from the culture medium of Aspergillus fumigatus isolated as one of the strains that can grow in a medium containing eugenyl -primeveroside as a sole carbon sauce. The -diglycosidase (pI 6.0; 47 kDa in SDS–PAGE) hydrolyzes pNP -primeveroside and pNP -gentiobioside to yield the corresponding disaccharides with a relative hydrolysis rate of 100 and 41, respectively.36 Other Diglycosidases
Occurrences of a few other kinds of diglycosidases have been reported, although they have not yet been purified. Heteroglycosidase A -glycosidase (a homodimer of 120–140 kDa; optimum pH 5.5), which hydorolyzes an isoflavone -glycoside (biochanin A), has been purified from Cicer arietinum.37 It certainly hydrolyzes disaccharide glycosides to give 34
Z. Gu¨ nata, C. Blondeel, M. J. Vallier, J. P. Lepoutre, J. C. Sapis, and N. Watanabe, J. Agric. Food Chem. 46, 2748 (1998). 35 H. Imaseki and T. Yamamoto, Arch. Biochem. Biophys. 92, 467 (1961). 36 S. Yamamoto, M. Okada, T. Usui, and K. Sakata, Biosci. Biotechnol. Biochem. 66, 801 (2002). 37 W. Ho¨ sel, Hoppe-Seyler’s Z. Physiol. Chem. 357, 1673 (1976).
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active site mapping wih substrate analogs
459
disaccharides, but also can hydrolyze -glucopyranosides with the same aglycons at a similar rate. This may be ascribed to a contaminant -glucosidase. This enzyme shows fairly high affinity toward aromatic aglycon structures. Linustatinase One of the cyanogenic glycosides hydrolyases of flax seeds (Linum usitatissimum) was characterized as a diglycosidase present as a heterodimer that can hydrolyze -bis-glucosides with 1,6 (linustatin)- and 1,3-linkages into disaccharides and aglycons.38,39 Concluding Remarks
A -primeverosidase, with which we encountered in studying the molecular basis of tea aroma formation, is one of the disaccharide-specific glycosidases, ‘‘diglycosidases.’’ Knowledge of this group of glycosidases in limited. We hope this paper will lead to a new page of glycosidase study. Acknowledgment This work was partly supported by a grant-in-aid [(B)(2)13460049 for K. S.] from the Ministry of Education, Science, Sports, and Culture of Japan. 38 39
T. W. Fan and E. E. Conn, Arch. Biochem. Biophys. 243, 361 (1985). J. E. Poulton, in ‘‘ -Glucosidases: Biochemistry and Molecular Biology’’ (A. Esen, ed.), ACS Symposium Series 533, p. 170. American Chemical Society, Washington, DC, 1994.
[34] Use of Synthetic Oligosaccharide Substrate Analogs to Map the Active Sites of N-Acetylglucosaminyltransferases I and II By Harry Schachter, Folkert Reck, and Hans Paulsen Introduction
Enzymes are the work horses of metabolism and have been a favorite topic of investigation by biochemists since the term ‘‘enzyme’’ was coined by Ku¨ hne in 1878.1 Many enzymes have now been purified to homogeneity, the genes that encode them have been cloned and expressed, and their 1
W. Ku¨ hne, Unters. Physiol. Institut. Univ. Heidelberg 1, 291 (1878).
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enzymes and cells
is not bound significantly to the adsorbent 6, but is inhibited very strongly by the soluble ligand 8a with a Ki of as low as 8 nM.44 Several factors affect the adsorptive characteristics of the affinity gel such as the unfavorable steric interaction between the protein and the matrix.46 The adsorptive characteristics of the affinity gel 6 might be optimized by taking several factors into consideration, such as the structure of the aglycon moiety of the ligand and the nature of the counteranion of the amidinium group, as well as the physicochemical properties of the matrix and spacer. Finally, the affinity adsorbent with a glycosylamidine as a ligand can be used not only for the purification of a target enzyme, but also for elimination of an undesirable activity from a crude enzyme preparation. This mode of application also holds for the soluble -glycosylamidines as inhibitors to suppress an undesirable activity selectively in a crude enzyme sample. The glycosylamidines thus have potential as a versatile tool for glycosidase studies. Acknowledgment This work was supported in partly by a Grant-in-Aid for Scientific Research [(B)(2) 13480187 for J.H.] from Japan Society for the Promotion of Science.
