Amino acids in conserved region II are crucial to substrate specificity, reaction velocity, and regioselectivity in the transglucosylation of Honeybee GH-13 α-glucosidases

Article abstract of DOI:10.1271/bbb.120473

Honeybees, Apis mellifera, possess three α-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-III preferred sucrose to maltose as substrate and formed only α-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the α-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) α-glucosidases and related enzymes.

Full text of DOI:10.1271/bbb.120473

Biosci. Biotechnol. Biochem., 76 (10), 1967–1974, 2012
Amino Acids in Conserved Region II Are Crucial to Substrate Specificity,
Reaction Velocity, and Regioselectivity in the Transglucosylation
of Honeybee GH-13 ꢀ-Glucosidases
*
Lukana NGIWSARA, Gaku IWAI, Takayoshi TAGAMI, Natsuko SATO, Hiroyuki NAKAI,
Masayuki OKUYAMA, Haruhide MORI, and Atsuo KIMURA
y
y
Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan
Received June 19, 2012; Accepted July 18, 2012; Online Publication, October 7, 2012
[doi:10.1271/bbb.120473]
Honeybees, Apis mellifera, possess three ꢀ-glucosidase
HBG-III.4,5) HBG-I is localized in the ventriculus.
HBG-II is present in ventriculus and the hemolymph.
HBG-III is secreted from the hypopharyngeal grand into
isozymes, HBG-I, HBG-II, and HBG-III, which belong
to glycoside hydrolase family 13. They show high
sequence similarity, but clearly different enzymatic
properties. HBG-III preferred sucrose to maltose as
substrate and formed only ꢀ-1,4-glucosidic linkages by
transglucosylation, while HBG-II preferred maltose and
formed the ꢀ-1,6-linkage. Mutation analysis of five
amino acids in conserved region II revealed that
Pro226-Tyr227 of HBG-III and the corresponding
Asn226-His227 of HBG-II were crucial to the discrim-
inating properties. By replacing these two amino acids,
the substrate specificities and regioselectivity in trans-
glucosylation were changed drastically toward the
other. The HBG-III mutant, Y227H, and the HBG-II
mutant, N226P, which harbor HBG-I-type Pro-His at
the crucial positions, resembled HBG-I in enzymatic
properties with marked increases in reaction velocities
on maltose and transglucosylation ratios. These findings
indicate that the two residues are determinants of the
enzymatic properties of glycoside hydrolase family 13
6
)
the nectar and is involved in honey formation. These
enzymes are clearly different in substrate specificity,
ability to catalyze transglucosylation, and regioselectiv-
ity in transglucosylation. HBG-I shows little or no
7
)
activity toward nigerose, isomaltose, or soluble starch.
It catalyzes transglucosylation at higher rates than the
others with ꢀ-1,4 regioselectivity to produce erlose
8)
(ꢀ-Glc-(1!4)-ꢀ-Glc-(1$2)-ꢁ-Fru) from sucrose. The
substrate specificity of HBG-II is so broad that gluco-
bioses having ꢀ-1,2, ꢀ-1,3, and ꢀ-1,6-glucosidic link-
ages, phenyl ꢀ-glucoside, and sucrose are substrates of
9
)
HBG-II besides maltooligosaccharides. HBG-I and
HBG-II exhibit non-Michaelis-Menten kinetics, which
has been considered allosteric behavior of the enzymes
7
,9)
even though they are monomeric enzymes.
instance, HBG-I cleaves the linkage of maltose, sucrose,
For
and 4-nitrophenyl ꢀ-glucoside in a mode of negative
)
7
cooperativity, whereas HBG-II shows positive coop-
9
)
(
GH-13) ꢀ-glucosidases and related enzymes.
erativity. HBG-III, on the other hand, follows typical
Michaelis-Menten kinetics. It preferentially hydrolyzes
sucrose. The values of kcat and Km of the enzyme on
several substrates are higher than those of HBG-I and
Key words: honeybee ꢀ-glucosidase; glycoside hydro-
lase family 13; substrate specificity; trans-
glucosylation
5
)
HBG-II. ꢀ-Glucosidase isozymes are also found in a
Japanese subspecies of eastern honeybee, Apis cerena
japnonica. One of them, JBG-I, displays non-Michaelis-
ꢀ
-Glucosidase (EC 3.2.1.20, ꢀ-D-glucoside glucohy-
drolase) catalyzes the hydrolysis of the non-reducing
1
0)
Menten type kinetic behavior, as observed for HBG-I.
