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Plant Physiol, May 2000, Vol. 123, pp. 275-286
Purification, Enzymatic Characterization, and Nucleotide Sequence
of a High-Isoelectric-Point
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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High-isoelectric-point (pI) 
-glucosidase was purified 7,300-fold
from an extract of barley (Hordeum vulgare) malt by
ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose
chromatography. The enzyme had high activity toward maltose
(kcat = 25 s
1), with an
optimum at pH 4.5, and catalyzed the hydrolysis by a retaining
mechanism, as shown by nuclear magnetic resonance. Acarbose was a
strong inhibitor (Ki = 1.5 µM). Molecular recognition revealed that all OH-groups in
the non-reducing ring and OH-3 in the reducing ring of maltose formed
important hydrogen bonds to the enzyme in the transition state complex.
Mass spectrometry of tryptic fragments assigned the 92-kD protein to a
barley cDNA (GenBank accession no. U22450) that appears to encode an
-glucosidase. A corresponding sequence (HvAgl97; GenBank
accession no. AF118226) was isolated from a genomic phage library using
a cDNA fragment from a barley cDNA library. HvAgl97 encodes
a putative 96.6-kD protein of 879 amino acids with 93.8% identity to
the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a
maximum 48 h after the start of germination.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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-Glucosidases
(EC 3.2.1.20) release -D-Glc from the non-reducing ends
of -glucosides, oligosaccharides, and starch. Type I enzymes prefer
aryl glucosides and Suc, type II prefer maltose and isomaltose, and
type III resemble type II but also attack starch (Chiba, 1988
, 1997;
Frandsen and Svensson, 1998). Plant -glucosidases have been purified
to homogeneity from buckwheat (Kanaya et al., 1976), corn (Chiba and
Shimomura, 1975), pea (Sun et al., 1995), rice (Takahashi et al.,
1971), spinach (Sugimoto et al., 1995), sugar beet (Chiba et al.,
1978), and broccoli (Monroe et al., 1999), and genes were cloned from
barley (Hordeum vulgare) (Tibbot and Skadsen, 1996), sugar
beet (Matsui et al., 1997), spinach (Sugimoto et al., 1997), potato
(Taylor et al., 1998), and Arabidopsis (GenBank accession no. AF014806;
Monroe et al., 1999). The sequences belong to glycoside hydrolase
family 31, which includes fungal -glucosidases; mammalian
sucrase-isomaltase, maltase-glucoamylase, and lysosomal
-glucosidase; and -glucosidase II in N-linked
sugar biosynthesis (Henrissat, 1991).
In conjunction with 
-amylase, limit dextrinase, and -amylase,
-glucosidase in germinating seeds was proposed to mobilize endosperm
starch (MacGregor, 1987). Early work addressed the purification and
specificity of these enzymes in malt (Jørgensen, 1963, 1964; Jørgensen and Jørgensen, 1963, 1967). -Glucosidase I and II (50 and 130 kD, respectively) have high activity on
p-nitrophenyl -D-glucoside, and
maltase I and II (14 and 66 kD, respectively) were described
subsequently (Stark and Yin, 1987). Malt -glucosidase was found to
exert an initial attack on and, with barley -amylase, to make a
synergistic 11-fold enhanced digestion of starch granules (Sun and
Henson, 1990). A similar study showed a 2-fold synergy (Sissons and
MacGregor, 1994), suggesting that the former -glucosidase preparation or perhaps both preparations may have contained traces of
amylolytic enzymes.
Cloning of a gibberellin-induced putative barley -glucosidase cDNA
provided new knowledge on this enzyme (Tibbot and Skadsen, 1996). The
encoded 97-kD protein belonged to glycoside hydrolase family 31 (Tibbot
and Skadsen, 1996) and was larger than the 33-kD enzyme reported
previously (Im and Henson, 1995). Recombinant inactive and active
-glucosidases were produced in Escherichia coli
and Pichia pastoris (Tibbot et al., 1998); however, the
active form had approximately 300 times lower specific activity
than the malt enzyme. The reason for this discrepancy is unknown,
but the recombinant enzyme lacked an N-terminal region. Moreover, immunoblotting with antibodies raised against the inactive E. coli form indicated that the full-length protein was processed in
germinating seeds (Tibbott et al., 1998).
In the present study, a high-pI -glucosidase was purified 7,300-fold
from 6-d old barley malt. Matrix-assisted laser desorption ionization
(MALDI) mass spectrometry (MS) of tryptic fragments of this 92-kD
protein showed that its sequence and that deduced from the cDNA (Tibbot
and Skadsen, 1996) were very similar. This enzyme had the highest
activity reported for the high-pI -glucosidase from barley. Acarbose
and 5'-thio-4-N--maltoside (Svensson and Sierks, 1992;
Sigurskjold et al., 1994; Andrews et al., 1995) were strong inhibitors.
Glucoside bonds were hydrolyzed with retention of the anomeric
configuration characteristic of family 31 enzymes (Frandsen and
Svensson, 1998). Recognition of deoxy maltosides indicated that all
OH-groups of the non-reducing ring and OH-3 of the reducing ring
contributed to transition state stabilization. The sequence of a
corresponding barley -glucosidase encoding DNA (cv Igri) of 7.1 kb
contained four exons.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Purification and Identification of Barley Malt High-pI
-Glucosidase
A protocol was established for the purification of a highly active
high-pI -glucosidase from barley malt. The progress of purification
was monitored by SDS-PAGE (Fig. 1), and
the degree of purification and yields at individual steps are given in
Table I. The protocol took advantage of
the fact that high-pI -glucosidase, in contrast to low-pI
-glucosidase, did not bind to DEAE-Fractogel at pH 7.5. The ratio of
high- to low-pI enzymes in the ammonium sulfate precipitate of malt
extract was approximately 0.25, based on the activity for maltose of
the eluted low-pI (not shown) and high-pI -glucosidases.
