Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Plant Cell Physiol. 48(7): 938–947 (2007) doi:10.1093/pcp/pcm065, available online at www.pcp.oxfordjournals.org ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Vicianin Hydrolase is a Novel Cyanogenic b-Glycosidase Specific to b-Vicianoside (6-O-a-L-Arabinopyranosyl-b-D-Glucopyranoside) in Seeds of Vicia angustifolia Young Ock Ahn 1, Hiromichi Saino, Masaharu Mizutani *, Bun-ichi Shimizu and Kanzo Sakata Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011 Japan Introduction The cyanogenic disaccharide glycoside, vicianin [mandelonitrile b-vicianoside (6-O-a-L-arabinopyranosyl-b-D-glucopyranoside)], is accumulated in seeds of Vicia angustifolia var. segetalis. Vicianin hydrolase (VH) catalyzes the hydrolysis of vicianin into mandelonitrile and a disaccharide vicianose. VH was purified from the seeds using DEAE-, CM- and Con A-Sepharose chromatography, and the molecular mass of the purified VH was estimated to be 56 kDa on SDS–PAGE. The N-terminal amino acid sequence of the purified VH was determined, and a cDNA encoding VH was obtained. The deduced VH protein consists of a 509 amino acid polypeptide containing a putative secretion signal peptide. It shares about 50% identity with various kinds of plant b-glycosidases including tea leaf b-primeverosidase and furcatin hydrolase, and is classified in family 1 of the glycosyl hydrolases. The VH transcript was detected abundantly in seeds and moderately in flowers, but only slightly in leaves, stems and roots, indicating that the organ distribution of VH expression is similar to that of the substrate vicianin. The recombinant VH was produced in insect cells with a baculovirus system, and was compared with the native VH in terms of substrate specificity. Both enzymes hydrolyzed vicianin to release vicianose, demonstrating that VH is a disaccharide-specific b-glycosidase. VH also hydrolyzed the mandelonitrile b-glucoside prunasin to some extent but did not hydrolyze the gentiobioside amygdalin, both of which contain the same aglycone as vicianin. Thus, VH is a unique cyanogenic glycosidase showing high glycone specificity for the disaccharide vicianoside. Cyanogenic glucosides are composed of an a-hydroxynitrile-type aglycone and a glucose moiety (McFarlane et al. 1975). Cyanogenic glucosides are biosynthesized from several kinds of L-amino acids. The amino acids are N-hydroxylated, converted to aldoximes, and then into nitriles. The nitriles are further hydroxylated to a-hydroxynitriles, and then glucosylated to form cyanogenic glucosides (Vetter 2000). The generation of HCN from cyanogenic glucosides is a two-step process involving degradation by a specific b-glucosidase and release of HCN by a-hydroxynitrilase (Nahrstedt 1988, Poulton 1988). Thus, cyanogenesis is a defense process by which plants produce HCN against herbivores. The catabolism of cyanogenic glucosides by b-glucosidases has been investigated in detail. In sorghum (Sorghum bicolor L. Moench), the cyanogenic glucoside dhurrin is hydrolyzed by a specific b-glucosidase, dhurrinase (Hosel et al. 1987), and in cassava (Manihot esculenta Crantz), the cyanogenic glucoside linamarin accumulated in the tubers is hydrolyzed by a specific b-glucosidase, linamarase (Koch et al. 1992). Cassava linamarase is a 70 kDa glycoprotein having high-mannose-type N-asparagine-linked oligosaccharides, and its cDNA has been isolated (Hughes et al. 1992). The cDNA of sorghum dhurrinase encodes a mature protein of 514 amino acid residues with a signal peptide of 51 amino acid residues (Cicek and Esen 1998). On the basis of their amino acid sequences, dhurrinase and linamarase show significant sequence similarities to various plant b-glucosidases, and they belong to family 1 of glycosyl hydrolases. In addition, several plant species contain cyanogenic disaccharide glycosides, in which the sugar moiety is replaced by gentiobiose, primeverose or vicianose (Vetter 2000). The hydrolysis of cyanogenic disaccharide glycosides occurs by two distinct mechanisms, namely a stepwise and sequential mechanism by two b-monoglucosidases, and, alternatively, a simultaneous mechanism by a single Keywords: Cyanogenesis — Cyanogenic glycoside — Disaccharide glycoside — Disaccharide glycoside-specific glycosidase — family 1 glycosyl hydrolase — Vicianin. Abbreviations: Dhr, dhurrinase; FH, furcatin hydrolase; pNP, para-nitrophenyl; TLC, thin-layer chromatography; VH, vicianin hydrolase. Sequence data from this article have been deposited in the EMBL/GenBank data libraries under accession number DQ371927. 1 Present address: Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. *Corresponding author: E-mail, [email protected]; Fax, þ81-774-38-3229. 938 Vicianin hydrolase from Vicia angustifolia Table 1 939 Summary of purification of VH from V. angustifolia Protein (mg) Total activity (U) Specific activity (U mg1) Yield (%) Purification factor (fold) Crude enzyme DEAE-Sepharose CM-Sepharose Con A-Sepharose 198.0 124.7 13.6 5.1 209.8 204.5 84.4 55.3 disaccharide-specific glycosidase. Prunus species contain the cyanogenic gentiobioside amygdalin [(R)-mandelonitrile b-gentiobioside (6-O-b-D-glucopyranosyl-b-D-glucopyranoside)] especially in their mature seeds, and the catabolism of amygdalin in black cherry (Prunus serotina) seeds occurs by the former stepwise mechanism. First, amygdalin hydrolase hydrolyzes the inter-glycosidic bond of the gentiobioside, in which two glucose units are linked by a b-1,6 glycosidic bond, to release glucose and a mandelonitrile b-D-glucopyranoside (prunasin). Then prunasin hydrolase hydrolyzes the glycosidic bond between glucose and mandelonitrile. Both enzymes share about 70% amino acid identity with each other (Kuroki and Poulton 1986, Yemm and Poulton 1986, Kuroki and Poulton 1987, Zheng and