Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues

Article abstract of DOI:10.1016/j.phytochem.2008.10.004

Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Boβfruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K_m values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k_cat values than the wild-type enzyme, but an increase in the k_cat value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.

Full text of DOI:10.1016/j.phytochem.2008.10.004

Phytochemistry 70 (2009) 25–31
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Insights into the catalytic properties of bamboo vacuolar invertase through
mutational analysis of active site residues
1
1
*
*
Tai-Hung Chen , Yu-Chiao Huang , Chii-Shen Yang, Chien-Chih Yang, Ai-Yu Wang , Hsien-Yi Sung
Institute of Microbiology and Biochemistry and Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2008
Received in revised form 27 September
Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to fam-
ily 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Bobfruct3 using
Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to
GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active
site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid res-
idues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved
Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L,
E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant
2
008
Available online 17 November 2008
Keywords:
Bambusa oldhamii
Poaceae
E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K
m
values of the four
Bamboo
mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants
W159L and E316Q exhibited lower kcat values than the wild-type enzyme, but an increase in the kcat value
was observed for the mutants W159F and C317A. The results of this study demonstrate that these resi-
dues have individual functions in catalyzing sucrose hydrolysis.
Homology modeling
Site-directed mutagenesis
Vacuolar invertase
Ó 2008 Elsevier Ltd. All rights reserved.
1
. Introduction
For example, both vacuolar and cell-wall-associated invertases are
glycoproteins, but the neutral/alkaline invertases are not glycosyl-
ated. Heavy metal ions such as Hg and Ag inhibit the activities of
acid invertases but not neutral/alkaline invertases. Additionally,
both types of acid invertases are b-fructofuranosidases, but neu-
tral/alkaline invertases are not (Sturm, 1999).
2
+
+
Sucrose is a principal product of photosynthesis, a major trans-
port form of carbohydrates, and an important source of carbon and
energy in higher plants. Utilization of sucrose for either growth or
maintenance of cell activities depends on the breakdown of its gly-
cosidic bond. Invertase (EC 3.2.1.26), which catalyzes the hydroly-
sis of sucrose to glucose and fructose, and sucrose synthase (EC
In addition to their differing biochemical properties, the se-
quences of acid invertases show a low level of homology with neu-
tral/alkaline invertases. Instead, acid invertases are highly
homologous to fructan-synthesizing fructosyltransferases and
fructan-degrading exohydrolases/fructosidases. They share several
conserved regions such as the NDPNG motif, RDP motif and EC mo-
tif (Vijn and Smeekens, 1999; Ritsema and Smeekens, 2003). The
importance of the conserved amino acid residues in these motifs
have been proposed by a number of mutational and crystallization
studies including yeast and bacterial invertases (Reddy and Maley,
1990, 1996; Alberto et al., 2004), bacterial and plant fructan-
degrading hydrolases (Nagem et al., 2004; Verhaest et al., 2005),
plant fructan-synthesizing fructosyltransferases (Ritsema et al.,
2005; Altenbach et al., 2005), and plant cell-wall-associated inver-
tases (Goetz and Roitsch, 2000; Verhaest et al., 2006; Le Roy et al.,
2
.4.1.13), which converts sucrose and UDP into fructose and
UDP–glucose, are the enzymes responsible for sucrose cleavage
in plants. Both enzymes are crucial for plant development, growth
and carbon partitioning (Sturm and Tang, 1999; Koch, 2004).
Plants possess a group of invertase isozymes that have been cat-
egorized as acid and neutral/alkaline invertases based on their pH
optima (Tymowska-Lalanne and Kreis, 1998; Sturm, 1999; Roitsch
and Gonzalez, 2004). The acid invertases are either vacuolar or cell-
wall-associated enzymes, while the neutral/alkaline invertases are
located in the cytoplasm (Tymowska-Lalanne and Kreis, 1998;
Sturm, 1999), mitochondria or plastids (Murayama and Handa,
2
007). The biochemical properties of the two types of acid inverta-
ses are similar, but differ from those of neutral/alkaline invertases.