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High-pI -glucosidase was purified 250-fold after
COO-Fractogel rechromatography at pH 5.5 concomitant with removal of abundant proteins (Fig. 1, lanes 2-4):
-amylase/subtilisin inhibitor (21 kD; Svendsen et al., 1986),
(1,3;1,4)--glucanase (33 kD; Woodward and Fincher, 1982),
-D-glucan exohydrolase (69 kD; Hrmova et al., 1996),
and lipoxygenase 2 (90 kD; Doderer et al., 1992), as identified by
N-terminal sequencing, western blotting using specific
antibodies, or in-gel trypsin digestion and MALDI-MS peptide mapping.
The N-terminal sequence AXPKTVGVYELTKGDFSAKVTNLGATVTDD of a
38-kD protein (Fig. 1, lanes 3 and 4) has 54% identity to aldose-1-epimerase-like protein from tobacco (Nicotiana
tabacum; GenBank G2739168). The 21-, 33-, and 38-kD proteins could
be removed by gel filtration (Sephacryl S-200 HR), but butyl-Sepharose
chromatography separated both these proteins and lipoxygenase 2 (90 kD)
from -glucosidase. The butyl-Sepharose eluate contained high-pI
-glucosidase and -D-glucan exohydrolase,
which were partially separated on COO-Fractogel
at pH 7.3, resulting in further 4.5-fold purification of the
-glucosidase (Fig. 1, lane 6). The high-pI -glucosidase of 92 kD
was thus purified 7,300-fold from malt extract (Table I) in 2% yield
taking into account that the low-pI enzyme possessed 80% of the
activity in the extract. The modest recovery is considered to stem from
the large number of purification steps, the hydrophobic nature, and the
small quantities present of the 92-kD high-pI -glucosidase, which
was reported to be processed to a predominant 81-kD form after 5 to
7 d of germination (Tibbott et al., 1998).
N-terminal sequencing identified -D-glucan
exohydrolase (69 kD; Hrmova et al., 1996) in the highly purified
-glucosidase. The 92-kD protein was N-terminally blocked. MALDI-MS
of the mixture of tryptic fragments generated by in-gel digestion of
the 92-kD protein indicated that it was likely encoded by a putative
barley -glucosidase gene for which a cDNA was cloned (Tibbot and
Skadsen, 1996). Protein alignment showed 93% sequence identity
between the two deduced protein sequences. Peptide masses determined by MALDI-MS were used in database searches, and the pattern (Fig. 2A) contained little noise information in
the form of significant unmatched components. The matches gave
(Fig. 2B) about 20% coverage of the sequence deduced from the
-glucosidase cDNA of cv Morex (Fig. 2A; Tibbot and Skadsen, 1996)
and cv Igri (Fig. 2A; the present work; GenBank AF118226). A
majority (14 of 22) of peptides (Fig. 2) matched both the Igri and
Morex sequences, while five matched only Igri and three only Morex.
This variation was expected as evident from the alignment of the
Igri and Morex sequences comprising 46 differences and eight gaps. Five
unmatched peaks in Figure 2A (two of medium and three of lower
intensity) probably stem from parts of the Alexis -glucosidase
that vary from the known sequences. MALDI-MS gives accurate masses but
provides no sequence information, so peptides from Alexis with a single
amino acid substitution relative to known sequences will escape
identification in the search for a match.
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Organization of the Gene Encoding Barley -Glucosidase
A single clone hybridizing to the cDNA clone AGL2790 (Tibbot and
Skadsen 1996) was identified by rescreening two primary isolated clones
from a barley genomic library at high stringency with a 2.3-kb cDNA
from a barley EST library (cv Himalaya). This 15- to 18-kb genomic
clone was characterized by restriction mapping, subcloned for
sequencing, and found to contain the entire AGL97 gene flanked by
non-coding regions. The organization of the -glucosidase gene
(HvAgl, cv Igri) and the localization of the protein
coding regions by identification of exon/intron boundaries are shown in
Figure 3. The gene from start to stop
codon has 7,113 bases and four exons separated by introns of varying
length. Exons have from 218 to 1,529 bp, whereas introns range from 86 to 4286 bp. All nucleotide sequences at exon/intron boundaries were
consistent with the consensus GT/AG sequence at the donor and acceptor
sites of RNA splicing. The nucleotide sequence was named
HvAgl97 (GenBank accession no. AF118226). The four exons
encode a protein of 879 amino acids with a calculated molar mass of
96.558 D. The theoretical pI is 6.93 (http://www.expasy.ch/tools/pi_tool.html), whereas isoelectric
focusing gave an experimental value of 
8.
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Expression of the Barley -Glucosidase Gene during
Germination
The transcription of the -glucosidase gene in germinating seeds
was followed from the grain (0 h) to 144 h (7 d) after the start
of germination in the malt house. While dry grain has no detectable
transcript, a very weak -glucosidase transcription signal was
detected 12 h after steeping. The signal increased during the next
12 h and expression reached a maximum after 48 h. The
transcript decreased at d 3 to 4 and disappeared at d 5 to 6. In a
parallel experiment, seeds (cv Alexis) were subjected to micromalting
and hybridized to RNA prepared from samples taken at the same time
points as those from the industrial malting. Micromalting and
industrial malting gave similar expression profiles (Fig.
4), with a transcription maximum at
48 h. This demonstrated excellent agreement for a particular gene
expression between performance in the malthouse and during
micromalting.