Poulton 1995). Vicianin [(R)-mandelonitrile b-vicianoside (6-O-a-Larabinopyranosyl-b-D-glucopyranoside)] found in seeds of several Vicia species is another cyanogenic disaccharide glycoside composed of a disaccharide vicianose and mandelonitrile (Kasai et al. 1981, Lizotte and Poulton 1988). In contrast to amygdalin catabolism, the hydrolysis of vicianin seems to occur by the latter simultaneous mechanism, by which a single disaccharide-specific glycosidase hydrolyzes to form mandelonitrile and vicianose. It has been reported that the seeds of Vicia angustifolia var. segetalis contain vicianin as well as vicianin hydrolase (VH) (Kasai et al. 1981). VH activity was also reported in ferns (Davallia trichomanoides Blume) (Lizotte and Poulton 1988). However, molecular studies of the amino acid and/or DNA sequence of VH have never been published. In this report, we describe the purification of VH from the seeds of V. angustifolia, cloning of VH cDNA, heterologous expression of the recombinant VH protein, and its biochemical characterization. Results Purification of VH in plants The crude extract of V. angustifolia seeds was applied to a CM-Sepharose column, and VH was eluted with a 100–140 mM sodium chloride gradient. Fractions containing VH activity were pooled and subjected to Con A-Sepharose chromatography. Active fractions were bound to the column and eluted with 0.4 M methyl 1.05 1.63 6.21 9.86 100 97.5 40.2 26.4 M A 205 1 2 1 1.55 5.91 9.39 3 4 1 B 116 97 84 66 55 45 36 29 24 C D Fig. 1 SDS–PAGE (A and B) and Western blotting (C and D) profiles of various VH preparations. (A) Native VH preparations from V. angustifolia seeds. Lane M, molecular markers (Sigma); lane 1, crude extract; lane 2, DEAE-Sepharose flow-through; lane 3, CM-Sepharose; lane 4, Con A-Sepharose. (B) Recombinant VH preparation from expression in insect cells. Lane 1, purified recombinant VH. (C) and (D) Western blots of gels identical to those shown in (A) and (B), respectively, but immunostained with anti-primeverosidase antibody. a-D-mannopyranoside, suggesting that VH is a glycoprotein. Table 1 summarizes the purification factor and yield values estimated for each purification step. Overall, about 9-fold purification was achieved with 26% recovery. Fig. 1A shows an SDS–PAGE of the same amount of protein from each purification step and Western blot analysis (Fig. 1C), which confirmed that the native VH was immunoreactive with the polyclonal antibody against b-primeverosidase of Camellia sinensis var. sinensis cv. Yabukita. This result shows that the molecular weight of the VH monomer is about 56 kDa, similar to that of most plant b-glucosidases. The N-terminal amino acid sequence of the purified protein was determined as GTGTPSQEVHPSHYA (Fig. 2, underlined). cDNA cloning of VH Based on the previous findings that b-primeverosidase and furcatin hydrolase (FH), catalyzing hydrolysis of 940 Vicianin hydrolase from Vicia angustifolia Fig. 2 Alignment of representative sequences from family 1 of the glycosyl hydrolases of various plants. The amino acids [identical (), strong conservation (:) and weak conservation (.)] of VH (V. angustifolia, GenBank accession No. DQ371927), FH (V. furcatum, AB122081), b-primeverosidase (C. sinensis, AB088027), amygdalin hydrolase (P. serotina, U26025), prunasin hydrolase (P. serotina, AF221526), linamarase (T. repens, X56733) and indican hydrolase (P. tinctorium, AB003089) are indicated under the sequence alignment. Conserved sequence motifs among family 1 glycosyl hydrolases are shown in bold. Regions corresponding to the sequence of purified VH in plants are shown by underlining. disaccharide glycosides, belong to family 1 of the glycosyl hydrolases (Mizutani et al. 2002, Ahn et al. 2004), reverse transcription–PCR (RT–PCR) was performed with degenerate primers for the family to obtain a cDNA encoding VH using total RNA of V. angustifolia seeds as a template. A fragment of the expected size (1,100 bp DNA) was amplified from a primer set of AHN-2 and -4. This cDNA fragment was cloned into a pGEM-Teasy vector (Promega), and the inserts of 21 clones were sequenced. The sequenced cDNA fragments represented three different glucosidases. Of these, two clones showed high sequence homology to white clover linamarase (70 and 79%; GenBank accession No. X56733), respectively (data not shown). The third clone showed the highest homology to amygdalin hydrolase (57%; U26025). Since vicianin and amygdalin contain the same aglycone moiety, mandelonitrile, the third clone was thought to encode VH. Thus, the V. angustifolia cDNA library was screened by plaque hybridization using the third clone as a probe. The probe hybridized with six plaques from approximately 300,000 screened. All of these clones contained identical cDNA terminal sequences, and one of the clones was completely sequenced. The N-terminal amino acid sequence deduced from the cloned cDNA showed a perfect match for the N-terminal sequence of the purified VH. At the end of the cloning procedure, 1.76 kb of a nearly full-length VH cDNA clone was obtained. The closest match found in the data bank was with putative b-glucosidase of Medicago truncatula (DFCI Medicago truncatula Gene Index no. TC94476) which shares 79% amino acid sequence identity (data not shown), but a Vicianin hydrolase from Vicia angustifolia functional characterization of the encoded protein of M. truncatula has not yet been performed. Sequence alignment of the two cDNAs revealed that the deduced protein sequence encoded by the cloned VH cDNA lacked the first three amino acid residues including the initiator methionine at the N-terminus. The nucleotide sequences of the cDNA for VH consist of a presumptive coding sequence of 1,530 nucleotides, and 233 nucleotides of untranslated sequence including a poly(A) tail at the 30 end (data not