2
007; Matrai et al., 2008; Lammens et al., 2008). However, such
studies have not been performed with plant vacuolar invertases.
In this paper, we report the construction of a theoretical structure
of bamboo vacuolar invertase by homology modeling and changes
of conserved amino acid residues by site-directed mutagene-
sis. The effect of each mutation on the enzymatic activity was
*
Corresponding authors. Fax: +886 2 23634729 (H.-Y. Sung); +886 2 23660434
(
A.-Y. Wang).
E-mail addresses: aywang@ntu.edu.tw (A.-Y. Wang), sunghy@ntu.edu.tw (H.-Y.
Sung).
1
Equal contribution to this paper.
0
031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.10.004

2
6
T.-H. Chen et al. / Phytochemistry 70 (2009) 25–31
characterized by expression and purification of the recombinant
vacuolar invertases in yeast Pichia pastoris.
residues Q118-N631 of Bobfruct3. The leader sequence and both
the N-terminus and C-terminus of the mature protein were omit-
ted for better accuracy. The primary model was further structurally
aligned with Arabidopsis AtcwINV1 D239A mutant in complex
with sucrose (PDB id: 2QQU, Lammens et al., 2008) to predict the
theoretical position of sucrose when binding Bobfruct3. As shown
in Figs. 2A and B, the overall structure of Bobfruct3 (residues
Q118-N631) with substrate is similar to that observed for GH32
enzymes, with the N-terminus composed of a five-bladed b-pro-
peller module while a b-sandwich structure can be observed in
the C-terminus. The three major conserved motifs, namely NDPNG,
RDP and EC, are located in the active site of the enzyme (Fig. 2C).
2
. Results
2.1. Sequence alignment and homology modeling
Plant vacuolar invertases belong to the glycoside hydrolase
family 32 (GH32) in the carbohydrate-active enzymes database
http://www.cazy.org). Fig. 1 shows the multiple sequence align-
(
ment of bamboo vacuolar invertase Bobfruct3 with other GH32 en-
zymes whose crystal structures have been resolved (Alberto et al.,
2
004; Nagem et al., 2004; Verhaest et al., 2005, 2006). As expected,
several conserved regions can be clearly noted. In order to obtain a
reasonable theoretical structure of Bobfruct3, protein homology
modeling was performed by using the cell-wall invertase Atc-
wINV1 from Arabidopsis thaliana (PDB id: 2AC1, Verhaest et al.,
2.2. Mutagenic replacement and expression of recombinant mutant
proteins
To assess the role of the conserved amino acid residues in or
2
006) as a template. The modeling procedure was only applied to
around the active site of Bobfruct3, site-directed mutagenesis
Fig. 1. Alignment of the deduced amino acid sequences of bamboo vacuolar invertase Bobfruct3 and four other GH32 enzymes with known structures. The sequences
compared are Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1, accession number NP566464), Cichorium intybus fructan 1-exohydrolase IIa (CiFEHIIa, CAC37922),
Aspergillus awamori exo-inulinase (AaInul, CAC44220) and Thermotoga maritime invertase (TmINV, NP229215). Identical residues are shown on a black background. The
characteristic conserved motifs of acid invertases and fructan-related fructosyltransferases are indicated by brackets below the sequences. The amino acid residues selected
for site-directed mutagenesis are denoted by asterisks.

T.-H. Chen et al. / Phytochemistry 70 (2009) 25–31
27
was performed on Asp135, Trp159, Arg259, Asp260, Glu316 and
Cys317. The plasmids containing either the mutated or wild-type
Bobfruct3 cDNA were transformed into Pichia pastoris for expres-
sion of the recombinant proteins in a secreted extracellular form.
After induction by methanol for 24 h, His-tagged recombinant pro-
teins were detected in the growth medium of each transformed
different levels of acid invertase activity (data not shown). How-
ever, no invertase activity was observed either for the mutant
E316A or for mutants with changes at Asp135, Arg259, or Asp260.