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Enzymic Properties of the Barley High-pI -Glucosidase
The pH optimum for hydrolysis of the preferred substrate maltose
was at pH 4.0 to 4.5, and no activity was detected at a pH 8.0. The low amount of highly purified -glucosidase allowed determination
of kinetic parameters for only selected substrates. Maltose was the
best substrate, with a kcat of 24.8 s1 and a Km of
2.2 mM. For the -1,6-linked isomaltose,
kcat was 5 s1
and Km was 11.0 mM, resulting in a 25-fold reduction in
kcat/Km compared with maltose. p-Nitrophenyl
-D-glucoside was hydrolyzed with a
kcat of 2.2 s1
and a Km of 0.6 mM, resulting in a 3-fold decrease of
kcat/Km. Preliminary tests indicated that the activity was lower on longer substrates, which agrees with results from other studies (Im and Henson, 1995). The -glucosidase was free from contamination by -amylase, -amylase, and limit dextrinase in assays specific for
these malt enzymes performed at a level of sensitivity allowing detection of amounts corresponding to 0.1% of the purified
-glucosidase.
Acarbose, a pseudotetrasaccharide strongly inhibiting glucoamylase,
-amylase, and other retaining or inverting -glucoside-specific hydrolases and transferases (Legler, 1990; Sinnott, 1990;
Svensson and Sierks, 1992; Sigurskjold et al., 1994; Svensson et al.,
1995; Frandsen and Svensson, 1998), was a competitive inhibitor with a
Ki of 1.5 µM
for the high-pI -glucosidase. This
Ki value is 2- to 3-fold lower than
that for barley -amylase (Søgaard et al., 1993). Surprisingly, the
maltose analog methyl 5'-thio-4-N--maltoside inhibited
the high-pI -glucosidase with Ki = 0.9 µM, which is extremely efficient compared
with the Km of 2.5 mM for maltose. A similar relation was found for
this inhibitor in maltose hydrolysis by A. niger
glucoamylase (Andrews et al., 1995). The two -glucosidase inhibitors
were thus roughly 100-fold more potent than castanospermine, which was
reported to give a Ki of 0.11 mM for barley high-pI -glucosidase (Henson and
Sun, 1995). Kinetic parameters and substrate specificity of different
plant -glucosidases have been reviewed (Frandsen and Svensson,
1998). Very recently, a recombinant potato enzyme was found to have a
Km of 17 mM for
maltose and a Vmax of 3.25 nM Glc formed h1
µg1 protein (Taylor et al., 1998), which was
recalculated to a kcat of 0.09 s1. It is remarkable that this catalytic
constant was as low as the value determined for the recombinant barley
enzyme produced in P. pastoris (Tibbot et al., 1998).
Although kcat was not reported for the
natural enzyme from potato, this coincidence may indicate that these
large recombinant proteins were not obtained in proper functional form
or that the sequence from potato corresponds to an
-glucosidase with a different functional role, possibly in the biosynthesis of glycoconjugates.
Stereochemistry of Methyl -Maltoside Hydrolysis
The stereochemistry of the catalytic hydrolysis of the glucoside
bond was determined by 1H-NMR spectroscopy. The
NMR spectra of methyl -maltoside before (Fig.
5A) and 7 or 120 min after the addition
of enzyme (Figs. 5, B and C) showed a doublet centered at 5.22 µL
L1 and assigned to H-1 of free -Glc.
The doublets at 4.37 and 4.64 µL L1 were
assigned to H-1 of the products, -methyl Glc and -Glc, respectively. The spectrum recorded 7 min after addition of the enzyme
(Fig. 5B) indicated a ratio of 65% -Glc to 35% -Glc. The latter
arose by mutarotation of initially formed -Glc. Because high-pI
-glucosidase released -Glc from the maltoside, it catalyzed glycoside bond hydrolysis with retention of the anomeric configuration, in agreement with the stereospecificity of the mechanism in glycoside hydrolase family 31 (Frandsen and Svensson, 1998).
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Molecular Recognition of Maltose Analogs
The specificity constant was determined for the -glucosidase
catalyzed hydrolysis of -methyl maltoside and its seven monodeoxy analogs. The results made it possible to identify and assess the strength of individual intermolecular enzyme-substrate OH-hydrogen bonds implicated in stabilization of the transition state in the hydrolysis. Deoxygenation at OH-4' or -6' thus resulted in about 1.2 × 103-fold reduction in
Vmax/Km,
and at OH-3', -2', and -3 in modest 14- to 60-fold reductions (Table
II), but no significant effect occurred
when OH-1 or -2 were replaced by hydrogen (Table II). The difference in
the transition state stabilization energy
(
G
) was calculated from the
Vmax/Km
values determined for analog and parent substrates. This molecular
recognition approach is an established procedure for probing binding
energy contributed by individual carbohydrate OH-groups to
transition-state stabilization. G values of approximately 19 kJ mol1 were correlated with the elimination of
OH-4' and -6', thus indicating that these OH-groups participate in
strong charged hydrogen bonds to the enzyme (Fersht et al., 1985). The
OH-2' and -3' at the non-reducing, and the OH-3 at the reducing end
ring each contributed 7 to 11 kJ mol1 to
transition-state stabilization (Table II), which is compatible with the
formation of neutral hydrogen bonds between the OH-groups and the
enzyme.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Barley high-pI -glucosidase was purified 7,300-fold from an
extract of 6-d-old malt, resulting in a specific activity of 16.5 units
mg1, corresponding to a
kcat = 25 s1.