46
P. Cuatrecasas and C. B. Anfinsen, Annu. Rev. Biochem. 40, 259 (1971).
[33]
Diglycoside-Specific Glycosidases
By Kanzo Sakata, Masaharu Mizutani, Seung-Jin MA, and Jun Hiratake Introduction
A -primeverosidase was reported for the first time as a glycosidase that hydrolyzes a -primeveroside (6-O- -d-xylopyranosyl- -d-glucopyranoside) to a disaccharide (primeverose) and an aglycon (anthraquinone) by Bridel et al. in 1925.1 Although it was suggested that this enzyme occurs widely in plants and microorganisms,2 no -primeverosidase had been purified until Sakata and colleagues isolated it from juvenile tea leaves as a key 1
W. Ho¨sel, ‘‘Glycosidation and Glycosidases,’’ Vol. 7, p. 725. Academic Press, New York, 1981. 2 V. Plouvier, Comp. R. Hebd. Seanaces Acad. Sci. Ser. D 286, 1071 (1980).
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glycosidase deeply involved in floral tea aroma formation in oolong tea and black tea.3,4 Gene cloning of the tea leaf -primeverosidase and its overexpression have now been accomplished.5,6 Floral tea aroma such as geraniol, linalool, and 2-phenylethanol of oolong tea and black tea were shown to be contained mainly as aroma precursors of diglycosides such as -primeverosides.7,8 A -primeverosidase responsible for the formation of aroma from the glycosidic aroma precursors was purified from the crude enzyme extract prepared from fresh juvenile tea leaves (Camellia sinensis).3,9,10 The purified enzyme shows high substrate specificity toward -primeverosides to yield aglycons and primeverose.11 Since then, many types of -primeverosides having various kinds of aglycons have been isolated from many varieties of plants not only as aroma precursors in flowers and fruits,11–13 but also as cyanogenic glycosides14–16 and as polar constituents.17–23 This fact strongly suggests that -primeverosidases are widely distributed among the plant kingdom. Research to confirm the wide occurrence of -primeverosidases and their physiological roles in plants is now in progress. 3
W. Guo, K. Yamauchi, N. Watanabe, T. Usui, S. Luo, and K. Sakata, Biosci. Biotech. Biochem. 59, 962 (1995). 4 K. Sakata, in ‘‘Caffeinated Beverages, Health Benefits, Physiological Effects, and Chemistry’’ (T. H. Parliament, C. Ho, and P. Schieberie, eds.), ACS Symposium Series 754, p. 327. American Chemical Society, Washington, DC, 2000. 5 M. Mizutani, H. Nakanishi, and K. Sakata. in ‘‘2001 International Conference on O-cha (Tea) Culture and Science,’’ Vol. Session II, p. 15. The Organizing Committee of the Conference, Shizuoka, Japan, 2001. 6 M. Mizutani, H. Nakanishi, J. Ema, S.-J. Ma, E. Noguchi, M. Fukuchi-Mizutani, K. Ochiai, Y. Tanaka, and K. Sakata, Plant Physiol. 130, 2164 (2002). 7 K. Sakata, N. Watanabe, and T. Usui, in ‘‘Food for Health in the Pacific Rim, Proceedings of the 3rd International Confrence of Food Science and Technology’’ (J. R. Whitaker et al. eds.), p. 93. Foods Nutrition Press, Inc., Trumbull, CT, 1999. 8 D. Wang, E. Kurasawa, T. Yoshimura, Y. Yamaguchi, K. Kubota, and A. Kobayashi, J. Agric. Food Chem. 49, 1900 (2001). 9 K. Ogawa, Y. Ijima, W. Guo, N. Watanabe, T. Usui, S. Dong, Q. Tong, and K. Sakata, J. Agric. Food Chem. 45, 877 (1997). 10 Y. Ijima, K. Ogawa, N. Watanabe, T. Usui, M. Ohnishi-Kameyama, T. Nagata, and K. Sakata, J. Agric. Food Chem. 46, 1712 (1998). 11 References on glycosidic aroma precursors are cited herein: S. J. Ma, M. Mizutani, J. Hiratake, K. Hayashi, K. Yagi, N. Watanabe, and K. Sakata, Biosci. Biotech. Biochem. 65, 2719 (2001). 12 M. Straubinger, H. Knapp, N. Watanabe, N. Oka, H. Washio, and P. Winterhalter, Natl, Prod. Lett. 13, 5 (1999). 13 H. Mayorga, C. Duque, H. Knapp, and P. Winterhalter, Phytochemistry 59, 439 (2002). 14 D. S. Seigler, Phytochemistry 14, 9 (1975). 15 D. Selmar, R. Lieberei, N. Junqueira, and B. Biehl, Phytochemistry 30, 2135 (1991). 16 J. Rockenbach, A. Nahrstedt, and V. Wray, Phytochemistry 31, 567 (1992).