2.3. Purification and characterization of the recombinant invertases
P. pastoris strain by western analysis using an anti-(His)
6
antibody
To avoid the effect of differential protein expression by various
strains on the observed activities of crude enzyme preparations,
the recombinant wild-type and mutant invertases were all purified
from the growth media of methanol-induced cultures using
(
data not shown). Expression levels for different mutants varied,
but each was obtained in soluble form. The wild-type enzyme
and the mutants W159F, W159L, E316Q and C317A possessed
Fig. 2. Theoretical structure of bamboo vacuolar invertase Bobfruct3 constructed by homology modeling: (A) Theoretical model of Bobfruct3 (Q118-N631) based on the
known structure of cell-wall invertase AtcwINV1 of Arabidopsis. The conserved motifs NDPNG, RDP and EC are highlighted with red, blue and cyan, respectively. (B)
Superposition of Bobfruct3 (green), AtcwINV1 (yellow) and Cichorium intybus fructan 1-exohydrolase IIa (red). (C) Close-up view of the conserved residues at and around the
active site. The primary model of Bobfruct3 in panel A was further structurally aligned with Arabidopsis AtcwINV1 D239A mutant in complex with sucrose. The residues in
Bobfruct3, wild-type AtcwINV1 and AtcwINV1 D239A mutant are shown in green, yellow and red, respectively.

2
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T.-H. Chen et al. / Phytochemistry 70 (2009) 25–31
Table 1
Specific activities of the purified wild-type and mutant invertases.
Enzyme
Specific activitya (nkatal/mg)
Wild type
D135N
D135E
W159F
W159L
R259K
R259L
109.7
0
0
512.3
12.8
0
0
D260N
D260E
E316Q
E316A
C317A
0
0
73.9
0
271.7
a
Sucrose was used as the substrate to determine the activity.
Table 2
Kinetic parameters for the wild-type and mutant invertases.
cat (sꢀ1)
k
ꢀ1 ꢀ1
Enzyme
K
m
(mM)
k
m
cat /K (mM s )
Wild type
W159F
W159L
E316Q
53.68
169.77
213.58
142.56
140.66
501.7
2342.2
57.86
338.41
1242.6
9.35
13.80
0.27
2.37
8.83
C317A
Fig. 2 (continued)
ities toward raffinose were 41.0%, 33.2%, 24.3%, 15.5% and 20.7% of
sucrose for wild-type enzyme, mutant W159F, W159L, E316Q and
C317A, respectively. The effects of mutation on the K
crose and the turnover number (kcat) of the enzyme were also
determined (Table 2). The apparent K values of the four mutants
possessing invertase activity were all higher than that of the wild-
type enzyme. The mutants W159L and E316Q exhibited lower kcat
values than the wild-type enzyme, but an increase in kcat value was
m
value for su-
ammonium sulfate fractionation and cobalt-based IMAC (Fig. 3).
The chromatograms of cobalt-based IMAC for the wild-type and
mutant enzymes were similar (data not shown). The specific activ-
ities of the purified enzymes are shown in Table 1. As observed in
the crude preparations, mutagenesis at Asp135 (in the NDPNG mo-
tif), Arg259 and Asp260 (both in the RDP motif) or replacement of
Glu316 (in the EC motif) with alanine generated inactive enzyme,
and only mutants W159F, W159L, E316Q and C317A possessed
invertase activity. The mutants W159L and E316Q retained 11.7%
and 67.3% of wild-type activity, respectively, while the specific
activities of W159F and C317A were 4.7-fold and 2.5-fold, respec-
tively, of that of the wild-type enzymes.
m
observed for mutants W159F and C317A. The kcat/K
W159F, W159L, E316Q and C317A were 148%, 2.9%, 25.3% and
4.4% of the wild-type enzyme, respectively (Table 2).
m
ratios of
9
3. Discussion
The four mutants possessing invertase activity exhibited the
same pH optimum (pH 5.0) and substrate specificity as the wild-
type enzyme did. They all hydrolyzed sucrose and raffinose but
could not use cellobiose, maltose, lactose and inulin as substrates.