A cDNA clone encoding barley -glucosidase was identified previously (Tibbot and Skadsen, 1996), and a corresponding recombinant protein was
produced in Pichia pastoris (Tibbot et al., 1998). The
Vmax of this enzyme toward maltose was
0.054 µmol min1 mg1
(Tibbot et al., 1998), which is equal to a
kcat of 0.08 s1, as recalculated using the theoretical size
of the recombinant enzyme of 89 kD. However, this protein was not
purified prior to characterization. Earlier kinetic analysis of a malt
-glucosidase resulted in a kcat of
12.6 µmol min1 mg1
(Im and Henson, 1995), recalculated to
kcat = 20 s1
using 97 kD, the theoretical enzyme molar mass, rather than the 33 kD
reported by the authors. These authors determined the enzyme concentration by active site titration with the inhibitor
castanospermine, and thus got a correct value for
kcat even though the preparation contained other proteins.
The recombinant enzyme from P. pastoris was constructed to
lack part of the N-terminal sequence, which perhaps resulted in low
activity (Tibbot et al., 1998). However, adverse post-translational modification or misfolding may also be the cause. This comparison makes
it clear that highly active, high-pI -glucosidase of the correct
size was purified in the present work. The low yield of the 92-kD
-glucosidase may in part stem from important processing to smaller
forms during germination, as indicated by immunoblotting of aleurone
and seed extracts using antibodies directed against the inactive
recombinant enzyme produced in E. coli (Tibbot et al.,
1998). The larger form was claimed to be a minor form in malt (Tibbott
et al., 1998). It is not known if the forms have the same activity.
Purification of barley high-pI malt -glucosidase of 92 kD was
extremely difficult, and resulted in about 30 µg enzyme
kg1 malt. Peptide mapping by MALDI-MS of
tryptic fragments showed that the sequence of this protein was very
similar to the deduced cDNA sequences from cv Morex (Tibbot and
Skadsen, 1996; GenBank U22450) and cv Igri (the present study) of
mutual identity of 93.8% (Fig. 6). The mass of a few tryptic fragments
did not match the cv Morex sequence, but did match the cv Igri sequence and vice versa. Some did not match either of these sequences, as
expected because the protein was from a third cultivar, cv Alexis.
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The DNA region encoding high-pI -glucosidase spanned 7.1 kb, of
which 37.2% is protein coding sequence. This is comparable to genes of
barley -amylase and limit dextrinase in which coding regions
occupied 42.6% and 29.2%, respectively (GenBank accession nos.
AF061203 and AF022725). In contrast, the coding region of barley
-amylases covered 87.3% and 73.7% of the Amy1/6-4 and Amy32b genes (GenBank accession nos. K02637 and X05166). The
-glucosidase cDNA encodes a putative 96.9-kD polypeptide (Tibbot and
Skadsen, 1996), which agrees with the 92 kD found for the present malt
enzyme by SDS-PAGE.
Previous expression analysis showed that transcription from an
-glucosidase gene increased during laboratory germination to a
maximum at 3 to 5 d after imbibition. Increasing -glucosidase activity was found up to d 7 in a series covering 10 d (Tibbot and
Skadsen, 1996). The present study on gene expression during d 0 to 6 revealed that transcription of the gene encoding the high-pI enzyme
reached a maximum at 48 h, both in micromalting and industrial
malting. Because we found that the low-pI -glucosidase was
predominant in malt, it was not meaningful to attempt to correlate the
present temporal transcription with the increase in enzyme activity in
extracts made during germination. Recently, transcription of the barley
limit dextrinase gene in the same samples used here was found to reach
a maximum 72 to 96 h after imbibition (Kristensen et al., 1999).
Thus, the -glucosidase transcript preceded that of limit dextrinase
and followed or coincided with that of -amylase (Tibbot and Skadsen,
1996).
Barley high-pI -glucosidase of glycoside hydrolase family
31 catalyzes glucoside hydrolysis with
retention of the anomeric configuration resulting from a
double-displacement mechanism through transition states with
substantial oxocarbenium ion character (Davies and Henrissat, 1995;
Frandsen and Svensson, 1998). Confirmation of this stereochemistry of
methyl -maltoside hydrolysis by 1H-NMR adds
further support to the identification of the 92-kD protein. In this
mechanism one carboxylic acid acts as a general acid/base catalyst and
a second as a catalytic nucleophile (Sinnott, 1990; Svensson and
Søgaard, 1993; McCarter and Withers, 1994; Tanaka et al., 1994; Davies
and Henrissat, 1995). The pH activity dependence for the high-pI
-glucosidase (see also Henson and Sun, 1995; Tibbott et al., 1998)
is compatible with a mechanism suggesting that the enzyme in the pH
range around 4.5 of optimal activity has a deprotonated and a
protonated carboxylic acid group at the active site. Mechanism-based
active site labeling of sugar beet -glucosidase by the inhibitor
conduritol B epoxide led to identification of an essential Asp (Iwanami
et al., 1995). The prominent sequence similarity with sugar beet
-glucosidase (Quaroni and Semenza, 1976; Chiba, 1997; Frandsen and
Svensson, 1998) suggests that Asp-437 is the catalytic nucleophile in
the barley enzyme. This agrees with site-directed mutagenesis to Asn of
Asp-481 in -glucosidase from Schizosaccharomyces pombe,
leading to inactivation (Mori et al., 1999), and mechanism-based
labeling of Asp-214 as a catalytic nucleophile in -glucosidase from
S. cerevisiae (McCarter and Withers, 1996a, 1996b).
Enzyme-substrate interactions through hydrogen bonds are essential in
specificity and for transition state stabilization. Deoxygenated sugar
analogs are widely used to identify the critical sugar OH groups and to
quantitate the associated individual energies in complexes of
carbohydrate-binding enzymes (Bundle and Young, 1992; Sierks and
Svensson, 1992; Sierks et al., 1992; Frandsen et al., 1996; Lemieux et
al., 1996). The transition-state stabilization energy
(G) was calculated from
hydrolysis of a series of deoxy maltosides by -glucosidase to be 19 kJ mol1, which was attributed to charged
hydrogen-bonds with OH-4' and -6'. Neutral hydrogen bonds, contributing
7 to 11 kJ mol1, were proposed to form with
OH-2', -3', and -3 in maltose (Fig. 7).