shown). PSORT analysis (http://psort.nibb. ac.jp) predicted that the VH cDNA encoded a 509 amino acid precursor polypeptide containing a mature protein of 490 amino acids and a 19 amino acid long putative secretion signal peptide at the N-terminal region. The molecular mass of the enzyme monomer was calculated to be 55,616, reasonably close to that of the native VH purified from seeds (55 kDa). The calculated pI of the enzyme monomer was 8.69. Characterization of VH compared with other plant b-glucosidases The amino acid sequence deduced from the VH cDNA shared 450% identity with family 1 glycosyl hydrolases from various plants such as amygdalin hydrolase from P. serotina (55% identity; GenBank accession No. U26025), indican b-glucosidase from Polygonum tinctorium (55%; AB003089), FH from Viburnum furcatum (54%; AB122081), linamarase from Trifolium repens (54%; X56733), b-primeverosidase from C. sinensis (53%; AB088027) and prunasin hydrolase from P. serotina (52%; AF221526). The deduced VH contained several sequence motifs that are characteristic and highly conserved among family 1 b-glucosidases (Fig. 2). Two glutamate residues were found at positions 204 and 416, which act as an acid–base catalyst and a catalytic nucleophile, respectively. The residues involved in the binding of the glycone (glucose) moiety are highly conserved in all family 1 glycosidases (Barrett et al. 1995, Rye and Withers 2000, Zechel and Withers 2000), and these residues are also found at Q56, H158, N203, E204, E472 and W473 in the VH sequence. Taken together, all these data confirmed that the VH is a member of glycosyl hydrolase family 1. VH in plants Evaluation of the organ specificity of VH in the plant was performed by RT–PCR. The VH transcript was detected abundantly in seeds and moderately in flowers, but only slightly in roots, stems and leaves (Fig. 3). Such organ-specific expression of VH is consistent with the trend observed for VH activity and the localization of its substrate, vicianin, which is mainly accumulated in the seeds (Kasai et al. 1981). 941 1 2 3 4 5 VH 25S rRNA Fig. 3 RT–PCR analysis to detect the VH transcript in different tissues of V. angustifolia. Total RNA (1 mg) was used to synthesize the first-strand cDNA using an oligo(dT) primer, and PCR was performed with the VH-specific primers, VH3 and VH4, and with 25S rRNA. Lane 1, seed; lane 2, flower; lane 3, leaf; lane 4, stem; lane 5, root. As a control, the result of RT–PCR of 25S rRNA (GenBank accession No. X61082) is shown in the bottom panel. Expression of the recombinant VH in insect cells To investigate the potential catalytic activity, the cloned cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. However, the recombinant enzyme did not have activity toward para-nitrophenyl (pNP) b-primeveroside and furcatin, which are hydrolyzed by the native VH, even though it did not show any glucosidase activity toward pNP b-D-glucopyranoside. The reason why the recombinant enzyme was not active on mono- and disaccharides is that VH is predicted to be a glycoprotein and it needs further post-translational modification (glycosylation), which does not occur in the E. coli expression system. As an alternative approach, the full-length cDNA of VH was subcloned into the pFastBac1 plasmid for expression in insect cells using a baculovirus expression system. VH activity was detected in the culture medium, confirming that the signal peptide sequence encoded by the VH cDNA was a secretion signal peptide. Thus, we selected a Trichoplusia ni (Tn) cell, which is known to be an efficient cell line for the production of glycoproteins, for further expression studies. Recombinant VH produced by insect cells was purified by Con A-Sepharose and CM-Sepharose chromatography (Fig. 1B). Western blot analysis confirmed that the recombinant VH also cross-reacted with the anti-b-primeverosidase antibody (Fig. 1D). Enzyme activities b-Vicianosidase activity for VHs was measured with the natural substrate vicianin. The specific activities for both native and recombinant enzymes against vicianin were 9.86 and 8.97 U mg1 protein, respectively. To confirm further the hydrolysis of vicianin, a reaction mixture containing vicianin and either the native VH or the recombinant VH was analyzed by thin-layer chromatography (TLC; Fig. 4, lanes 5 and 6). The spot corresponding to vicianin disappeared, and a spot corresponding to a 942 Vicianin hydrolase from Vicia angustifolia 1 2 3 4 5 6 Fig. 4 TLC of the products of hydrolysis of vicianin by VH. The enzyme was incubated with substrate at a concentration of 5 mM vicianin. TLC was carried out on silica gel 60 F254 plates using a solvent system of butanol : water : acetic acid (3 : 2 : 1, by vol.). Glycosides and sugars were detected by heating at 1208C after spraying with 0.2% (w/v) naphthoresorcinol in H2SO4 : ethanol (1 : 19, v/v). Lane 1, arabinose; lane 2, glucose; lane 3, cellobiose; lane 4, vicianin; lane 5, the reaction products of native purified VH; lane 6, the reaction products of the recombinant VH. disaccharide, which showed an Rf value similar to those of cellobiose, was detected. The products from the reaction mixtures of native VH and recombinant VHs were identical, and neither arabinose nor glucose was detected in the reaction mixture. The reaction products were confirmed to be a disaccharide (vicianose) and mandelonitrile. Thus, the VH cDNA isolated in this study was shown to encode VH of V. angustifolia. The results also demonstrate that VH is a disaccharide-specific glycosidase hydrolyzing the b-glycosidic bond between mandelonitrile and vicianose without cleaving the inter-glycosidic bond between arabinose and glucose. Substrate specificity of VH Various kinds of disaccharide glycosides and monosaccharide glucosides were used to determine the substrate specificity of