However, the relative rates of hydrolysis of raffinose by the mu-
tants were all lower than that of the wild-type enzyme. The activ-
Based on the stereochemical outcomes of the hydrolysis reac-
tion, glycoside hydrolases can be classified as either inverting or
retaining enzymes. The inverting enzymes act via a general acid/
base-catalyzed direct displacement mechanism which results in
overall inversion of the anomeric configuration at the site of cleav-
age. The retaining enzymes operate through a double-displace-
Fig. 3. SDS–PAGE analysis of the purified wild-type and mutated invertases. The recombinant wild-type and mutant invertases, purified by ammonium sulfate fractionation
and cobalt-based IMAC, were separated on 10% SDS–polyacrylamide gels, which were then stained with Coomassie blue R-250. Lanes 1 and 8, molecular mass markers; lanes
2
and 9, wild-type Bobfruct3; lanes 3–7, mutants W159F, W159L, E316Q, E316A and C317A, respectively; lanes 10–15, mutants D135N, D135E, D260N, D260E, R259K and
R259L, respectively.

T.-H. Chen et al. / Phytochemistry 70 (2009) 25–31
29
ment mechanism with a net retention of the anomeric configura-
tion. The retaining mechanism involves two active site residues
acting as a nucleophile and an acid/base catalyst. Attack of the ano-
meric center by the nucleophile results in the formation of a cova-
lent glycosyl-enzyme intermediate, which is subsequently
hydrolyzed in a general acid/base-catalyzed manner (Sinnott,
positioning of a water molecule, rather than to provide general
base assistance. The positioning of the water molecule is sufficient
to hydrolyze the glycosyl-enzyme intermediate. Ascorbate, a cofac-
tor for myrosinase, can substitute for the function of the catalytic
base (Burmeister et al., 1997, 2000). We propose that the Glu316
in wild-type Bobfruct3 functions as an acid/base catalyst that en-
hances the rate of sucrose hydrolysis. When Glu316 is replaced
with another amino acid residue that can provide the hydrogen-
bond network for positioning the water, the hydrolysis reaction
still can occur, but at lower rate, in acidic condition in which pro-
tonation of the glycosidic oxygen of sucrose by an acid/base cata-
lyst could be unnecessary. However, it requires further
investigation.
In addition to the two amino acid residues that may be directly
involved in catalysis in the active site, other conserved residues in
or around the active site were also examined, including Trp159,
Arg259, Asp260 and Cys317.
Trp159 is located between the NDPNG and RDP motifs. The cor-
responding residue in T. maritime invertase was reported to form a
hydrogen-bond with the hydroxyl group of the C6 of fructose
(Alberto et al., 2004). Replacing Trp159 with either Leu or Phe re-
1
990; Davies and Henrissat, 1995; White and Rose, 1997). Acid
invertases are retaining enzymes. By affinity labeling and site-di-
rected mutagenesis, Reddy and Maley (1990, 1996) first proposed
that the nucleophile and the acid/base catalyst in the active site
of yeast invertase were the Asp residue in the NDPNG motif and
the Glu residue in the EC motif, respectively. The proposed cata-
lytic mechanism is as follows: the Glu residue protonates the gly-
cosidic oxygen of sucrose and the nucleophile Asp attacks C-2 of
the fructose moiety, forming a fructosyl-enzyme intermediate
and releasing glucose. Subsequently, the Glu abstracts a proton
from water, which enables the hydroxyl group to displace fructose
and restore the original active center (Reddy and Maley, 1996). The
NDPNG and EC motifs are highly conserved across invertases and
fructan-related fructosyltransferases. Replacing the homologs Asp
with Asn or Glu with Ala, which resulted in inactive enzymes,
has also been reported for other invertase and fructosyltransferas-
es (Goetz and Roitsch, 2000; Ozimek et al., 2004; Ritsema et al.,
sulted in increases in K
m
values for sucrose, confirming its role in
fructose binding. Interestingly, the two mutations had opposite ef-
fects on the turnover number and specificity constant of the en-
zyme (Table 2). There were 8.7-fold and 34.6-fold reductions in
2
005). Analyses of crystal structures confirmed that these two ami-
no acid residues, together with the Asp residue of RDP motif, are
located in the active site of invertases and fructan-metabolizing
enzymes (Alberto et al., 2004; Nagem et al., 2004; Verhaest et al.,
the kcat and kcat/K
creases in the kcat and kcat/K
m
values of W159L, but 4.7-fold and 1.5-fold in-
values of W159F as compared to
m
2
005; Verhaest et al., 2006; Lammens et al., 2008). By homology
the wild-type enzyme. The results indicated that the aromatic
side-chain of the residue in this position was important for effi-
cient catalysis. The increase in the kcat value of W159F can be ex-
plained by assuming that the interaction between Phe and
fructose is weaker than that between Trp and fructose. Therefore,
once the enzyme is saturated with sucrose and the catalytic reac-
tion has occurred, the second product, fructose, can be released
more readily from the active site of W159F than from that of the
wild-type enzyme.