Remarkably, all four OH-groups of the non-reducing sugar ring participate in intermolecular hydrogen bonds in the enzyme-substrate transition state complex, whereas in the reducing end ring only OH-3
contributed to stabilization and did so less strongly than each of the
OH-groups from the other ring. This pattern indicates that the enzyme
is of type II, having poor activity on oligosaccharide substrates
and starch. The reasonable activity on 4-nitrophenyl -D-glucopyranoside, which lacks a hydrogen
bond donor equivalent to OH-3, was explained by 4-nitrophenol
being a good leaving group that compensated for the lack of the
OH-3-mediated hydrogen bond to the enzyme.
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In summary, highly active, high-pI -glucosidase was purified
from barley malt and classified by peptide mapping and nucleotide sequencing to glycoside hydrolase family 31. This work contributes to
the knowledge about genes and molecular properties of plant -glucosidases, and forms a basis for future studies of the role of
these enzymes during germination.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Materials
Isomaltose, maltose, p-nitrophenyl
-D-glucopyranoside, Glc oxidase
(Aspergillus niger), and the Glc oxidase kit were from Sigma-Aldrich, St. Louis). Red-Pullulan was from Megazyme
(Ireland). Insoluble Blue starch, butyl-Sepharose 4 FF, SDS-PAGE, and
isoelectric focusing gels were from Amersham-Pharmacia Biotech
(Uppsala), and the -amylase assay kit was from Behring
Diagnostics (American Hoechst, Charlotte, NC). Fractogels EMD
DEAE 650 (S) and EMD COO 650 (S) were from
Merck (Darmstadt, Germany). Chemicals for
Tricine-SDS-polyacrylamide gels, Ready gels, and Bio-Gel P-6 DG
were from Bio-Rad Laboratories (Hercules, CA). A barley (Hordeum
vulgare, cv Igri) genomic library in 
FIX II was obtained from
Stratagene (La Jolla, CA). Nylon Hybond-N+ and nitrocellulose membranes
were from Amersham (Buckinghamshire, UK), and Schleicher & Schuell (Dassel, Germany), respectively. [32P]dCTP was from DuPont-NEN
(Stevenage, UK), and the XAR5 x-ray film was from Eastman-Kodak
(Rochester, NY). The DNA sequencing kit was from Perkin-Elmer Applied
Biosystems (Foster City, CA). The ProtoBlot kit, restriction enzymes,
and sequencing grade trypsin were from Promega Biotechnology (Madison,
WI). Ultrapure water was from a Milli-Q system (Millipore, Bedford,
MA). Other chemicals were of analytical grade. Monodeoxy maltosides
(Bock and Pedersen, 1987; Sierks et al., 1992), methyl
5'-thio-4-N--maltoside (Andrews et al., 1995), and
acarbose were the kind gifts of Dr. K. Bock (Carlsberg Laboratory), Dr.
B.M. Pinto (Simon Fraser University, Burnaby, British Columbia,
Canada), and Dr. E. Möller (Bayer AG, Wuppertal, Germany),
respectively. Antibodies against barley (1,3;1,4)--glucanase and
lipoxygenase 1/2 were kindly provided by Drs. O. Olsen and J. Rouster
(Carlsberg Research Laboratory).
Plant Material
Six-day-old barley (Hordeum vulgare cv Alexis; Carlsberg Maltings) malt was dried (30 kg) in a kiln (5 d) at 28°C (residual weight 17 kg) to 8% (w/w) water content. Industrial malt samples were from Carlsberg Maltings (batch 339), and micromalt samples of the same variety were prepared in a micromalt apparatus (Automated Malting System, Phoenix-Biosystems, Australia) using European Brewery Convention standard conditions.