In spite of the differences in enzymatic properties,
terminal ꢀ-glucosidic linkage of substrates to produce
,2)
ꢀ
-glucose.1 It catalyzes transglucosylation as well,
honeybee ꢀ-glucosidases belong to GH-13 and exhibit
1
1,12)
depending on the kinds of substrate and their concen-
trations. It is ubiquitous, and is found in a wide variety
of organisms, from microorganisms to higher mammals.
The enzymatic properties of ꢀ-glucosidases are diverse,
considerable sequence similarity to each other.
HBG-III shares 57% and 64% identical amino acids
with HBG-I and HBG-II respectively.
GH-13 includes a wide variety of ꢀ-glucoside-active
enzymes, which catalyze hydrolysis and transglucosy-
lation with retention of the anomeric configuration of the
1
)
particularly in substrate specificity. Some act prefera-
bly on sucrose and aryl ꢀ-glucosides, and others prefer
maltooligosaccharides as substrate. From the structural
point of view, most ꢀ-glucosidases fall into glycoside
hydrolase families 13 (GH-13) and 31 according to the
CAZy classification of glycoside hydrolases.2,3)
3
,13)
substrates.
For instance, ꢀ-amylase (EC 3.2.1.1),
pullulanase (EC 3.2.1.41), cyclodextrin glucanotransfer-
ase (EC 2.4.1.19), and neopullulanase (EC 3.2.1.135)
are GH-13 enzymes. Exo-glucoside-acting enzymes,
such as oligo-1,6-glucosidase (EC 3.2.1.10), dextran
glucosidase (EC 3.2.1.70), amylosucrase (EC 2.4.2.4),
The European honeybee (Apis mellifera) possesses
three ꢀ-glucosidase isozymes, HBG-I, HBG-II, and
y
To whom correspondence should be addressed. Haruhide MORI, Tel: +81-11-706-2816; Fax: +81-11-706-2808; E-mail: haru@abs.agr.
hokudai.ac.jp; Atsuo KIMURA, Tel/Fax: +81-11-706-2808; E-mail: kimura@abs.agr.hokudai.ac.jp
Present address: Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan
*
Abbreviations: GH-13, glycoside hydrolase family 13; HPAEC-PAD, high performance anion-exchange chromatography-pulsed amperometric
detection

1968
L. NGIWSARA et al.
sucrose phosphorylase (EC 2.4.1.7), and isomaltulose
synthase (EC 5.4.99.11), are also included in the family.
GH-13 enzymes share three domains, A, B, and C, in
protein structure. The active sites are in a cleft or a
Strains and plasmid. Escherichia coli DH5ꢀ (Stratagene, La Jolla,
CA) was used for plasmid preparation. Pichia pastoris GS115 (his4,
þ
Mut ) (Invitrogen, Carlsbad, CA) was used as host for the enzyme
production. For HBG-III production, a pPICZꢀA derivative (Invitro-
gen) harboring the cDNA coding for mature HBG-III (Ala18-Phe567)
between the EcoRI and NotI sites was used. The ORF encoded mature
HBG-III equipped with an 89-amino acid long ꢀ-factor signal
sequence, which was linked to its N-terminus by two amino acids,
E-F, at the joint. The HBG-II production plasmid was a pPICZA
derivative (Invitrogen) harboring the HBG-II cDNA between the
EcoRI and NotI sites of the plasmid. The start codon and the stop
codon of the HBG-II cDNA were placed adjacent to the EcoRI site and
the NotI site of pPICZA respectively.