modeling, we have shown that the theoretical structure of bamboo
vacuolar invertase Bobfruct3 (residues Q118-N631) is similar to
the resolved crystal structures of Arabidopsis cell-wall invertase
and the other three GH32 enzymes. The homologs of the nucleo-
phile Asp and the acid/base catalyst Glu in Bobfruct3 are Asp135
and Glu316, respectively. The critical requirement of Asp135 was
supported by the results of site-directed mutagenesis. No catalytic
activity was detected in mutant D135N (Table 1). Even when
Asp135 was replaced with Glu, a residue with only one more
Both Arg259 and Asp260 are part of the RDP motif. The Asp res-
idue is conserved and occurs in the active site of invertases and
fructan-metabolizing enzymes. However, it was suggested that
this residue does not have a direct function during catalysis but
may affect the nucleophilicity of the nucleophile Asp in the NDPNG
motif (Nagem et al., 2004; Verhaest et al., 2006). Alberto et al
(2004) proposed that the Arg and Asp in the RDP motif (Arg137
and Asp138 in T. maritime invertase) play crucial roles in substrate
binding and recognition since the former is hydrogen-bonded to
the glucose O4 and the latter forms hydrogen-bonds to O3 and
O4 of the fructose unit. In AtcwINV1, the Asp149 of the RDP motif
was reported to have a role in stabilization of the fructose ring;
Arg148, together with W82, D239 and K242, have an influence
on the orientation of glucose (Lammens et al., 2008). In this study,
the Arg259 was changed to Lys or Leu and Asp260 was replaced
with Asn or Glu. The mutations all resulted in the complete loss
of enzymatic activity, in spite of the retention of positive and neg-
ative charges by Lys and Glu, respectively. Since the RDP motif is
connected by two flexible coils and turn structures, mutations at
Arg259 or Asp260 may cause conformational disturbances and a
less stable local structure, which consequently lead to changes in
substrate binding orientation and prevention of catalysis.
–
CH
2
in the side-chain, the enzymatic activity was not retained
(Table 1), indicating the importance of the structural distance
and orientation for nucleophilic attack.
The results of mutagenesis at Glu316 also suggested its impor-
tance in the active site but its specific role remains uncertain.