native and recombinant VHs. The recombinant protein showed the same pattern of substrate specificity as native VH (data not shown). The substrate specificity of VH, compared with that of FH, is shown in Fig. 5. VH showed the highest activity toward the natural substrate vicianin. VH also hydrolyzed pNP b-primeveroside, 2-phenylethyl b-primeveroside and furcatin. These results indicate that VH was able to hydrolyze not only vicianoside but also the other disaccharide glycosides tested. On the other hand, VH showed 51% activity toward mandelonitrile-, 2-phenylethyl- and pNP b-gentiobioside. These data clearly indicate that VH shows Fig. 5 Substrate specificities of VH and FH toward several monosaccharide and disaccharide glycosides. Reaction rates are expressed as a percentage of that observed with vicianin (100% ¼ 9.7 U mg protein1). Data are from Ahn et al. (2004). a strict substrate specificity for b-vicianoside, b-acuminoside and b-primeveroside, but not for b-gentiobioside. The substrate specificity of VH toward disaccharide glycosides was consistent with that of FH (Ahn et al. 2004) and tea b-primeverosidase (Mizutani et al. 2002), which do not hydrolyze b-gentiobiosides. It is interesting to note that VH showed 34% of its vicianin hydrolase activity toward prunasin, which is b-D-glucopyranoside with the same aglycone mandelonitrile as that of vicianin, but VH showed only 0.2–3.4% activity toward 2-phenylethyl- and pNP b-D-glucopyranoside. This result indicates that VH possess a pronounced specificity for the aglycone as well as the glycone moiety. VH did not hydrolyze other monosaccharide glycosides such as pNP b-D-xylopyranoside, pNP a-L-arabinopyranoside and pNP b-D-galactopyranoside (0.8, 0.7 and 0.8% of the activity of vicianin, respectively). Discussion VH is a unique cyanogenic glycosidase specific to a disaccharide vicianoside Cyanogenesis is a chemical defense in response to injury of cells and tissues against herbivores and pathogens, Vicianin hydrolase from Vicia angustifolia and 42,500 plant species including ferns, gymnosperms and angiosperms contain cyanogenic glucosides, indicating that the defense system is ancient in the plant kingdom (Vetter 2000, Zagrobelny et al. 2004). HCN is poisonous not only to animals and insects that feed on plants, but also to plants themselves. To prevent poisoning themselves, plants store cyanogenic glucosides and degrading enzymes in a separate compartment such as the endoplasmic reticulum, cytosol and plastid (Hughes et al. 1992, Cicek and Esen 1999, Matsushima et al. 2003). When the cells are damaged by attack, the compartments are breached to make the substrate and enzyme come together, resulting in the hydrolysis of substrate. In this way, the release of HCN is regulated by physical and spatial mechanisms (Kojima et al. 1979, Nahrstedt 1988, Poulton 1988). In addition, the hydrolysis of cyanogenic glucosides is biochemically controlled by a specific b-glucosidase, which shows a narrow spectrum of substrate specificity for cyanogenic glucosides. b-Glucosidases belong to a multigene family, glycosyl hydrolase family 1, in which various b-glucosidases are distributed through entire plants. Therefore, enzymatic degradation of each substrate glucoside should have different spatial and temporal regulation and/or a narrow spectrum of substrate for each b-glucosidase. These substrate specificities can be explained by several examples such as amygdalin hydrolase and prunasin hydrolase in P. serotina, BGLU 45 and 46 in Arabidopsis thaliana, Glu1 and Glu2 in Zea mays, and Dhr1 and Dhr2 in S. bicolor (Cicek and Esen 1998, Cicek and Esen 1999, Zhou et al. 2002, Escamilla-Trevino et al. 2006). Each set of enzymes shares 467–71, 78, 90 and 88% sequence identity, respectively, but shows different substrate specificity in terms of the aglycone moiety. Therefore, they seem to have evolved for altered aglycone specificity. Alternatively, a marked specificity for the glycone moiety can be achieved by disaccharide-specific glycosidases. The best known cyanogenic diglucoside is amygdalin which is responsible for the bitter taste of almonds and other rosaceous seeds, and amygdalin degradation requires two b-glucosidases called amygdalin hydrolase and prunasin hydrolase, indicating that this is different from the case of disaccharide-specific glycosidases. Thus, VH is a unique disaccharide-specific cyanogenic glycosidase, which has evolved toward strict glycone specificity for the disaccharide vicianoside. Although both VH and the substrate vicianin are accumulated in seeds, their compartments at the tissue or cellular level must be different to prevent unnecessary release of toxic HCN. VH is a glycoprotein, and the putative secretion signal at the N-terminal sequence acts as a secretion signal in insect cells, suggesting that VH may be localized towards the outside of the cells (e.g. cell walls). Upon seed disruption by herbivores, vicianin is specifically hydrolyzed by VH. Furthermore, vicianin seems to be 943 resistant to hydrolysis by most b-glucosidases because of the modification of the glycone moiety with a 6-O-arabinosyl group, and VH is probably the only enzyme to hydrolyze vicianin efficiently in Vicia seeds. Biochemical specificity as well as spatial separation between substrate and enzyme controls the timing and place of the release of toxic HCN in V. angustifolia. Thus, the ccurrence of vicianin and VH in V. angustifolia seems to be an evolutionarily sophisticated strategy as a chemical defense. VH as a disaccharide-specific glycosidase We have been interested in the disaccharide glycosidespecific glycosidases (diglycosidases) that hydrolyze disaccharide glycoside into a disaccharide unit and an aglycone because of their pronounced preference for disaccharide glycosides and a one-step reaction mechanism