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enzymes and cells
[33]
-Primeverosidase (EC 3.2.1.149)
Enzyme Assays -Primeverosidase activity is examined by assay using p-nitrophenyl (pNP) -primeveroside or 2-phenylethyl -primeveroside as a substrate. The activity is determined by measuring the liberation of p-nitrophenol or 2-phenylethanol from each glycoside. pNP -Primeveroside is synthesized enzymatically by transglycosylation between xylobiose and pNP -d-glucopyranoside with a -xylosidase from Aspergillus pulverulentus.24 2-Phenylethyl -primeveroside is synthesized chemically by glycosidation of 2-phenylethanol with 2,3,4,20 ,30 ,40 -hexa-O-benzoylprimeverosyl trichloroacetimidate.11 Stock solutions of each substrate (50 mM) are prepared by dissolving the glycosides in deionized water. The enzyme is dissolved in 20 mM citrate buffer (pH 6.0) containing 0.1% bovine serum albumin (BSA). One unit of the -primeverosidase activity is defined as the amount of enzyme liberating 1 mol of p-nitrophenol from pNP -primeveroside per minute in 20 mM citrate buffer (pH 6.0) at 37 . Each reaction mixture (150 l) contains 10 mM substrate, 20 mM citrate buffer (pH 6.0), 0.1% BSA, and 60 l of the enzyme solution. A mix ture without the enzyme is preincubated at 37 , and the reaction is initiated by adding the enzyme. An aliquot (20 l) of each enzyme reaction mixture is withdrawn after various time intervals during the incubation for analyses as follows. Assay with p-Nitrophenyl b-Primeveroside. An aliquot (20 l) of a reaction mixture is added to 80 l of 0.2 M Na2CO3 solution (pH 12), and the liberated p-nitrophenol is determined by measuring the absorbance at 405 nm in a spectrophotometer (UV-3101PC UV-VIS-NIR scanning spectrophotometer, Shimadzu, Japan).
17
L. H. W. van der Plas, M. J. M. Hargendoorn, and D. C. Jamar, J. Plant Physiol. 152, 235 (1998). 18 G. C. H. Derksen, T. A. van Beek, A. Groot, and A. Capelle, J. Chromatogr. A 816, 277 (1998). 19 Y. Lu, P.-J. Xu, Z.-N. Chen, and G.-M. Liu, Phytochemistry 49, 1135 (1998). 20 S. D. Petrovic, M. S. Gorunovic, V. Wray, and I. Merfort, Phytochemistry 50, 293 (1999). 21 A. Tamaki, H. Otsuka, and T. Ide, J. Nat. Prod. 62, 1074 (1999). 22 S. Yamamura, K. Osawa, K. Ohtani, R. Kasai, and K. Yamasaki, Phytochemistry 48, 131 (1998). 23 J. W. Jaroszewski, A. B. Rasmussen, H. B. Rasmussen, C. E. Olsen, and L. B. Jfrgensen, Phytochemistry 42, 649 (1996). 24 T. Murata, M. Shimada, N. Watanabe, K. Sakata, and T. Usui, J. Appl. Glycosci. 46, 431 (1999).
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Assay with 2-Phenylethyl b-Primeveroside. For the reaction with 2phenylethyl glycosides, an aliquot (20 l) of a reaction mixture is added to 2 l of 1 N NaOH, and then 8 l of an MeCN solution containing 3 g of benzyl alcohol is added as an internal standard. A sample (20 l) from each mixture (30 l) is injected to a HPLC. Analysis of liberated 2-phenylethanol is performed under the following HPLC conditions: column, YMC-pack ODS-AQ (4.6 250 mm) (YMC Co., Japan); detection, 210 nm with a Waters 996 photodiode array detector; column temperature, 40 ; mobile phase, 33% MeCN; and flow rate, 1.0 ml/min. Benzyl alcohol and 2-phenylethanol are detected at tR 6.7 and 8.7 min, respectively. The hydrolysis rate is determined from the linear portion (within 10% hydrolysis of the substrate) of each progress curve. Standard curves can be prepared by measuring the value of various concentrations of p-nitrophenol or 2-phenylethanol. Protein concentration is determined by the Bradford method25 using the Coomassie protein assay reagent (Pierce, Rockford, IL) with BSA as a standard. Purification of A b-Primeverosidase from a Tea Plant (Camellia sinensis var. sinensis cv. Yabukita) Fresh tea leaves are finely chopped, crushed in dry ice-acetone by a homogenizer, and filtered in vacuo. The residue is washed with chilled acet one (20 ) until the filtrate becomes nearly colorless. The residue is spread on filter paper and is then placed in vacuo to remove acetone. The residual acetone powder is stored at 20 before use. The acetone powder (100 g, equivalent to 600 g of the fresh leaves) is suspended in 0.1 M citrate buffer (pH 6.0, 2 liters), stirred for 4 h at 4 , and centrifuged at 14,000 g for 20 min. To the supernatant, an equal volume of chilled acetone (20 ) is added gradually with stirring, and the mixture is left overnight at 4 . The precipitate obtained by centrifugation at 14,000 g for 20 min is dissolved in 0.1 M citrate buffer (pH 6.0, 500 ml). Ammonium sulfate is added up to 40% saturation, and the mixture is centrifuged at 14,000 g for 20 min. The supernatant is subjected to a butyl-Toyopearl 650M column (Tosoh Corp., Tokyo, Japan) equilibrated with 20 mM citrate buffer (pH 6.0) containing 40% ammonium sulfate. The enzyme fractions are eluted by a linear gradient of ammonium sulfate from 40 to 0% saturation in 1 liter each of 20 mM citrate buffer (pH 6.0) with a flow rate of 5 ml/min. -Primeverosidase fractions are combined and concentrated by ultrafiltration (Amicon PM-10, Grace Japan Co., Ltd., Tokyo, Japan) and are dialyzed against 20 mM citrate buffer (pH 6.0) containing 50 mM NaCl. 25