Replacement of Glu316 with Ala generated an inactive enzyme;
however, the mutant E316Q retained 67% activity of the wild-type
enzyme (Table 1). In Arabidopsis AtcwINV1, Glu203 was identified
as the acid/base catalyst in the active site. The mutant E203A
exhibited a drastic decrease in kcat value (Le Roy et al., 2007) and
mutant E203Q was inactive (Matrai et al., 2008; Lammens et al.,
2
008). The X-ray crystallographic structure of the E203Q in com-
plex with sucrose showed that the sugar binding and the catalytic
pocket arrangement is significantly altered in this mutant (Matrai
et al., 2008; Lammens et al., 2008). Although the theoretical struc-
ture of Bobfruct3 (residues Q118-N631) is similar to the revolved
structures of AtcwINV1, the difference in enzymatic activity be-
tween Bobfruct3 E316Q mutant and AtcwINV1 E203Q mutant sug-
gests that the two homologous residues may not have exactly
same role in the two enzymes. The reaction catalyzed by the
retaining glycoside hydrolases usually involves two carboxyl
groups acting as a nucleophile and an acid/base catalyst (Sinnott,
Cys317 and the preceding Glu316 (the acid/base catalyst) reside
in the EC motif. The cysteine in the EC motif is thought to play a
role in transition state stabilization and/or the catalytic residue
microenvironment in T. maritime invertase, since it forms hydro-
gen-bonds to the two Asp residues in the active site (Alberto
et al., 2004). However, replacement of this conserved cysteine with
other residues has different effects on the activity of invertases
from different species. In yeast, the mutant C205A exhibited
1
990; Davies and Henrissat, 1995; White and Rose, 1997) but vari-
ants of the acid/base catalyst exist in some enzymes (Zechel and
Withers, 2001; Burmeister et al., 1997). For example, in plant
myrosinase (thioglucoside glucohydrolase), which belongs to fam-
ily 1 of glycoside hydrolases and also is a retaining enzyme, the Glu
residue at the usual position of the acid–base catalyst is naturally
replaced by Gln. The role of this Gln is thought to ensure a precise

3
0
T.-H. Chen et al. / Phytochemistry 70 (2009) 25–31
decreased affinity for sucrose and kcat, and a 6-fold reduction in the
value (Reddy and Maley, 1996), while in C. rubrum, the mu-
tant C237S was inactive (Goetz and Roitsch, 2000). In this study,
the mutant C317A of Bobfruct3 was found to exhibit higher K
and kcat values than the wild-type enzyme, but the specificity con-
stant was not significantly changed. The results indicate that the
function of this residue in transition state stabilization and/or the
catalytic residue microenvironment was not significant, and it
may instead have a role in substrate binding.
Recent studies on the AtcwINV1 mutants showed that next to
the highly conserved residues of the NDPNG, EC and RDP motif,
Trp82, Asp239 and Lys242 are additional key residues in stabiliza-
tion of the substrate molecule (Le Roy et al., 2007; Matrai et al.,
Table 3
Primers used for the mutagenesis of Bobfruct3 cDNA.
kcat/K
m
Mutation
D135N
Nucleotide sequence
m
0
0
S: 5 -CAGAAGAACTGGATGAACAACCCTAACGGGCCTGTG-3
0
As: 5
-CCCGTTAGGGTTGTTCATCCAGTTCTTCTGAGGCTG-3
0
0
D135E
W159F
W159L
R259K
R259L
D260N
D260E
E316Q
E316A
C317A
S: 5 -CAGAAGAACTGGATGAACGAGCCTAACGGGCCTGTG-3
0
0
As: 5 -CCCGTTAGGCTCGTTCATCCAGTTCTTCTGAGGCTG-3
0
0
S: 5 -GAGGGCGCGGTGTTCGGCAACAAGATCGCCTGGG-3
0
0
As: 5 -CTTGTTGCCGAACACCGCGCCCTCCGGGTTGTAC-3
0
0
S: 5 -GGCGCGGTGCTGGGCAACAAGATCGCCTGGG-3
0
0
As: 5 -GATCTTGTTGCCCAGCACCGCGCCCTCCGG-3
0
0
S: 5 -GCCAAGGACTTCAAGGACCCCACCACCGCCTGG-3
0
0
As: 5 -GGTGGGGTCCTTGAAGTCCTTGGCGCCGATGGC-3
0
0
S: 5 -GCCAAGGACTTCCTGGACCCCACCACCGCCTGG-3
0
0
As: 5 -GGTGGGGTCCAGGAAGTCCTTGGCGCCGATGGC-3
2
008; Lammens et al., 2008). The Asp239 can interact directly with
S: 5
0
-AAGGACTTCCGCAACCCCACCACCGCCTGGC-3
0
0
0
the glucose moiety of sucrose. Substitution of Asp239 with alanine
alters substrate specificity of AtcwINV1 into a 1-kestose (Le Roy
et al., 2007). In Bobfruct3, the Trp82, Asp239 and Lys242 homologs
are Trp159, Asp355 and Arg358, respectively. Whether the func-
tions of these residues are conserved in different invertases or
not will be investigated in the future.