pocket formed by ðꢁ=ꢀÞ barrel-shaped catalytic domain
8
A and protruding domain B inserted between the third
-stand and the third ꢀ-helix of domain A. The four
ꢁ
conserved sequence regions, I–IV, which are located
1
3)
in ꢁ-strands 3, 4, 5, and 7 of the ðꢁ=ꢀÞ barrel or their
8
C-terminal extensions, are deeply involved in catalysis
and substrate binding in that they form subsite ꢀ1. In
particular, region II and region III include Asp (Asp223
of both HBG-II and HBG-III) and Glu (Glu292 of HBG-
Mutagenesis. Site-directed mutagenesis was done using a PrimeS-
TAR Mutagenesis Kit (Takara Bio, Otsu, Japan). Specific oligonu-
cleotides were designed following the manufacturer’s instructions to
generate mutations, as follows: In HBG-III, Leu225 (CTG) ! Ile
1
2)
II and Glu286 of HBG-III), which are considered to
play the role of nucleophile and that of general acid/
base in the catalytic reaction, respectively. The residues
following the two catalytic amino acids are thought to
play important roles, in substrate binding at the þ1 and
(
ATC)/Val (GTA), Pro226 (CCT) ! Asn (AAC), Tyr227 (TAC) !
His (CAC), Ile228 (ATT) ! Met (ATG)/Leu (TTA), and Cys229
(
TGC) ! Phe (TTC). In HBG-II, Asn226 (AAC) ! Pro (CCA), and
1
4–18)
þ2 subsites in GH-13 enzymes.
His227 (CAC) ! Tyr (TAC). The PCR mixture was used directly in
the transformation of E. coli DH5ꢀ. Plasmids were prepared, and the
sequences of the ORFs and the connections were verified using a
BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM
In this study, to identify the amino acids responsible
for the specific enzymatic properties of HBGs, we
focused on the amino acid residues in region II (Fig. 1).
Using HBG-II and HBG-III, which have been success-
3
10 DNA sequencer (Applied Biosystems, Foster City, CA).
1
2)
fully produced heterologously in Pichia pastoris, the
residues were replaced by the corresponding amino
acids of the other isozymes. The site-directed mutation,
particularly at positions equivalent to Pro226-Tyr227 of
HBG-III, caused drastic changes in substrate specificity.
Their drastic change in regioselectivity in transglucosy-
lation is also described below, together with the nature
of the wild-type HBG-II and HBG-III.
Transformation of P. pastoris. The expression plasmids were
linearized by restriction digestion with SacI and used in the trans-
formation of P. pastoris GS115. Competent cells were prepared by the
method of Lin-Cereghino et al.,19) with some modifications. Briefly,
P. pastoris was grown in 10 mL of YPD (1% yeast extract, 2%
peptone, and 2% glucose) until A600 reached 1. First the cells were
ꢁ
incubated for 5 min at 30 C in 1.8 mL of BEDS solution (10 mM
Bicine-NaOH pH 8.3, 3% v/v ethylene glycol, 5% v/v dimethyl
sulfoxide, and 1 M sorbitol), supplemented with 0.2 mL of 1 M
dithiothreitol (DTT), and then resuspended in 50 mL of BEDS solution
without DTT. The linearized plasmid DNA was mixed with the
competent cells (55 mL) in a 1-mm gapped cuvette, and an electro-
poration pulse was applied at 1.5 kV, 200 ꢀ, and 25 mF (GenePulser II,
Bio-Rad, Hercules, CA). The transformants were screened on YPDS
agar plates (1% yeast extract, 2% peptone, 2% glucose, 1 M sorbitol,
and 2% agar) containing 100 mg/mL of zeocin. Single colonies were
streaked onto an MDH agar plate (1.34% yeast nitrogen base with
ammonium sulfate and without amino acids (Difco, Franklin Lakes,
NJ), 4 mg/mL of D-biotin, 2% glucose, and 1.5% agar) and stored
Materials and Methods
Substrates. Maltose and sucrose were purchased from Nacalai
Tesque (Kyoto, Japan). Kojibiose, nigerose, maltotriose, and isomal-
tulose were from Wako Pure Chemical Industries (Osaka, Japan).