M. Bradford, Anal. Biochem. 72, 248 (1976).
448
[33]
enzymes and cells TABLE I Purification of -Primeverosidase from C.
Purification step
SINENSIS
cv. Yabukita
Total Total Yield Specific activity Purification protein (mg) activity (unit) (%) (unit/mg protein) fold (-fold)
Buffer extract Acetone ppt 40% (NH4)2SO4 sup Butyl-Toyopearl CM-Toyopearl Mono S
1000 220 110 7.7 0.54 0.30
40 38 32 3.8 0.70 0.51
100 95 80 9.3 1.8 1.3
0.04 0.17 0.29 0.49 1.3 1.7
1.0 4.3 7.3 12 33 43
The fraction is applied to a CM Toyopearl column (22 130 mm, Tosoh), and -primeveroidase fractions are eluted by a linear gradient of NaCl from 50 to 150 mM in 500 ml citrate buffer (pH 6.0). The -primeverosidase fraction is placed on a column (5 50 mm) of Mono S HR (Amersham Pharmacia Biotech Co. Ltd., Tokyo, Japan) equilibrated with 20 mM citrate buffer (pH 6.0). The -primeverosidase is eluted with a linear gradient of NaCl from 0.1 to 0.25 M at a flow rate of 1 ml/min. Because -primeverosidase is nearly coeluted with -glucosidases in each of the column chromatographis, -glucosidases are thoroughly eliminated from the -primeverosidase fractions by tracing both the activities using pNP -d-glucopyranoside and -primeveroside as substrates during the whole purification process. The purification procedures are summarized in Table I. -Primeverosidases from fresh leaves of two other cultivars for black tea (C. sinensis var. assamica) and oolong tea (C. sinensis var. sinensis cv. Shuixian) are also purified in the same way.9,10 Enzymatic Characteristics The purified -primeverosidase from cv. Yabukita gives a single band with an apparent molecular mass of 61 kDa on SDS–PAGE. The molecular weight has been determined to be 60,480 by TOFMS analysis.10 The enzymatic properties of -primeverosidases from three different cultivars for green tea, oolong tea, and black tea are summarized in Table II.9,10 Substrate Specificity In order to clarify the substrate specificity of the -primeverosidase with respect to the glycon moiety, nine kinds of diglycosides (1–9) and a glucoside (10) of 2-phenylethanol were synthesized (Fig. 1).11 These glycosides are designed by considering the structures of the natural aroma precursors isolated from plants along with the ease of synthesis.
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TABLE II Summary of Enzymatic Properties of -Primeverosidase -Primeverosidase Black tea MW pI Optimum temperature ( )a Stable temperatureb Optimum pHc pH stabilityd Specific activity (unit/mg)e
60,310 9.5 45 40 4 4–5 0.99
Oolong tea 60,480 9.5 45 40 4 3–5 0.98
Green tea 60,170 9.4 45 45 4 4–5 0.90
a
The optimum temperature of the enzyme was measured with pNP -primeveroside in 20 mM citrate buffer (pH 6.0) at various temperatures (30–70 ). b The thermal stability of the enzyme was estimated from the residual activities after incubation in 20 mM citrate buffer (pH 6.0) at various temperatures (30–60 ). c The optimum pH of the enzyme was examined in buffers with various pH values (2.0–9.0) at 37 for 1 h. d The pH stability of the enzyme was estimated from residual activities after incubation in the buffers with various pH values (2.0–9.0) at 37 for 1 h. The buffers used were 20 mM Clark and Lubs buffer (pH 2.0), 20 mM citrate buffer (pH 3.0–6.0), and 20 mM Tris–HCl buffer (pH 7.0–9.0). e -Primeverosidase activity was measured with pNP -primeveroside in 20 mM citrate buffer (pH 6.0) at 37 .