to release aglycone from substrates. Since the reaction mechanism of VH is quite similar to that of b-primeverosidase from tea plants (C. sinensis) (Mizutani et al. 2002) and FH from V. furcatum (Ahn et al. 2004), VH is the third example of disaccharide-specific glycosidases. The native and recombinant VH have proved that VH hydrolyzes vicianin into vicianose and mandelonitrile. Cloning of VH cDNA has shown that VH is classified into family 1 of b-glycosyl hydrolases, consistent with the findings that b-primeverosidase and FH also belong to family 1. The native and recombinant VH showed the highest activity toward the natural substrate vicianin, followed by other disaccharide glycosides such as b-primeverosides and b-acuminoside. Importantly, VH hydrolyzed these disaccharide glycosides to release the corresponding disaccharose units, indicating that VH is a disaccharide glycoside-specific glycosidase. The substrate specificity of VH has been determined with four sets of glycosides containing four types of aglycones. The data indicated that VH had much greater activity toward disaccharide glycosides (b-vicianoside, b-primeveroside and b-acuminoside), except for b-gentiobiosides, when compared with the monosaccharide b-glucosides containing the same aglycones (Fig. 5). Although VH showed significant activity toward prunasin containing the same aglycone as the natural substrate vicianin, the activity toward prunasin was 34% compared with that for vicianin. The results also indicate that VH prefers disaccharide glycosides rather than monosaccharide b-glucosides in terms of the glycone moiety. The presence of a pentose unit on the C-6 hydroxy group of the glucose is important for substrate recognition and binding by VH. Surprisingly, VH could not hydrolyze b-gentiobiosides including amygdalin. The second sugar moiety of b-gentiobioside is glucose, which has an extra bulky hydroxymethyl group at the C-6 position, and the bulky group at C-6 is likely to hinder the binding and the hydrolysis of b-gentiobioside by VH. Kasai et al. (1981) 944 Vicianin hydrolase from Vicia angustifolia Fig. 6 A neighbor-joining phylogenetic tree of members of glycosyl hydrolase family 1 from various plants. The entire amino acid sequences of VH (V. angustifolia, GenBank and/or Swiss-Prot/TrEMBL accession No. A2SY66), FH (V. furcatum, Q75W17), tea leaf b-primeverosidase (C. sinensis, Q7X9A9), amygdalin hydrolase (P. serotina I, Q40984), prunasin hydrolase (P. serotina II, Q9M5X4), dhurrinase (S. bicolor, Q41290), linamarase of white clover (T. repens I, P26205) and cassava (M. esculenta, Q41172), b-glucosidase of indigo (P. tinctorium, Q9XJ67), thai rosewood (Dalbergia cochinchinensis, Q9SPK3), cicer (Cicer arietinum, Q9FSY8), serpentwood (Rauvolfia serpentina, Q9SPP9), madagascar periwinkle (Catharanthus roseus, Q9M7N7), maize (Z. mays, P49235), Cucurbita pepo (Q9FVL4), lodgepole pine (Pinus contorta, Q9ZT64), Costus speciosus (Q42707), arabidopsis (Arabidopsis thaliana, Q8GY78) and human (Homo sapiens, Q9H227), putative b-glucosidase of medicago (M. truncatula, DFCI Medicago truncatula Gene Index no. TC94476) and rice (Oryza sativa, Q7XKV5), and myrosinase of white mustard (Sinapis alba, P29736) and rape (Brassica napus, Q00326) were subjected to phylogenetic analysis. The bootstrap probability of the clustering at a node is indicated when it is greater than 50%. also showed that a crude enzyme from V. angustifolia seeds hydrolyzed vicianin but did not hydrolyze amygdalin. On the other hand, VH purified from D. trichomanoides hydrolyzes amygdalin at 27% of the rate of vicianin hydrolysis (Lizotte and Poulton 1988). Vicia VH is also distinct from fern VH in terms of physical properties such as different pI and presence or absence of glycosylation. Thus it could be concluded that VH of V. angustifolia and VH of D. trichomanoides are variants of b-glucosidases that have diversified to show an altered substrate specificity and physical properties to fulfill the common role of the VH activity in the respective plant species. Evolution of disaccharide-specific glycosidase in family 1 of glycosyl hydrolases The phylogenetic tree based on the full amino acid sequences of b-glycosidases was constructed using a neighbor-joining method (Fig. 6). VH clustered with the homologous clone of putative b-glucosidase from M. truncatula, which shares 79% amino acid sequence identity with VH. The high similarity suggests that Medicago b-glucosidase may be a disaccharide glycosidespecific glycosidase, although there is no report showing that Medicago plants contain either vicianin or disaccharide glycosides. VH also lies in the same clade with indican b-glucosidase and also lies at the next cluster of b-primeverosidase and FH. However, VH clustered with neither amygdalin hydrolase nor prunasin hydrolase whose natural substrates have the same aglycone moiety, mandelonitrile. This observation indicates that VH and amygdalin hydrolase have evolved horizontally from different b-glucosidases in original plants. In this study, we described a unique cyanogenic glycosidase, VH, from V. angustifolia as the third example of a disaccharide glycoside-specific glycosidase in higher plants. Because a wide variety of plant species are found to have disaccharide glycosides, this new class of disaccharide glycoside-specific glycosidases belonging to glycosyl hydrolase family 1 is expected to be widely distributed in plants. Structural biological studies including Vicianin hydrolase from Vicia angustifolia X-ray crystallographic analysis of these diglycosidases are now in progress to clarify the characteristic substrate recognition of these enzymes. A preliminary survey of these diglycosidases is also in progress to determine their distribution throughout the plant kingdom. Materials and Methods Chemicals Vicianin