Download Vicianin Hydrolase is a Novel Cyanogenic b

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.
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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.
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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.
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(Received November 27, 2006; Accepted May 15, 2007)