-Primeverosides, -vicianosides, -acuminosides, -gentiobiosides, and 6-O- -l-arabinofuranosyl- -d-glucopyranoside have been found in tea leaves as well as in several kinds of flowers and fruits.11 The aglycon moiety is fixed as 2-phenylethyl group, as 2-phenylethanol is one of the most common aroma compounds and its diglycosides ( -primeveroside, accuminoside, and 6-O- -l-arabinofuranosyl- -d-glucopyranoside) have been isolated from tea leaves as well as from several kinds of flowers and fruits as aroma precursors.11 The hydrolysis rate for the -primeverosidase purified from tea leaves is determined by using 10 mM of each synthetic 2-phenylethyl -glycoside (1–10) and pNP -glycoside (11–15) (Table III). The purified enzyme shows highly selective activity toward the diglycosidie substrates, -primeverosides (1 and 11). Interestingly, all the diglycosides (1–5 and 11) hydrolyzed by the enzyme are naturally occurring and have a (1–6) or an (1–6) glycosidic linkage in the glycon moiety. None of the unnatural diglycosides (6–9) with a (1–4)- or an (1–4)-glycosidic linkage are hydrolyzed. The 2-phenylethyl group seems to be preferred more as an aglycon for the -primeverosidase than pNP, as the hydrolysis rate of 1 was about twice
450 enzymes and cells
Fig. 1. 2-Phenylethyl glycosides designed to study the substrate specificity of the -primeverosidase from tea leaves (cv. Yabukita).
[33]
[33]
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diglycoside-specific glycosidases
TABLE III Substrate Specificity of -Primeverosidase Purified from Juvenile Tea Leaves
Substrate 2-Phenylethyl -primeveroside (1) 2-Phenylethyl -vicianoside (2) 2-Phenylethyl -acuminoside (3) 2-Phenylethyl -gentiobioside (4) 2-Phenylethyl 6-O- -l-arabinofuranosyl -d-glucopyranoside (5) 2-Phenylethyl -lactoside (6) 2-Phenylethyl -cellobioside (7) 2-Phenylethyl -maltoside (8) 2-Phenylethyl -melibioside (9) 2-Phenylethyl -d-glucopyranoside (10) pNP -Primeveroside (11) pNP -d-Glucopyranoside (12) pNP -d-Galactopyranoside (13) pNP -d-Xylopyranoside (14) pNP -d-Arabinopyranoside (15) a
Hydrolysis activitya (mol/min/mg protein)
Relative activity (%)
39.1 1.16 0.301 0.098
100 2.97 0.60 0.34
0.027 NDb ND ND ND ND 21.3 0.091 ND ND ND
0.05 ND ND ND ND ND 54.4 0.23 ND ND ND
Hydrolysis activity is expressed by initial velocity (mol/ml/min) per protein concentration (mg/ml). The activity was determined by the liberation of 2-phenyethanol or p-nitrophenol from each glycoside. The liberated 2-phenyethanol was analyzed by HPLC, and p-nitrophenol was determined at 405 nm. Each reaction mixture contained 10 mM of each substrate, tea leaf -primeverosidase (0.22 unit/ml), and 20 mM citrate buffer (pH 6). The mixtures were incubated at 37 . [S.-J. Ma et al., Biosci. Biochem. Biotechnol. 65, 2719 (2001)].
as high as that of 11. This is due in part to the fact that pNP glycoside is not a natural substrate in tea leaves. To define substrate specificity with respect to the glycon moiety more quantitatively, the kinetic parameters (Km and kcat) for 1 and 2 are determined by the reaction of various concentrations of 1 (0.5–4.0 mM) or 2 (5–25 mM) with the enzyme under the respective assay condition as described earlier. The Michaelis constant of 1 (Km ¼ 2.00 mM) is 7.5-fold higher than that of 2 (Km ¼ 14.9 mM), and the specificity constant for 1 (kcat/Km ¼ 21.9 s1 mM) is 56-fold higher than that for 2 (kcat/Km ¼ 0.391 s1mM). These results indicate that the substrate specificity of the tea leaf -primeverosidase is highly specific for -primeverosides, although this enzyme hydrolyzes 2-phenylethyl -vicianoside and -acuminoside as well as -primeveroside.