Isomaltose and panose (ꢀ-Glc-(1!6)-ꢀ-Glc-(1!4)-Glc) were from
Tokyo Chemical Industry (Tokyo). Turanose (ꢀ-Glc-(1!3)-Fru) was
from ICN Biomedicals (Irvine, CA), maltulose was from Alfa Aesar
(Ward Hill, MA), and erlose was from Hayashibara Biochemical
Laboratories (Okayama, Japan).
ꢁ
at 4 C.
Enzyme preparation. P. pastoris transformants were grown in
BMGY medium (as MDH, but 1% glycerol replaced 2% glucose, and
with the addition of 1% yeast extract, 2% peptone, and 0.1 M potassium
HBG-III
HBG-II
HBG-I
223-
223-
230-
206-
176-
280-
ꢁ
phosphate buffer, pH 6.0) with shaking at 30 C for 24 h. Then the cells
were resuspended in BMMY (as BMGY, but 0.5% methanol replaced
ꢁ
Taka-amylase
B. sub -amylase
Sucrose hydrolase (SUH)
Sucrose phosphorylase (SucP) 192-
Amylosucrase
1% glycerol) and shaken continuously for 72 h at 30 C. The
recombinant enzymes were purified from the culture supernatant by
ammonium sulfate precipitation at 90% saturation and anion-exchange
chromatography on a DEAE Sepharose Fast Flow column (3 ꢂ 15 cm,
1
00 mL) and a 6-mL Resource-Q column (GE Healthcare, Little
286-
200-
194-
Chalfont, UK) with
a BioCAD 700E HPLC system (Applied
Trehaluose synthase (MutB)
SmDG
Biosystems). Both columns were pre-equilibrated with 20 mM sodium
phosphate buffer (pH 7.8). The protein samples were dialyzed against
the same buffer in advance and loaded onto the columns. The adsorbed
proteins were eluted with a linear gradient of 0 to 0.12 M NaCl in the
first chromatography, and with combined gradients of NaCl (0 to
Fig. 1. Partial Amino Acid Sequences of Conserved Region II.
The catalytic Asp, indicated by an asterisk, and the adjacent
amino acids are shown. Amino acids mutated in this study are
underlined. Pro226-Tyr227 of HBG-III and their equivalents are
indicated by bold-faced letters. The partial sequences, listed from
the top, are of ꢀ-glucosidase isozymes III, II, and I from Apis
0
.12 M) and pH (pH 7.8 to 6.0) in the second. In both chromatog-
raphies, active fractions were pooled. Using Centriprep YM-30
Millipore, Billerica, MA), the protein was concentrated and the
(
6
,12)
mellifera (HBG-III, HBG-II, and HBG-I respectively),
2
buffer was replaced with 20 mM sodium phosphate buffer (pH 7.8).
The homogeneity of the purified enzymes was confirmed by SDS–
PAGE20) on 8% polyacrylamide gel. Proteins were stained by Rapid
CBB Kanto (Kanto Chemical, Tokyo).
5)
Taka-amylase from Aspergillus oryzae, Bacillus subtilis ꢀ-
amylase, sucrose hydrolase SUH from Xanthomonas axonopo-
26)
27)
28)
dis, sucrose phosphorylase from Bifidobacterium adolescentis,
3
0)
amylosucrase from Neisseria polysaccharea, trehalulose synthase
3
1)
(MutB) from Psuedomonas mesoacidophila, and dextran gluco-
sidase (SmDG) from Streptococcus mutans.
Enzyme and protein assays. The protein concentrations were
determined by quantification of the various amino acids of protein
2
9)

Crucial Amino Acids for Specific Properties of GH-13 HBGs
1969
ꢁ
hydrolysate (6 N HCl at 110 C for 24 h) by the ninhydrin colorimetric
640 mM sodium hydroxide as isocratic mobile phase. The reaction
mixture was adequately diluted with water, and 10 mL was injected.