was kindly provided by Professor Ritsuo Nishida (Department of Applied Life Science, Kyoto University). pNP b-Dglucopyranoside was a gift from Nihon Shokuhin Kako. 2-Phenylethyl b-D-glucopyranoside, 2-phenylethyl b-primeveroside and 2-phenylethyl b-gentiobioside were synthesized by the method of Ma et al. (2001). pNP b-primeveroside was kindly provided by Amano Enzyme. p-Allylphenyl b-D-glucopyranoside was kindly provided by Professor Koji Kato (Ly et al. 2002). Furcatin was isolated from leaves of V. furcatum according to the method by Hattori and Imaseki (1959) with slight modifications. p-Allylphenol (chavicol) was synthesized from estragole by the method of Ohigashi and Koshimizu (1976). Other chemicals were purchased from Sigma-Aldrich, Nacalai Tesque or Wako Pure Chemicals. Plant materials Vicia angustifolia plants were sampled in the mountainous area west of the Biwa Lake, Japan. Leaves, stems, roots, seeds and flowers were immediately frozen in liquid nitrogen and stored at 808C. Purification of VH from the seeds of V. angustifolia All procedures were carried out at 48C. Frozen seeds (approximately 30 g) of V. angustifolia were ground in a chilled mortar with a pestle in liquid nitrogen in the presence of 3.0 g of polyvinylpolypyrrolidone until a fine powder was produced. The powder was suspended in 300 ml of an extraction buffer (100 mM citrate buffer, pH 6.0) and stirred overnight at 48C. The slurry was centrifuged at 22,000g for 40 min, at 48C. The supernatant was filtered through four layers of filter paper (Grade 4, Whatman, Maidstone, UK). The filtrate was dialyzed overnight against 3.5 liters of buffer A (20 mM citrate, pH 6.0). This enzyme preparation was applied onto a DEAESepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with buffer A. After washing the column with the same buffer to remove unbound proteins, VH activity was found in the flow-through fractions. The flow-through fractions were applied onto a CM-Sepharose column (Amersham Pharmacia Biotech), and VH was eluted with a linear gradient of 0–250 mM NaCl in buffer A at a flow rate of 2.5 ml min1. Each fraction was tested for VH activity and the active fractions were pooled. Then, the preparation was applied to a Con A-Sepharose 4B column (Amersham Pharmacia Biotech) pre-equilibrated with buffer A. After extensive washing with buffer A, bound proteins were eluted with 0.4 M methyl a-D-mannopyranoside in buffer A. Fractions containing VH activity were pooled, dialyzed overnight against 2 liters of buffer A, and stored at 48C. The N-terminal sequence of the purified VH was determined by automated Edman degradation on a Shimadzu PPSQ-21 protein sequencer. 945 Reverse transcription–PCR Tissue samples were ground in a mortar with a pestle in the presence of liquid nitrogen. Total RNA of leaves, stems, roots, flowers and seeds was prepared using the MagExtractor-RNA total RNA purification kit (Toyobo, Tokyo, Japan), or RNAeasyÕ plant mini-kit (QIAGEN, Hilden, Germany). Four degenerate primers (AHN1, AHN2, AHN3 and AHN4), as described in Ahn et al. (2004), were employed to obtain cDNA fragments encoding family 1 glycosidases from seeds. A 1 mg aliquot of total RNA was used to synthesize the first-strand cDNA using an RNA PCR Kit (Toyobo). Reverse transcription was carried out at 428C for 60 min. The PCR was carried out at 948C for 2 min, followed by 45 cycles of 30 s at 948C, 50 s at 418C and 90 s at 728C. PCR products were analyzed on a 1% agarose gel and a cDNA fragment of the expected size was recovered from the agarose gel using a Max extract gel extraction kit (Toyobo) followed by cloning into the pGEM-T easy vector (Promega, Madison, WI) according to the manufacturers’ instructions. DNA sequencing and phylogenetic analysis The dideoxy chain termination method using an ABI prism BigDye termination Cycle Sequencing Reaction Kit (Applied Biosystems, Warrington, UK) and an ABI prism 377 Sequencer (Applied Biosystems) was carried out to determine the nucleotide sequences. Nucleotide sequences were analyzed using the DNASIS software system (Hitachi Software, Tokyo, Japan). A multiple alignment of selected members of glycosyl hydrolase family 1 from various plants was created using ClustalW 1.83. A phylogenetic tree was constructed using the neighbor-joining method with bootstrap analysis (1,000 replicates) and Kimura’s correction for protein distances. Isolation of a full-length cDNA clone coding for VH Total RNA isolated from the seeds of V. angustifolia plants was used for cDNA library construction. Poly(A)þ RNA was isolated using a PolyATtrack mRNA isolation systems from Promega. The cDNA library was constructed using the SuperScriptTM Lambda System for cDNA Synthesis and Cloning (Life Technologies, Gaithersburg, MD). Approximately 300,000 cDNA-containing phages were screened on nylon filters (Hybond-N, Amersham Pharmacia Biotech) using an alkaline phosphatase-labeled probe based on a PCR product obtained with degenerate primers. A total of six positive phages were isolated and converted to plasmids. Sequence analysis showed that all six clones were identical, including their length. A clone, whose insert was 1,763 bp in length, was selected for further analysis. Electrophoresis (SDS–PAGE) The purity and molecular weight of the protein were checked by SDS–PAGE on a 10% polyacrylamide gel. The proteins were detected by staining with Comassie brilliant blue R-250. To estimate the molecular weight, a standard molecular marker mixture was also run (Sigma-Aldrich, St Louis, MO). Western blot analysis using an anti-b-primeverosidase polyclonal antibody was done as described by Mizutani et al. (2002). Heterologous expression of VH in insect cells The VH open reading frame was amplified by PCR using a set of two primers: 50 primer (sense) VH1, 50 -GGATCCATGGGA GCTATAAGTCCTTCCCTC-30 , containing a BamHI site (underlined); and 30 primer (antisense) VH2, 50 - CTCGAGTCATTTGA GAAGAAACTTTTGGAG-30 , containing an XhoI site 946 Vicianin hydrolase from Vicia angustifolia (underlined). The amplification product was cloned into the pCR 2.1-TOPO vector, and its sequence was checked for introduced PCR errors. The entire coding region of the VH cDNA was excised by digestion with BamHI/XhoI, purified and cloned into the pFastBac1 donor plasmid, and the recombinant plasmid was transferred into DH10Bac competent cells. Colonies containing recombinant bacmids were identified by blue–white selection and PCR. Recombinant bacmid DNA was isolated from the selected E. coli. The insect cell lines Spodoptora frugiperda (Sf9) and T. ni (Tn4) were maintained at 278C in Sf-900 II Serum Free Medium (SFM) and Express-V, respectively. Purified bacmid DNA was transfected into Sf9 cells to produce a recombinant baculovirus by standard procedures described in the Bac-to-Bac Baculovirus expression systems manual (Life Technologies). Briefly, 6 ml of Cellfectin (Invitrogen, Carlsbad, CA) and 1 mg of bacmid DNA were used to transfect 9 105cells per well in a 35 mm well plate, and the medium was harvested 72 h postinfection. To prepare the recombinant VH protein, the Tn cells were infected with the recombinant VH virus, and the culture medium containing the secreted VH protein was harvested 96 h post-infection. Purification of recombinant VH Harvested culture media were centrifuged at 1,500g for 10 min, and the supernatant was concentrated 10-fold by ultrafiltration through a YM-50 membrane (Millipore, Bedford, MA). The concentrated solution was applied to a 5 ml Con ASepharose column pre-equilibrated with buffer A (20 mM citrate buffer, pH 6.0), at a flow rate of 0.5 ml min1. The column was washed with two bed volumes of buffer A, and the recombinant protein was eluted with five bed volumes of buffer A containing 400 mM methyl-a-D-mannopyranoside (Wako, Osaka, Japan) at a flow rate of 0.5 ml min1. Active fractions were pooled and applied to a CM-Sepharose column pre-equilibrated with buffer A. The column was washed with two bed volumes of buffer A, and the recombinant enzyme was eluted with three bed volumes of buffer A containing 200 mM NaCl. Active fractions were then pooled and dialyzed overnight against 1 liter of buffer A. The slurry was concentrated 10-fold by ultrafiltration through a YM-50 membrane. Protein was resolved by 10% SDS–PAGE and stained with Coomassie brilliant blue R-250. The purified VH was stored at 48C for further analysis. Organ-specific expression of VH transcript A 1 mg aliquot of total RNA from each of leaves, stems, roots, seeds and flowers was used to synthesize the first-strand cDNA using an RNA PCR Kit Ver. 2.1 (Takara-Bio, Ohtsu, Japan). Reverse transcription was carried out at 428C by using an oligo(dT) primer, and the PCR with the primer set VH3, and 50 -GAAGCTCGTAAAGATGGTATTAGGATCAGT-30 ; VH4, 50 -GCACTCATTTGAGAAGAAACTTTTGGAGCC-30 was carried out for 2 min at 948C, followed by 30 cycles of 30 s at 948C, 50 s at 558C and 90 s at 728C. As a quantitative control, 25S rRNA of V. angustifolia (GenBank accession No. X61082) was detected with the primer set 25SF, 50 -AAGCTACCGTGCGT TGGATTATGACTGAAC-30 ; and 25SR, 50 -TATTTAAGTCGT CTGCAAAGGATTCTACCC-30 by PCR, 20 cycles of 30 s at 948C, 50 s at 558C and 90 s at 728C. PCR products were analyzed on a 1% (w/v) agarose gel. Enzyme assays Enzyme activity was determined by measuring the liberation of aglycones from each glycoside. Each reaction mixture (50 ml) contained 10 mM substrate, 20 mM citrate buffer (pH 6.0) and 10 ml of the enzyme solution. A mixture without the enzyme was pre-incubated at 378C, and the reaction was started by adding the enzyme and stopped by the addition of 50 ml of 1 M Na2CO3. To determine the activity of a reaction mixture with vicianin, amygdalin and prunasin, each 20 ml of reaction mixture was subjected to HPLC, and benzaldehyde, which is a product of mandelonitrile liberated from the substrates, was detected at 250 nm. One unit was defined as the amount of enzyme liberating 1 mmol benzaldehyde min1 under the assay conditions. In the reactions with pNP b-glycosides, the activity was determined spectrophotometrically by measuring the liberated p-nitrophenol at 405 nm. For the reactions with furcatin, 20 ml of reaction mixture was subjected to HPLC, and p-allylphenol (retention time, 7.6 min) liberated from the substrate was detected at 277 nm. The activity toward 2-phenylethyl b-glycosides was measured by the amount of 2-phenylethanol liberated from the substrate using HPLC. Protein concentration was determined by the Bradford method (1976) using the Coomassie protein assay reagent (Pierce, Rockford, IL). Analysis of liberated benzaldehyde and 2-phenylethanol was performed under the following conditions: column, COSMOSIL 5C18-AR-II (4.6 50 mm) (NACALAI TESQUE, Kyoto, Japan); detection at 250 and 210 nm for benzaldehyde and 2-phenylethanol, respectively, using a SHIMADZU SPD-10AUP UV-VIS detector; column temperature, 408C; mobile phase, 50% (v/v) methanol in water; flow rate, 0.8 ml min1. The liberated p-allylphenol was detected at 277 nm with mobile phase, 60% (v/v) methanol in water containing 0.1% (v/v) acetic acid; flow rate, 0.75 ml min1. TLC was carried out on silica gel 60 F254 plates (Merck 5715, 0.25 mm) (Merck, Darmstadt, Germany), using a solvent system of 1-butanol : water : acetic acid (3 : 2 : 1, by vol.). Glycosides and sugars were detected by heating at 1208C after spraying with 0.2% (w/v) naphthoresorcinol in H2SO4 : ethanol (1 : 19, v/v). Acknowledgments This work was supported in part by the Ministry of Education, Science, Sports, and Culture of Japan (grant-in-aid no. 13460049 and 16380079). We thank Professor Asim Esen, Dr. Farookahmed