452
enzymes and cells
[33]
Cloning of Tea Leaf b-Primeverosidase The purified -primeverosidase described previously is applied to reversed-phase HPLC using a CAPCELL PAK C18 SG300 column (4.6 150 mm, Shiseido, Tokyo, Japan), and a single peak fraction is collected and directly analyzed to determine the N-terminal amino acid sequence. The fraction is further digested with a lysyl endopeptidase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) or trypsin (Wako), and the resulting peptide fragments are separated by reversed-phase HPLC using a CAPCELL PAK C18 SG120 column (4.6 250 mm, Shiseido). Amino acid sequences are analyzed by automated Edman degradation using an ABI 492 protein sequencer. The N-terminal and internal amino acid sequences thus determined are shown with an underline in Fig. 2. With the aid of the amino acid sequences determined from the purified primeverosidase, the cDNA encoding tea -primeveosidase is isolated from cv. Yabukita.5 Total RNA is isolated from fresh leaves of cv. Yabukita, and the mRNA is purified with an oligo(dT) cellulose column type 7 (Faversham Biosciences, Piscataway, New Jersey). The cDNA library is constructed from the mRNA with a double-stranded Uni-ZAP XR vector (Stratagene, San Diego, CA) following the manufacturer’s instructions. The oligonucleotide primer, BGLU1:50 -GGIGA(T/C)GTIGCIGA(T/C)GA(T/C)TT (T/C)TA (T/C)CA-30 is generated from the internal amino acid sequence, GDVADDFYH, determined from the purified -primeverosidase. By using a set of BGLU1 and an oligo(dT)16 primer, polymerase chain reaction (PCR) is performed through 30 cycles of 60 s at 94 , 90 s at 45 , and 60 s at 72 with the mass-excised plasmid library as templates. PCR products are separated by agarose gel (2%) electrophoresis, and the major band is cloned into the pGEM-T vector (Promega, Madison, WI). The cDNA fragment is labeled with [32P]dCTP by a random priming method, and about 500,000 plaques from the tea cDNA library are screened with the labeled fragment as a probe. The isolated cDNA consists of a 1524-bp open reading frame encoding a polypeptide of 507 amino acid residues, 8-bp 50 -untranslated region, a 179-bp 30 -noncoding region, and a poly(A) tail (Fig. 2). The deduced amino acid sequence of the -primeverosidase has high homology with -glucosidases from various plants such as amygdalin hydrolase (58%) from black cherry,26 -glucosidase for indoxyl -d-glucoside (55%) from the indigo,27 and linamarase from cassava (50%).28 Many 26
L. Zheng and J. E. Poulton, Plant Physiol. 109, 31 (1995). Y. Minami, Y. Shigeta, U. Tokumoto, Y. Tanaka, K. Yonekura-Sakakibara, H. Oh-oka, and Matsubara, Plant Sci. 142, 219 (1999). 28 M. A. Hughes, K. Brown, A. Pancoro, S. Murray, E. Oxtoby, and J. Hughes, Arch. Biochem. Biophys. 295, 273 (1992). 27
[33] diglycoside-specific glycosidases
453
Fig. 2. Nucleotide and predicted amino acid sequences of -primeverosidase cDNA from cv. Yabukita. Peptide sequences determined from the purified protein are underlined. The arrow indicates the position of the N-terminal amino acid sequence of the purified protein and also the possible cleavage site predicted by Psort analysis. Possible N-glycosylation sites are boxed. Catalytic residues in conserved sequence motifs in family 1 glycosyl hydrolases are double underlined.
454
enzymes and cells
[33]
glycoside hydrolases from bacteria to mammals have been classified into 83 families according to amino acid sequence similarity (http:// afmb.cnrs-mrs.fr/pedro/CAZY/ghf.html), and tea -primeverosidase is classified in a family 1 glycosyl hydrolase because -primeverosidase shows high similarities to the family 1 glycoside hydrolases from various plants. Expression in Escherichia coli The cDNA encoding the mature -primeverosidase is amplified by PCR with a set of primers (50 -TCTAGAGCTCAAATCTCCTCCTTCAAC and 50 -GTCGACCTACTTGAGGAGGAATTTCTT) and is ligated into the pMALc2 vector (New England Biolabs, Inc., Beverly, MA) to generate pMALc2-Pri. Escherichia coli (JM109) is transformed with pMALc2-Pri, and the expression of -primeverosidase fused with a maltose-binding protein is induced by the addition of 0.1 mM isopropyl -d-thiogalactoside (IPTG). The cells are further grown at 20 , 150 rpm, for 24 h. The cells are collected by centrifugation at 5000 g for 10 min and are resuspended in 50 mM citrate buffer, pH 6.0, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The suspension is sonicated 15 times for 30 s at 30-s intervals and is centrifuged at 28,000 g for 30 min. The cell extract is applied to the amylose resin column, and the fusion protein is purified according to the manufacturer’s instructions. When the recombinant fusion protein between a maltose-binding pro tein and the mature primeverosidase is expressed at 37 , all the recombinant proteins are found as inclusion bodies, and -primeverosidase activity is not detected in the cell lysates. However, when the transformed cells are grown at 20 in the presence of 0.1 mM IPTG, a part of the recombinant proteins is detected in the soluble fractions. The recombinant fusion protein purified by amylose resin affinity chromatography is able to hydrolyze either pNP -primeveroside or 2-phenylethyl -primeveroside. Mode of Hydrolysis by b-Primeverosidase pNP -Primeveroside is incubated with either the -primeverosidase purified from tea leaves or the recombinant protein produced in E. coli, and the hydrolysates are analyzed by TLC. TLC is carried out on silica gel 60 F254 plates using a solvent system of butanol–pyridine–water–acetic acid (6:4:3:1, v/v). Glycosides and sugars are detected by heating the plate at 120 after spraying with 0.2% naphthoresorcinol in H2SO4–EtOH (1:19, v/v). The spot corresponding to primeverose (Rf 0.39) is clearly observed in each of the hydrolysates, but no spots for glucose (Rf 0.48) and xylose (Rf 0.60) are detected (Fig. 3). This confirms that -primeverosidase is a unique diglycosidase specifically hydrolyzing the -glycosidic bond between an
[33]
diglycoside-specific glycosidases
455
Fig. 3. Thin-layer chromatogram of hydrolysis of pNP-primeveroside. pNP-Primeverosides at a final concentration of 5 mM were incubated in citrate buffer (pH 6.0) with either the tea -primeverosidase or the recombinant -primeverosidase expressed in E. coli. Products of the reaction were analyzed by TLC. Lanes 1, 2, 3, 4, and 7 were standard. Lane 1, pNPprimeveroside; lane 2, glucose; lane 3, xylose; lane 4, primeverose; lane 5, hydrolysis product by the tea -primeverosidase; lane 6, hydrolysis product by the recombinant enzyme; and lane 7, standard mixture (glucose, xylose, primeverose).