The reaction products were identified based on retention times and
were quantified from areas of peaks in the comparison with standard
carbohydrates (5–200 mM).
method using an amino acid analyzer (AminoTac JLC/500V, JEOL,
Tokyo).
Enzyme activity (1 U) was defined as the amount of enzyme that
ꢀ
1
catalyzes the formation of glucose at an initial rate of 2 mmol min
from 5.8 mM maltose under standard reaction conditions. The reaction
mixture (50 mL), composed of 5.8 mM maltose, 44 mM sodium acetate
buffer (pH 5.5), 0.022% Triton X-100, and an adequate concentration
Results
ꢁ
of the enzyme, was incubated at 37 C for 10 min. The enzymatic
reaction was terminated by the addition of 100 mL of 2 M Tris–HCl
Enzyme production and specific activity
Honeybee ꢀ-glucosidase isozymes II and III (HBG-II
and HBG-III) were heterologously produced in Pichia
pastoris. The ORFs encoded respectively the full-length
immature HBG-II containing the original N-terminal
signal sequence for secretion of the protein and mature
HBG-III (Ala18-Phe567 of immature HBG-III) with the
(
pH 7.0), which inhibited activity completely, and glucose concen-
1)
2
trations were measured by the glucose oxidase-peroxidase method
using Glucose C-II Test Wako (Wako Pure Chemical Industries).
The substrate specificities of the enzymes were analyzed by
measuring glucose-releasing velocities from various saccharides under
standard reaction conditions. The substrate concentrations were 1 mM
for HBG-II, and 5 mM for HBG-III. Isomaltose was 20 mM only for
HBG-III.
ꢀ
-factor signal sequence at the N-terminus and two
additional amino acids, Glu-Phe, in the joint part. After
2 h of induction, the enzymes were accumulated in 1L
7
Kinetic constant determination. The initial velocities of glucose
liberation from sucrose and maltose were determined over a wide
range of substrate concentrations up to 150 mM. The reactions were
done under standard assay conditions. The ranges of substrate
concentrations, in which a linear relationship was observed in
Lineweaver-Burk plots, were determined first, and then kinetic
parameters Km and kcat were determined by non-linear regression to
the Michaelis-Menten equation using KaleidaGraph software (Synergy
Software, Reading, PA). In each experiment, the enzymes were diluted
to an appropriate concentration to determine initial reaction velocities.
The sucrose concentrations used for the HBG-III derivatives were 10–
of the supernatant to 184 U for HBG-II and 83 U for
HBG-III, which were calculated to be 12 mg/L and
7.0 mg/L respectively by the specific activities of the
purified enzymes (Table 1).
Five amino acids in conserved region II, Leu225,
Pro226, Tyr227, Ile228, and Cys229, of HBG-III were
mutated. Seven single mutant HBG-III enzymes, L225I/
V, P226N, Y227H, I228L/M, and C229F, and four
double mutants, L225I-P226N, P226N-Y227H, and
Y227H-I228L/M, were generated. Counter-mutants of
HBG-II, N226P, H227Y, and N226P-H227Y, were
prepared. All the mutant enzymes were produced as
wild-type enzymes with similar yields (4.5 mg/L for
Y227H-I228L to 54 mg/L for P226N in the HBG-III
mutants, and 3.9 mg/L for H227Y to 12 mg/L for
N226P-H227Y in the HBG-II mutants). In the SDS–
PAGE analyses, all of the enzymes, purified from the
supernatant, showed a single protein band migrating to
the positions of 68 kDa and 76 kDa respectively, as
native HBG-III and HBG-II. The specific activities of
recombinant wild-type HBG-III and HBG-II for 5.8 mM
maltose were 5.90 U/mg and 7.75 U/mg respectively
1
50 mM (L225I, L225V, and L225I-P226N), 2–100 mM (the wild type,
P226N, P226N-H227Y, I228M, and C229F), 4–80 mM (I228L), 2–
0 mM (Y227H-I228L), 2–40 mM (Y227H), and 2–30 mM (Y227H-
I228M). Sucrose for the HBG-II derivatives was 5–60 mM (H227Y),
–20 mM (N226P), and 5–30 mM (wild-type HBG-II, N226P-H227Y).