Enzyme Purification
Malt Extraction and Ammonium Sulfate Precipitation
Dried malt (17 kg) was milled using 4-mm plate spacing (Carlsberg Technology Development), and the flour was stirred for 1 h in 200 L of 20 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 5 mM CaCl2, pH 7.5, at room temperature, followed by settling for 16 h at 4°C. The pH was kept at 7.5 by adding 1 M NaOH. The supernatant was concentrated to 10 L by ultrafiltration (DDS-RO GR81P membrane, 20-kD cutoff, Danisco, Copenhagen), and adjusted to pH 6.7. Ammonium sulfate was added to 20% (w/v) saturation, and the extract was centrifuged at 4,550g for 45 min at 4°C. Ammonium sulfate was added to the supernatant to 70% (w/v) saturation. The precipitate, collected by centrifugation in six portions of approximately 185 g each, was stored at 4°C.Chromatographic Separations
All steps were at 4°C. Ammonium sulfate precipitate (370 g) was stirred (1 h) into 700 mL of 20 mM HEPES and 0.5 M CaCl2, pH 7.5, centrifuged (4,550g, 30 min), and the supernatant was desalted on Bio-Gel P-6 (10 × 38 cm) in 10 mM HEPES and 2.5 mM CaCl2, pH 7.5 at a flow rate of 840 mL h1. Fractions with high
activity were pooled and applied at a flow rate of 108 mL
h1 to DEAE-Fractogel (2.6 × 30 cm) in 20 mM HEPES and 5 mM
CaCl2, pH 7.5. The pass-through and the first
wash (1 column volume) containing high-pI -glucosidase were then
pooled. Following continued washing (10 volumes), low-pI
-glucosidase (used in a separate study) was eluted by a linear
gradient (0
0.8 M NaCl) in the buffer (2 × 900 mL; rate: 120 mL h1). The high-pI
-glucosidase pool was dialyzed against 50 mM
sodium acetate and 5 mM
CaCl2, pH 5.5, applied to a
COO-Fractogel (2.6 × 25 cm; rate: 130 mL
h1) equilibrated in the buffer, and washed (10 volumes) and eluted using a linear gradient (00.4
M NaCl; 2 × 900 mL at 130 mL
h1). Fractions with activity were pooled,
dialyzed, and rechromatographed as above on
COO-Fractogel (1.6 × 5 cm; linear
gradient: 2 × 200 mL at 51 mL h1). The
pool with activity was mixed with 1 volume of 50 mM sodium acetate, 5 mM
CaCl2, and 4 M NaCl, pH
5.5, and applied to butyl-Sepharose (1.6 × 2 cm) equilibrated in
the same buffer containing 2 M NaCl. After a
buffer wash (10 volumes), the buffer was made to 1.5 M, 1.25 M, and 1 M NaCl to be used for stepwise elution (6 column volumes each; rate: 51 mL h1). The 1.25 and 1 M NaCl eluates containing activity were pooled, dialyzed against 20 mM HEPES and 5 mM CaCl2, pH 7.3, and
applied to COO-Fractogel (1.6 × 1 cm;
rate: 60 mL h1). After a buffer wash (10 volumes), the high-pI -glucosidase was eluted by a linear gradient
(00.2 M NaCl) in the buffer (2 × 20 mL at
60 mL h1), pooled, and dialyzed against 0.1 M sodium acetate, pH 4.5. The enzyme preparation
(E280 = 0.12) was stored at 4°C.
Enzymatic Procedures
Enzyme Activity Assays
Activity on maltose (15 mM) was assayed by adding 10-µL aliquots of fractions to 190 of µL 0.1 M of sodium acetate, pH 4.5, in 300-µL PCR tubes at 37°C. After a suitable reaction time (5-15 min) and enzyme inactivation (7 min, 100°C), the mixture was centrifuged. The supernatant was mixed in a microtiter plate with an enzyme-color solution (100 µL) containing 5 units mL1 Glc oxidase, 1 unit
mL1 peroxidase, and 0.23 mg
mL1 o-dianisidine in 1 M
Tris, pH 7.6. The A450 after 1 h
at room temperature was read in a microtiter plate reader (Ceres
Uv900Hdi, Bio-Tek) and standardized using
D-Glc. One activity unit was considered to be the
amount of enzyme that releases 2 µmol Glc from maltose min1 at 37°C.
Vmax and
Km were obtained by fitting initial
hydrolysis rates at 10 substrate concentrations (0.125 × Km 8 × Km) to the Michaelis-Menten equation.
The reaction was started by the addition of enzyme (10 µL) to
substrate, and aliquots (35 µL) were transferred at intervals to
Eppendorf tubes, heat inactivated, centrifuged, and added to
enzyme-color solution (200 µL). The kcat = Vmax/[E0],
where [E0] is the enzyme concentration
determined from amino acid analysis.
Ki was calculated using
Ki = (100 % inhibition)
[I]/% inhibition (1 + [S]/Km).
Activity for maltose (15 mM) was determined at 20 pH values (pH 2.5-7.3 in 0.1 M
citrate-phosphate; pH 7.6-9.2 in 0.1 M boric
acid/sodium tetraborate).
-Amylase and -amylase assays used Insoluble Blue Starch in 20 mM sodium acetate, 5 mM
CaCl2, pH 5.0, at 37°C (Juge et al., 1995) and
a mixture of 4-nitrophenyl -D-maltopentaoside (0.85 mM) and 4-nitrophenyl -D-maltohexaoside
(0.65 mM) in the presence of 800 units
L1 microbial -glucosidase in 47 mM sodium phosphate, pH 6.0, at 40°C (Mathewson and
Seabourn, 1993), respectively. A test for limit dextrinase used
2% (w/v) Red Pullulan in 0.2 M sodium acetate and 5 mM CaCl2, pH 5.0, at 40°C
(Kristensen et al., 1998).
Activity toward Deoxy-Maltose Analogs
Energetics of the enzyme transition state complex were mapped using seven deoxy maltose analogs. Because these were sparse, we did not determine Vmax and Km. Instead, the second-order rate constants Vmax/Km = vo/EoSo, where vo is the initial rate of hydrolysis and Eo and So are enzyme and substrate concentrations were measured. Activity was assayed at two So values near 0.1 × Km to confirm second-order conditions. Hydrolysis was started by adding enzyme (1.457 units
mL1) in 50- to 100-µL assays in 0.1 M sodium acetate, pH 4.5. A standard developing
solution (see above) was used for deoxy analogs in the reducing ring,
while the solution contained 60 units mL1 Glc
oxidase, 1 unit mL1 peroxidase, and 0.10 mg
mL1 o-dianisidine for analogs at the
non-reducing ring. The absorbance was read after 4 h at room
temperature, and quantitated using the relevant deoxy sugar or
D-Glc. The increase in activation energy for
hydrolysis by removal of a substrate OH-group was calculated using the
equation G = RT
ln[(Vmax/Km)a/(Vmax/Km)b]
(Wilkinson et al., 1983), where a and b refer to
the analog and parent substrates, respectively.