aglycon and a primeverose without cleaving the interglycosidic bond between the sugars. The stereochemistry for the enzymatic hydrolysis by -primeverosidase is analyzed by 1H-NMR (Fig. 4). The method is essentially the same as described by Wang et al.29 2-Phenylethyl -primeveroside (10 mg) is dissolved in 0.7 ml of D2O. -Primeverosidase (0.2 unit) purified from tea leaves is lypophilized and redissolved in 40 l of D2O. The enzyme (20 l) is added to an NMR tube containing the substrate, and the tube is incubated at 37 . The 1H-NMR measurement at the time intervals is per formed at 25 because the axial proton of -anomer of the released primeverose is overlapped with the HDO signal at 37 . NMR spectra are recorded on a JEOL JNM-AL 400 spectrometer. 1H-NMR spectra of the reaction mixture containing 2-phenylethyl -primeveroside and -primeverosidase reveal that the -anomer (Ha, 4.43, J ¼ 8.1 Hz) of primeverose is formed first. The -anomer (He, 5.04, J ¼ 3.8 Hz) of primeverose appears later as a 29
A. W. Wang, S. He, J. H. Grubb, W. S. Sly, and S. G. Withers, J. Biol. Chem. 273, 34057 (1998).
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Fig. 4. Time course of hydrolysis of 2-phenylethyl primeveroside catalyzed by the tea -primeverosidase, followed by 1H-NMR. (A) Postulated retaining hydrolysis of 2-phenylethyl primeveroside by the tea enzyme. (B) Spectra recorded 0, 5, 10, 20, 40, 90, and 180 min after addition of the enzyme. Ha and He indicate the H-1 resonances of - and -primeverose, respectively.
[33]
diglycoside-specific glycosidases
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consequence of mutarotation. Thus tea -primeverosidase is a retaining glycosidase as has been observed for other family 1 -glucosidases (Fig. 4). Diglycosidases from Other Plants and Microrganisms
A -primeverosidase has been found to be a real diglycoside-specific glycosidase, e.g., diglycosidase, which shows very high substrate specificity toward -primeverosides. In the early 1920s a few kinds of diglycosidases, which hydrolyzes diglycosides into aglycons and disaccharides, such as 6-O- -l-rhamnopyranosyl-d-glucose (rutinose), 6-O- -l-arabinopyranosyl-d-glucose (vicianose), and primeverose, were reported. However, most reports dealt with the crude enzyme, and only four kinds of diglycosidases have been purified and discussed in terms of their enzymic characteristics as shown in this section. Vicianin Hydrolase The occurrence of cyanogenic diglycosidases in Vicia angustifolla1 and Geumurbanaum30 has been reported. A vicianin hydrolase, which hydrolyzes vicianin to liberate vicianose and (R)-mandelonitrile, was purified from squirrel’s foot fern (Davallia tricomanoides).31 The enzyme was shown to have molecular weight of 340,000, a Km value of 4.9 mM for vicianin, and an optimum pH 5.5. SDS–PAGE analysis yielded three polypeptide bands at 56, 49, and 32.5 kDa. Rutinosidase (Rutinase) Occurrences of this enzyme in many kinds of plants have been reported for a long time. Two kinds of rutin hydrolases have been purified from the seeds of Fagopyrum tartaricum.32 They are both monomeric proteins of 68 kDa and show an optimum pH at 5.0. Km values for rutin are 130 and 120 mM, respectively. Quite recently, a rutinosidase has been purified from Penicillium rugulosum, which was cultured in a medium containing 2% rutin as a sole carbon sauce.33 This enzyme was shown to be a homotetramer with a molecular weight of 245,000 and an optimum pH at 2.2. An Endoglycosidase from Grape A diglycosidase (molecular weight 58,000; optimum pH, 4–5) was purified from Muscatt grape peel guided by rutinosidase activity measured with 30
M. Psenak, A. Jindra, P. Kovacs, and Dulovocova, Planta Med. 19, 154 (1970). P. A. Lizotte and J. E. Poulton, Plant Physiol. 86, 322 (1988). 32 T. Yasuda and H. Nakagawa, Phytochemistry 37, 133 (1994). 33 T. Narikawa, H. Shinoyama, and T. Fujii, Biosci. Biotechnol. Biochem. 64, 1317 (2000). 31