6
5
Maltose for HBG-III was ranging in 4–150 mM (L225I, L225V,
I228L), 4–100 mM (L225I-P226N, P226N, I228M), 4–50 mM (wild-
type HBG-III, C229F), and 0.2–7 mM (Y227H, P226N-Y227H,
Y227H-I228L/M). For HBG-II, maltose of 6–150 mM (N226P-
H227Y), 6–30 mM (H227Y), 0.5–5 mM (N226P), and 0.5–3 mM
(wild-type HBG-II) was used. Maltotriose for HBG-III was 0.2–
15 mM (Y227H, Y227H-I228M), 0.5–20 mM (Y227H-I228L), 2–50 mM
(
(
C229F), 2–80 mM (wild type, L225I/V, P226N, I228M), 5–80 mM
I228L, L225I-P226N), and 2–150 mM (P226N-Y227H). For HBG-II,
maltotriose was 0.5-1.5 mM (N226P), 0.5–3 mM (wild type), 1–10 mM
N226P-H227Y), and 0.5–150 mM (H227Y). All experiments were
performed in triplicate, and averages and standard deviations were
(Table 1).
The introduction of mutations in HBG-III markedly
(
calculated.
affected the specific activity. Single mutant HBG-III,
P226N, and two double mutants containing the
P226!N mutation showed lower activity than the wild
type. In contrast, Y227H and the combination of it
with I228L/M exhibited drastic increases in activity
The transglucosylation ratio, rtg, was defined as previously:22)
rtg ¼ vtg=ðvtg þ vhÞ
where vtg and vh are initial reaction velocities of transglucosylation and
hydrolysis respectively.
(Table 1). Two single mutants of HBG-II, N226P and
H227Y, showed very high activities, but double mutant
N226P-H227Y decreased in specific activity to a level
similar to wild-type HBG-III (Table 1).
Analysis of transglucosylation by TLC. Maltose and sucrose
ꢀ
1
(
150 mM) were incubated with 22 mg mL of enzyme under standard
reaction conditions for 15 min to 6 h, and the reaction mixtures were
ꢁ
heated at 95 C for 3 min to terminate the reaction. The solution was
diluted 5-fold, and 2 mL was spotted onto a Silica gel 60 plate (Merck,
Darmstadt, Germany). Carbohydrates were developed by a solvent
system of nitromethane:1-propanol:water (2:5:1.5, v/v), and were
visualized with a methanol solution containing 0.03% 1-naphthol and
Substrate specificity
The substrate preferences of the enzymes for
disaccharides were investigated (Fig. 2). Wild-type
HBG-III showed high preference for sucrose with the
highest initial reaction velocity among the substrates at
5 mM. Maltose was consumed with 24% of the velocity
of sucrose. Kojibiose, nigerose, isomaltose, turanose,
and maltulose were poor substrates. Wild-type HBG-II,
on the other hand, exhibited broad substrate specificity
at a fixed concentration of 1 mM (Fig. 2). The best
substrate for HBG-II was maltose, with 2.3-fold higher
initial velocity than sucrose. Kojibiose, isomaltose,
turanose, and isomaltulose were catalyzed as fast as
5% (w/v) sulfuric acid and heating.
Analysis of transglucosylation by HPAEC-PAD. The reaction
products of 150 mM maltose and sucrose were analyzed by high
performance anion-exchange chromatography-pulsed amperometric
detection (HPAEC-PAD). The enzyme reaction was done under
ꢁ
standard conditions, and was stopped by heating at 100 C for 3 min.
The reaction time was 3 min to 5 h. Quantification was done by
HPAEC-PAD using a Dionex ICS-3000 system equipped with a
CarboPac PA-1 column (ꢂ4 ꢂ 250 mm; Dionex, Sunnyvale, CA).