Stereochemistry of Glucoside Bond Hydrolysis
-Glucosidase (62 units) from
COO-Fractogel rechromatography at pH 5.5 was
passed on Sephacryl S-200 (1.6 × 85 cm) in 0.2 M
sodium acetate, pH 5.5, 5 mM CaCl2.
The sample (same specific activity as after butyl-Sepharose) was mixed
with 10 volumes of 0.1 M sodium acetate, pH 4.5, in
D2O (99%), concentrated 10-fold in a 10-kD
cutoff ultrafiltration unit (Centricon, Amicon, Beverly, MA) at 4°C,
and the solvent exchange was repeated. A substrate (45 mM
methyl--maltoside, 600 µL) 1H-NMR spectrum
was recorded at 310 K (AMX-600 spectrometer at 600 MHz, Bruker
Instruments, Billerica, MA), and the stereochemistry of hydrolysis was
followed by recording spectra at intervals after addition of enzyme.
Analysis of the Gene
Screening of Genomic Library
The barley genomic library was screened by plaque hybridization using a cDNA fragment from a barley expressed sequence tag (EST) library as a probe. This probe (LOK-PS333; cv Himalaya) corresponded to cDNA clone pAGL.2737 (GenBank U22450, cv Morex; Tibbot and Skadsen, 1996). Single plate lifts of approximately 0.5 × 106 plaques were prehybridized and hybridized at
42°C (16 h) in 5× SSC, 5× Denhart's solution, 50% (w/v)
formamide, 0.2% (w/v) SDS, 10 µg mL1
poly(A+), and 10 µL mL1
sheared salmon sperm DNA (9.7 mg mL1). Filters
were washed three times for 15 min at 22°C in 2× SSC and 0.2%
(w/v) SDS, then three times for 5 min at 65°C in 0.2× SSC and
0.1% (w/v) SDS, and exposed to XAR5 film (Eastman-Kodak) in
cassettes for 16 h at 80°C. Two positive primary plaque areas were re-screened. A single confirmed positive plaque was subjected to a
final screening.
Sequencing the Coding Region for the Barley -Glucosidase
-glucosidase -clone was digested by restriction
enzymes, and the DNA fragments were subcloned into pBlueScript SK. Subcloned fragments encoding protein were sequenced using a
dideoxynucleotide cycle sequencing kit (DyeDeoxy, Perkin-Elmer Applied
Biosystems, Foster City, CA) and a DNA sequencer (model 373, Perkin-Elmer Applied Biosystems). Each nucleotide in the sequence was
analyzed in at least two sequencing passes on both DNA strands. The
samples were prepared as described by Rasmussen (1994). The DNA contig assembly was performed with ABI Prism sequencing software 1.0 for Apple
Macintosh and Microgenie 6.0 (Beckman Instruments, Fullerton, CA).
Analysis of nucleotide sequences was done using GCG 9.0 (Genetics Computer Group, Madison, WI) and DNA Tools 5.0 (S. Rasmussen, Carlsberg Laboratory).
Northern Analysis
Total RNA was isolated from germinating seeds (cv Alexis) as described previously (Leah and Mundy, 1989). Seed samples were taken 0 to 144 h after imbibition. For northern blots, 10 µg of RNA was
separated in formaldehyde agarose gels, blotted onto Hybond-N+ membranes, and the blots were developed by a
32Plabeled DNA probe made using the Multiprime
Labeling System (Amersham). RNA blots were hybridized and washed as
described previously (Leah and Mundy, 1989). Electronic images of
32P-labeled blots were made and visualized using
phosphor imager cassettes and ImagesQuant version 3.3 software
(Molecular Dynamics, Sunnyvale, CA).
Malting and Micromalting of Barley
Uniform samples of standard malt were collected at 0 (dry grain), 12, 24, 48, 72, 96, 120, and 144 h after the start of steeping (i.e. the addition of water during an industrial malting of 65 tons of barley) (cv Alexis; Carlsberg Maltings). Samples of the same variety were prepared at the same time points using a microscale malting system on an 80-g scale (Carlsberg Technical Service). All samples were kept in liquid nitrogen and processed simultaneously.
Analytical Techniques
SDS-PAGE, Western Blotting, and Isoelectric Focusing
N-Tris(hydroxymethyl) methyl-Gly (Tricine) SDS-PAGE gels comprised a separation gel (16 × 12 × 0.075 cm) and a stacking gel (16 × 3 × 0.075 cm) of 8.25% (w/v) and 4% (w/v) acrylamide, respectively. Both were 1.5% (w/v) in bisacrylamide. Protein was denatured by boiling for 3 min in sample buffer (0.1 M Tris-HCl, pH 6.8, 8% [w/v] SDS, 21% [w/v] glycerol, and 0.02% [w/v] Coomassie Brilliant Blue G 250). The electrophoresis was run at 13°C for 16 h at 20 mA and 100 V (Bio-Rad II xi cell and power supply model 100/500; Schägger and von Jagow, 1987).
Proteins were blotted after SDS-PAGE (Phast gel, Pharmacia) to
nitrocellulose by diffusion (15 min) at 70°C (Phast system, Pharmacia). The membranes were soaked for 30 min at room temperature in
TBST (10 mM Tris-HCl, 0.15 M NaCl, and 0.05%
[w/v] Tween 20, pH 8.0) containing 1% (w/v) bovine
serum albumin, followed by TBST containing 0.1% (w/v) primary
rabbit antibody (30 min), a wash (three times for 10 min), and TBST
containing alkaline phosphatase-coupled goat IgG against rabbit IgG for
30 min. After washing three times for 10 min, the blot was developed in
5 mL of TBST with 21 µL of nitroblue tetrazolium (50 mg
mL1) and 16 µL of
5-bromo-4-chloro-3-indolyl-phosphate (50 mg
mL1).