As: 5 -GGTGGTGGGGTTGCGGAAGTCCTTGGCGCCG-3
0
0
S: 5 -AAGGACTTCCGCGAGCCCACCACCGCCTGG-3
0
0
As: 5 -GGTGGTGGGCTCGCGGAAGTCCTTGGCGCC-3
0
0
S: 5 -GCCACCGGCATGTGGCAGTGCATCGACTTCTACCCC-3
0
0
As: 5 -GAAGTCGATGCACTGCCACATGCCGGTGGCCGGGAC-3
0
0
S: 5 -GCCACCGGCATGTGGGCCTGCATCGACTTCTACCCC-3
0
0
As: 5 -GAAGTCGATGCAGGCCCACATGCCGGTGGCCGGGAC-3
0
0
S: 5 -GGCATGTGGGAGGCCATCGACTTCTACCCC-3
0
0
As: 5 -GAAGTCGATGGCCTCCCACATGCCGGTGGC-3
4
. Conclusions
The results of this study demonstrate that the conserved resi-
as a template DNA for the generation of mutated plasmids. Site-di-
rected mutagenesis was carried out using the QuickChange site-di-
rected mutagenesis kit and mutagenic primer pairs (Table 3).
dues in or around the active site of bamboo vacuolar invertase
Bobfruct3 have individual functions in catalyzing sucrose hydroly-
sis. Our results also suggest that, as long as the conformation of the
active site is properly maintained, decreasing the affinity of the en-
zyme for substrate by mutagenesis at Trp159 and Cys317 can in-
crease the turnover number of the enzyme, probably by
facilitating the release of reaction products from the enzyme.
Although this finding requires further investigation, it suggests a
possible way to engineer this enzyme for application.
5.4. Expression and purification of the wild-type and mutated
invertases in yeast
Pichia pastoris strain X-33 was transformed with plasmid
pBoIT3 or mutated plasmids by electroporation. The transformed
cells were grown in buffered glycerol-complex medium [0.1 M
potassium phosphate, pH 6.0, 1% (w/v) yeast extract, 2% (w/v) pep-
ꢀ5
5
. Experimental
tone, 1.34% (w/v) yeast nitrogen base (YNB), 4 ꢁ 10 % (w/v) biotin
and 1% (v/v) glycerol] at 30 °C until the A600 value reached 1.0. The
cells were collected by centrifugation and then resuspended in the
buffered MeOH-complex medium [0.1 M potassium phosphate, pH
6.0, 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB,
5
.1. General experimental procedures
The QuickChange site-directed mutagenesis kit was from Strat-
agene (La Jolla, CA). The cobalt-based immobilized metal affinity
chromatography (IMAC) resin was from BD Biosciences Clontech
Palo Alto, CA). Yeast extract, peptone and yeast nitrogen base were
obtained from Becton, Dickinson and Company (Sparks, MD). Other
chemicals were from Sigma-Aldrich (St. Louis, MO).
ꢀ5
4 ꢁ 10 % (w/v) biotin and 0.5% (v/v) MeOH] containing 1% casa-
mino acid, and were grown at 30 °C for 24 h.
(
For purification of the recombinant invertases secreted in the
medium, the methanol-induced cultures were centrifuged at
6000g for 30 min to remove cells. The proteins in the supernatants
were precipitated by 80% saturation of ammonium sulfate fol-
lowed by centrifugation. The precipitates were then dissolved in
PB-7.0 (50 mM sodium phosphate, pH 7.0) and dialyzed against
PB-7.0 containing 0.3 M NaCl. The resultant enzyme solution was
mixed with cobalt-based IMAC resin and incubated for 1 h at
4 °C. The enzyme–resin mixture was then packed into a column,
washed with PB-7.0 containing 0.3 M NaCl and 10 mM imidazole,
and eluted with PB-7.0 containing 0.3 M NaCl and 150 mM imidaz-
ole. Proteins in each fraction were analyzed by 12.5% SDS–PAGE.