ꢀ
1
ꢁ
Elution was performed at a flow rate of 0.8 mL min at 25 C using

1970
L. NGIWSARA et al.
Table 1. Kinetic Parameters of Hydrolysis at Low Concentrations of Substrates
Sucrose
Maltose
Maltotriose
Specific
activitya
Enzyme
kcat
Km
kcat=Km
ꢀ1 ꢀ1
kcat
ꢀ1
Km
kcat=Km
ꢀ1 ꢀ1
kcat
ꢀ1
Km
kcat=Km
(U/mg)
ꢀ1
ꢀ1
ꢀ1
(
s
)
(mM)
(s mM
)
(s
)
(mM)
(s mM
)
(s
)
(mM)
(s mM
)
HBG-III
Wild type
5.90
7.45
6.25
1.50
222 ꢃ 10
332 ꢃ 3
42:3 ꢃ 2:8
73:6 ꢃ 2:1
49:6 ꢃ 2:2
13:9 ꢃ 3:1
17:5 ꢃ 1:9
37:2 ꢃ 1:7
34:6 ꢃ 0:5
17:0 ꢃ 1:5
20:3 ꢃ 1:5
5:27 ꢃ 0:12
4:51 ꢃ 0:1
47:2 ꢃ 1:5
101 ꢃ 2
14:0 ꢃ 1:7
34:7 ꢃ 1:7
22:8 ꢃ 0:9
30:8 ꢃ 0:9
2:43 ꢃ 0:03
23:0 ꢃ 0:2
25:0 ꢃ 0:6
14:8 ꢃ 0:5
74:2 ꢃ 2:7
3:40 ꢃ 0:31
2:91 ꢃ 0:08
2:23 ꢃ 0:05
133 ꢃ 1
257 ꢃ 6
177 ꢃ 6
8:56 ꢃ 0:16
19:2 ꢃ 0:7
14:6 ꢃ 1:1
6:43 ꢃ 0:58
1:63 ꢃ 0:14
9:69 ꢃ 0:09
10:0 ꢃ 0:7
8:12 ꢃ 0:2
10:8 ꢃ 0:6
15:6 ꢃ 0:4
13:4 ꢃ 0:3
12:2 ꢃ 0:6
L225V
L225I
P226N
159 ꢃ 3
3:20 ꢃ 0:09
1:86 ꢃ 0:34
5:78 ꢃ 0:49
6:19 ꢃ 0:05
5:24 ꢃ 0:12
8:61 ꢃ 1:05
4:45 ꢃ 0:43
50:8 ꢃ 0:8
6:19 ꢃ 0:4
118 ꢃ 2
24:8 ꢃ 1:1
100 ꢃ 5
0:201 ꢃ 0:012 18:9 ꢃ 1:4
2:98 ꢃ 0:47
72:2 ꢃ 5:8
13:1 ꢃ 0:1
9:77 ꢃ 0:65
13:2 ꢃ 0:8
5:97 ꢃ 0:25
Y227H
I228L
I228M
56.5
4.55
3.73
12.0
1.30
48:3 ꢃ 0:4
1:65 ꢃ 0:04
1:04 ꢃ 0:05
4:68 ꢃ 0:44
117 ꢃ 4
127 ꢃ 0
231 ꢃ 12
181 ꢃ 2
37:6 ꢃ 0:5
26:1 ꢃ 0:9
69:0 ꢃ 6:3
24:0 ꢃ 1:4
97:4 ꢃ 0:4
107 ꢃ 4
C229F
L225I-P226N
145 ꢃ 14
89:8 ꢃ 2:2
0:324 ꢃ 0:019 64:4 ꢃ 2:0
P226N-Y227H 0.800 0:522 ꢃ 0:076 15:5 ꢃ 1:5 0:0341 ꢃ 0:0059 0:325 ꢃ 0:027 0:780 ꢃ 0:240 0:452 ꢃ 0:118 0:453 ꢃ 0:021 0:646 ꢃ 0:093 0:711 ꢃ 0:071
Y227H-I228L 66.5
Y227H-I228M 34.8
95:3 ꢃ 5:8
73:3 ꢃ 6:1
17:0 ꢃ 1:9
12:2 ꢃ 3:0
5:67 ꢃ 0:66
6:55 ꢃ 2:20
166 ꢃ 3
106 ꢃ 8
4:13 ꢃ 0:29
2:78 ꢃ 0:26
40:4 ꢃ 2:1
38:4 ꢃ 1:6
183 ꢃ 4
111 ꢃ 3
2:32 ꢃ 0:02
1:47 ꢃ 0:07
78:9 ꢃ 2:1
75:4 ꢃ 5:7
HBG-II
Wild type
N226P
7.75
20.6
15.4
87:6 ꢃ 12:5 30:6 ꢃ 5:7
2:91 ꢃ 0:34
14:8 ꢃ 0:5
4:65 ꢃ 0:36
9:34 ꢃ 0:63
37:7 ꢃ 2:1
108 ꢃ 9
4:02 ꢃ 0:37
2:74 ꢃ 0:19
26:4 ꢃ 2:9
24:8 ꢃ 0:2
9:43 ꢃ 0:41
39:4 ꢃ 2:1
5:57 ꢃ 0:18
3:93 ꢃ 0:13
87:2 ꢃ 3:3
137 ꢃ 8
3:82 ꢃ 0:14
2:16 ꢃ 0:15
16:9 ꢃ 1:1
14:5 ꢃ 0:9
22:8 ꢃ 0:1
63:2 ꢃ 1:6
14:5 ꢃ 1:1
14:0 ꢃ 0:3
199 ꢃ 3
211 ꢃ 6
231 ꢃ 11
13:4 ꢃ 0:4
45:6 ꢃ 2:2
24:9 ꢃ 2:7
H227Y