Isoelectric focusing was performed with Phast gels (43 × 37 × 0.45 mm, 5% [w/v] acrylamide, 1.5% [w/v] bisacrylamide;
Pharmacia), pH 3.0 to 9.0, and proteins were visualized by Coomassie
Brilliant Blue R 350 or silver staining according to the instructions
of the manufacturer. The gel was overlaid with 25 mM
maltose, 0.1 M sodium acetate, pH 4.5, and 1%
(w/v) agarose (8.5 mL) mixed with 60 mM Tris-HCl,
pH 7.5, 115 units mL1 Glc oxidase, 0.5 unit
mL1 peroxidase, 21% (w/v) glycerol (1 mL), and 2.5 mg mL1 o-dianisidine (0.5 mL) at 37°C for 15 min to give a brown-colored zymogram.
In Situ Gel Plug Trypsin Digestion
Coomassie-stained protein bands were cut from SDS polyacrylamide (8.25% [w/v] Tricine) gels and kept in water at18°C.
Prior to digestion, the gel plug was washed three times for 20 min in 40% (w/v) acetonitrile/60% (w/v) 50 mM
NH4HCO3, pH 7.8, at 37°C, and dried for 20 min in a vacuum centrifuge. Trypsin (5 µL; 0.33 µg
µL1) was added to the gel (sample:trypsin,
approximately 25:1 [w/w]). After reswelling of the gel, 15 µL of 50 mM NH4HCO3, pH
7.8, was added and digestion continued at 37°C for 16 h.
Peptides were extracted three times in 100 µL of 60% (w/v)
acetonitrile, lyophilized, and analyzed by MALDI time of flight (TOF) MS.
MALDI-MS and Protein Identification by Database Searches
Mass spectra were acquired on a MALDI-TOF mass spectrometer (Voyager-Elite, Perseptive Biosystems, Framingham, MA) equipped with delayed ion extraction technology. Lyophilized samples were dissolved in 20 µL of 0.1% (w/v) trifluoroacetic acid and prepared for MALDI-MS analysis by placing a 0.8-µL sample on a probe tip followed by 0.4 µL of matrix solution. The MALDI matrices were 2,5-dihydroxybenzoic acid (Aldrich, Milwaukee, WI) dissolved in a mixture of 0.1% (v/v) trifluoroacetic acid and acetonitrile 2:1 (v/v) (25 g L1), and -cyano-4-hydroxy-cinnamic
acid (Sigma-Aldrich) in 70% (v/v) acetonitrile (20 g
L1). All mass spectra were obtained in
reflector mode and calibrated using internal calibration. Data
processing was carried out using GRAMS/386 software (Galactic
Industries, Salem, NH). Protein identification was performed by
searching the European Molecular Biology Laboratory comprehensive
non-redundant protein sequence database (nrdb)
(ftp://ftp.embl-heidelberg.de/pub/databases/nrdb/) using the
PeptideSearch software
(http://www.mann.embl-heidelberg.de/Services/PeptideSearch/) (Mann et
al., 1993).
Amino Acid and Amino-Terminal Sequence Analyses
Amino acid analysis was performed on 200 to 500 pmol of protein after hydrolysis (6 M HCl; 24 h in sealed, evacuated tubes; 110°C) on a Pharmacia LKB Alpha Plus equipped with an ion-exchange column. N-terminal sequence analysis was performed on 50 to 500 pmol of protein on an sequenator (model 470A or 477A, Applied Biosystems) and a PTH analyzer (model 120, Applied Biosystems).| |
ACKNOWLEDGMENTS |
|---|
We are grateful to Sidsel Ehlers, Suksawad Vongvisuttikun, and Aksel Englyst for excellent technical assistance, and to Bodil Corneliussen, Lone Soerensen, and Dr. Ib Svendsen for N-terminal sequencing and amino acid analysis. Jean Sage and Dr. Søren W. Rasmussen are thanked for advice on DNA sequencing. Bent Ole Pedersen is acknowledged for performing the NMR spectroscopy experiments.
| |
FOOTNOTES |
|---|
Received November 9, 1999; accepted January 23, 2000.
1 This work was supported by the Danish Research Councils' Committee for Biotechnology (grant no. 95-02014 to P.R. and B.S.).
2 Present address: Enzyme Functionality, Novo Nordisk, Novo Allé, DK-2880 Bagsvaerd, Denmark.
3 Present address: Mass Spectrometry Resource, Department of Biochemistry and Biophysics, Boston University School of Medecine, Boston, MA 02118-2526.
* Corresponding author; e-mail bis{at}crc.dk; fax 45-33-27-47-08.
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LITERATURE CITED |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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-maltoside bound to glucoamylase and its activity as a competitive inhibitor.
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[Medline]-glucosidase (glucoamylase) in sugar beet seeds.
Agric Biol Chem
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-glucosidases of the glucoside hydrolase family 31: molecular properties, substrate specificity, reaction mechanism, and comparison with family members of different origin.
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[CrossRef][ISI][Medline]-glucosidases: their characteristics and roles in starch degradation.
ACS Symp Ser
618: 51-58
-D-glucan exohydrolase with -D-glucosidase activity: purification, characterization, and determination of primary structure from a cDNA clone.
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271: 5277-5286
-glucosidase from germinated barley seeds: substrate specificity, subsite affinities and active-site residues.
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[CrossRef]-glucosidase.
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59: 459-463
[Medline]-glucosidase II.
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43: 67-71
-Glucosidase from Barley
Malt
, cv
Morex; 
values for high-pI 