S. Kittur (Department of Biological Sciences, Virginia Polytechnic Institute and State University) and Professor Hema A. Bandaranayake (Department of Biology, Xavier University of Louisiana) for their careful reviews of the manuscript. We are grateful to Professor Ritsuo Nishida (Department of Applied Life Science, Kyoto University) and Professor Koji Kato (Faculty of Applied Biological Science, Gifu University) for kindly providing vicianin and p-allylphenyl b-D-glucopyranoside, respectively. References Ahn, Y.O., Mizutani, M., Saino, H. and Sakata, K. (2004) Furcatin hydrolase from Viburnum furcatum Blume is a novel disaccharide-specific acuminosidase in glycosyl hydrolase family 1. J. Biol. Chem. 279: 23405–23414. Barrett, T., Suresh, C.G., Tolley, S.P., Dodson, E.J. and Hughes, M.A. (1995) The crystal structure of a cyanogenic b-glucosidase from white clover, a family 1 glycosyl hydrolase. Structure 3: 951–960. Vicianin hydrolase from Vicia angustifolia Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72: 245–254. Cicek, M. and Esen, A. (1998) Structure and expression of a dhurrinase (b-glucosidase) from Sorghum. Plant Physiol. 116: 1469–1478. Cicek, M. and Esen, A. (1999) Expression of soluble and catalytically active plant (monocut) b-glucosidases in E. coli. Biotechnol. Bioeng. 63: 392–400. Escamilla-Trevino, L.L., Chen, W., Card, M.L., Shih, M.C., Cheng, C.L. and Poulton, J.E. (2006) Arabidopsis thaliana b-glucosidases BGLU45 and BGLU46 hydrolyse monolignol glucosides. Phytochemistry 67: 1651–60. Hattori, S. and Imaseki, H. (1959) A phenolic glycoside in Viburnum furcatum Blume. J. Amer. Chem. Soc. 81: 4424–4425. Hosel, W., Tober, I., Eklund, S.H. and Conn, E.E. (1987) Characterization of beta-glucosidases with high specificity for the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) moench seedlings. Arch. Biochem. Biophys. 252: 152–62. Hughes, M.A., Brown, K., Pancoro, A., Murray, B.S., Oxtoby, E. and Hughes, J. (1992) A molecular and biochemical analysis of the structure of the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Cranz). Arch. Biochem. Biophys. 295: 273–279. Kasai, T., Kisimoto, M. and Sin’itiro, K. (1981) On the free sugar and cyanogenic glycoside in the seed of Vicia angustifolia var. Segetalis. Kagawa Daigaku Nogakubu Gakujutsu Hokoku 32: 111–119. Koch, B., Nielsen, V.S., Halkier, B.A., Olsen, C.E. and Møller, B.L. (1992) The biosynthesis of cyanogenic glucosides in seedlings of cassava (Manihot esculenta Crantz). Arch. Biochem. Biophys. 292: 141–150. Kojima, M., Poulton, J.E., Thayer, S.S. and Conn, E.E. (1979) Tissue distributions of dhurrin and of enzymes involved in its metabolism in leaves of Sorghum bicolor. Plant Physiol. 63: 1022–1028. Kuroki, G.W. and Poulton, J.E. (1986) Comparison of kinetic and molecular properties of two forms of amygdalin hydrolase from black cherry (Prunus serotina Ehrh) seeds. Arch. Biochem. Biophys. 247: 433–439. Kuroki, G.W. and Poulton, J.E. (1987) Isolation and characterization of multiple forms of prunasin hydrolase from black cherry (Prunus serotina Ehrh) seeds. Arch. Biochem. Biophys. 255: 19–26. Lizotte, P.A. and Poulton, J.E. (1988) Catabolism of cyanogenic glycosides by purified vicianin hydrolase from squirrel’s foot fern (Davallia trichomanoides Blume). Plant Physiol. 86: 322–324. Ly, T.N., Yamaguchi, R., Shimoyamada, M. and Kato, K. (2002) Isolation and structural elucidation of some glycosides from the rhizomes of 947 smaller galanga (Alpinia officinarum Hance). J. Agric. Food Chem. 50: 4919–4924. Ma, S.J., Mizutani, M., Hiratake, J., Hayashi, K., Yaki, K., Watanabe, N. and Sakata, K. (2001) Substrate specificity of beta-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing. Biosci. Biotechnol. Biochem. 65: 2719–2729. Matsushima, R., Kondo, M., Nishimura, M. and Hara-Nishimura, I. (2003) A novel ER-derived compartment, the ER body, selectively accumulates a b-glucosidase with an ER retention signal in Arabidopsis. Plant J. 33: 493–502. McFarlane, I.J., Lees, E.M. and Conn, E.F. (1975) The in vitro biosynthesis of dhurrin, the cyanogenic glucoside of Sorghum bicolor. J. Biol. Chem. 250: 4708–4713. Mizutani, M., Nakanishi, H., Ema, J., Ma, S.J., Noguchi, E., Inohara-Ochiai, M., Fukuchi-Mizutani, M., Nakao, M. and Sakata, K. (2002) Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation. Plant Physiol. 130: 2164–2176. Nahrstedt, A. (1988) Cyanogenesis and the role of cyanogenic compounds in insects. CIBA Found. Symp. 140: 131–150. Ohigashi, H. and Koshimizu, K. (1976) Chavicol, as a larva-growth inhibitor, from Viburnum japonica Spreng. Agric. Biol. Chem. 40: 2283–2287. Poulton, J.E. (1988) Localization and catabolism of cyanogenic glycosides. CIBA Found. Symp. 140: 67–91. Rye, C.S. and Withers, S.G. (2000) Glycosidase mechanisms. Curr. Opin. Chem. Biol. 4: 573–580. Vetter, J. (2000) Plant cyanogenic glucosides. Toxicon 38: 11–36. Yemm, R.S. and Poulton, J.E. (1986) Isolation and characterization of multiple forms of mandelonitrile lyase from mature black cherry (Prunus serotina Ehrh) seeds. Arch. Biochem. Biophys. 247: 440–445. Zagrobelny, M., Bak, S., Rasmussen, A.V., Jorgensen, B., Naumann, C.M. and Lindberg Møller, B. (2004) Cyanogenic glucosides and plant–insect interactions. Phytochemistry 65: 293–306. Zechel, D.L. and Withers, S.G. (2000) Glycosidase mechanism: anatomy of a finely tuned catalyst. Acc. Chem. Res. 33: 11–18. Zheng, L. and Poulton, J.E. (1995) Temporal and spatial expression of amygdalin hydrolase and (R)-(þ)-mandelonitrile lyase in black cherry seeds. Plant Physiol. 109: 31–39. Zhou, J., Hartmann, S., Shepherd, B.K. and Poulton, J.E. (2002) Investigation of the microheterogeneity and aglycone specificityconferring residues of black cherry prunasin hydrolases. Plant Physiol. 129: 1252–1264. (Received November 27, 2006; Accepted May 15, 2007)