After electrophoresis, the proteins on the gels were stained with
Coomassie Blue R-250 or were transferred onto PVDF membranes
5
.2. Homology modeling and structure prediction
Residues of plant vacuolar invertases Bobfruct3 from Q118-
N631 were submitted to the Swiss-Model server (http://swissmod-
el.expasy.org) to perform sequence analysis, and a protein from A.
thaliana (PDB id: 2AC1) showing 47% sequence identity was thus
determined as the best template for model construction. The mod-
el returned from the server was further structurally aligned with
Arabidopsis AtcwINV1 D239A mutant in complex with sucrose
(
PDB id: 2QQU) with Pymol 0.99rc6 (DeLano Scientific, CA, USA)
in order to predict the theoretical position of sucrose when binding
with Bobfruct3. A final model was proposed after performing en-
ergy minimization with DeepView 3.7 (Guex and Peitsch, 1997).
for immunodetection using an anti-(His) antibody. Fractions con-
taining the recombinant invertase proteins were collected.
6
5.5. Acidic invertase assay and enzyme kinetics
5.3. Plasmid and site-directed mutagenesis
The reaction mixture contained 0.1 M sodium acetate, pH 5.0,
Plasmid pBoIT3, which was constructed by insertion of the cod-
0.1 M sucrose and an appropriate amount of enzyme. After incuba-
tion at 37 °C, the amount of reducing sugars (glucose and fructose)
produced was measured by the method of Somogyi-Nelson
(Nelson, 1944). For routine assay of invertase activity during puri-
ing region of Bobfruct3 cDNA into the EcoRI/XbaI site of the Pichia
expression vector pPICZ
B (Hsieh et al., 2006), was used for
expression of the wild-type recombinant invertase in yeast, and
a

T.-H. Chen et al. / Phytochemistry 70 (2009) 25–31
31
fication of the enzyme, the incubation time was 20 m. For determi-
nation of kinetic parameters, the reactions were performed for
various time periods to obtain initial velocities. The sucrose
concentrations used in kinetic analysis were 0, 20, 40, 60, 80,
Le Roy, K., Lammens, W., Verhaest, M., De Coninck, B., Rabijns, A., VanLaere, A., Van
den Ende, W., 2007. Unraveling the difference between invertases and fructan
exohydrolases:
a single amino acid (Asp-239) substitution transforms
Arabidopsis cell-wall invertase1 into a fructan 1-exohydrolase. Plant Physiol.
145, 616–625.
Matrai, J., Lammens, W., Jonckheer, A., Le Roy, K., Rabijns, A., Van den Ende, W., De
Maeyer, M., 2008. An alternate sucrose-binding mode in the E203Q Arabidopsis
invertase mutant: an X-ray crystallography and docking study. Proteins 71,
552–564.
Murayama, S., Handa, H., 2007. Genes for alkaline/neutral invertase in rice: alkaline/
neutral invertases are located in plant mitochondria and also in plastids. Planta
1
00, 150, 200 and 300 mM. One unit of activity was defined as
the amount of enzyme that catalyzed the formation of l mol of
l
reducing sugar from sucrose in 1 h at 37 °C. The kinetic constants
were determined from the Eadie-Hofstee plot using the program
Enzyme Kinetics!Pro (ChemSW Software, CA). The kcat values were
calculated on the basis of a molecular mass of 76,200 Da, as deter-
mined by gel filtration chromatography.
225, 1193–1203.
Nagem, R.A., Rojas, A.L., Golubev, A.M., Korneeva, O.S., Eneyskaya, E.V., Kulminskaya,
A.A., Neustroev, K.N., Polikarpov, I., 2004. Crystal structure of exo-inulinase
from Aspergillus awamori: the enzyme fold and structural determinants of
substrate recognition. J. Mol. Biol. 344, 471–480.
Acknowledgements
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Schutten, G.H., Dijkhuizen, L., 2004. Site-directed mutagenesis study of the
three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121.
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This work was supported by grants from the National Science
Council, the Republic of China (Taiwan).
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