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pNP -rutinoside.34 The glycosidase showed diglycosidase activity by hydrolyzing -vicianosides and -acuminosides with 2-phenylethanol as their aglycons to liberate the corresponding disaccharides and aglycons. The Km value of this enzyme for pNP -rutinoside is 1.69 mM. Furcatin Hydrolase A partially purified enzyme from Viburnum furcatum was confirmed to hydrolyze furcatin (p-allylphenyl -d-apiofuranosyl- -d-glucopyranoside) into acuminose and an aglycon.35 Quite recently the partial cDNA of the diglycosidase was isolated by the RT-PCR method using primers designed for the conserved regions of the glycosyl hydrolase family 1. A full-length cDNA clone encoding putative furcatin hydrolase was isolated by screening of a cDNA library constructed from the leaves of V. furcatum and 50 -race PCR (unpublished data). The amino acid sequence of furcatin hydrolase showed 64% sequence identity with that of the tea leaf -primeverosidase. This is the second example of cDNA cloning of a diglycoside-specific glycosidase. A b-Primeverosidase-like Diglycosidase A -diglycosidase was purified from the culture medium of Aspergillus fumigatus isolated as one of the strains that can grow in a medium containing eugenyl -primeveroside as a sole carbon sauce. The -diglycosidase (pI 6.0; 47 kDa in SDS–PAGE) hydrolyzes pNP -primeveroside and pNP -gentiobioside to yield the corresponding disaccharides with a relative hydrolysis rate of 100 and 41, respectively.36 Other Diglycosidases
Occurrences of a few other kinds of diglycosidases have been reported, although they have not yet been purified. Heteroglycosidase A -glycosidase (a homodimer of 120–140 kDa; optimum pH 5.5), which hydorolyzes an isoflavone -glycoside (biochanin A), has been purified from Cicer arietinum.37 It certainly hydrolyzes disaccharide glycosides to give 34
Z. Gu¨ nata, C. Blondeel, M. J. Vallier, J. P. Lepoutre, J. C. Sapis, and N. Watanabe, J. Agric. Food Chem. 46, 2748 (1998). 35 H. Imaseki and T. Yamamoto, Arch. Biochem. Biophys. 92, 467 (1961). 36 S. Yamamoto, M. Okada, T. Usui, and K. Sakata, Biosci. Biotechnol. Biochem. 66, 801 (2002). 37 W. Ho¨ sel, Hoppe-Seyler’s Z. Physiol. Chem. 357, 1673 (1976).
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active site mapping wih substrate analogs
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disaccharides, but also can hydrolyze -glucopyranosides with the same aglycons at a similar rate. This may be ascribed to a contaminant -glucosidase. This enzyme shows fairly high affinity toward aromatic aglycon structures. Linustatinase One of the cyanogenic glycosides hydrolyases of flax seeds (Linum usitatissimum) was characterized as a diglycosidase present as a heterodimer that can hydrolyze -bis-glucosides with 1,6 (linustatin)- and 1,3-linkages into disaccharides and aglycons.38,39 Concluding Remarks
A -primeverosidase, with which we encountered in studying the molecular basis of tea aroma formation, is one of the disaccharide-specific glycosidases, ‘‘diglycosidases.’’ Knowledge of this group of glycosidases in limited. We hope this paper will lead to a new page of glycosidase study. Acknowledgment This work was partly supported by a grant-in-aid [(B)(2)13460049 for K. S.] from the Ministry of Education, Science, Sports, and Culture of Japan. 38 39
T. W. Fan and E. E. Conn, Arch. Biochem. Biophys. 243, 361 (1985). J. E. Poulton, in ‘‘ -Glucosidases: Biochemistry and Molecular Biology’’ (A. Esen, ed.), ACS Symposium Series 533, p. 170. American Chemical Society, Washington, DC, 1994.
[34] Use of Synthetic Oligosaccharide Substrate Analogs to Map the Active Sites of N-Acetylglucosaminyltransferases I and II By Harry Schachter, Folkert Reck, and Hans Paulsen Introduction
Enzymes are the work horses of metabolism and have been a favorite topic of investigation by biochemists since the term ‘‘enzyme’’ was coined by Ku¨ hne in 1878.1 Many enzymes have now been purified to homogeneity, the genes that encode them have been cloned and expressed, and their 1
W. Ku¨ hne, Unters. Physiol. Institut. Univ. Heidelberg 1, 291 (1878).
METHODS IN ENZYMOLOGY, VOL. 363
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