146 ꢃ 11
97:4 ꢃ 2:3
244 ꢃ 3
N226P-N227Y 5.85
203 ꢃ 13
Parameters were determined in a range of substrate concentrations that gave a linear relationship between 1/s and 1/v.
aHalf of glucose-releasing velocity on 5.8 mM maltose.
30
25
20
15
10
5
0
1
2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
wild type
P226N Y227H P226N-Y227H wild type N226P H227Y N226P-H227Y
HBG-III
HBG-II
Fig. 2. Substrate Preference of Wild-Type and Mutant HBG-III and HBG-II.
Initial glucose-releasing velocities for various disaccharides are shown. 1, kojibiose; 2, nigerose; 3, maltose; 4, isomaltose; 5, sucrose;
, turanose; 7, maltulose; 8, isomaltulose. The substrate concentration was 5 mM for HBG-III and 1 mM for HBG-II.
6
sucrose. Nigerose and maltulose were also catalyzed,
with 20% and 10% of the initial rate of maltose
respectively.
with decreased velocities for isomaltose, and isomaltu-
lose by 4.3- and 1.7-fold respectively. In the mutants
related to H227Y, the velocities on maltose were lower,
and the sucrose-acting rates were increased. In partic-
ular, N226P-H227Y catalyzed sucrose with 158% of the
velocity of the wild type, while all of the other rates
decreased to 44% or less. For the double mutant, sucrose
was the most preferred substrate.
The sucrose preference of wild-type HBG-III was
considerably changed by the mutations (Fig. 2). The
P226N mutant showed significantly reduced reaction
rates for sucrose and maltose, while its rates for turanose
and isomaltose increased 2.4- and 1.8-fold respectively,
and hence turanose was a better substrate than maltose
for the P226N mutant. The Y227!H mutation reduced
the activity for sucrose to 57%, but caused a drastic
increase in the reaction rates for maltose, isomaltose,
and turanose, by 2.9- to 6.0-fold. At a concentration of
Kinetic parameters
The kinetic parameters for sucrose, maltose, and
maltotriose were determined at low ranges of substrate
concentrations, giving an apparently linear relationship
between 1/v and 1/[s] (Table 1). Maltotriose was the
best substrate for both wild-type HBG-III and HBG-II
among the three substrates with respect to the highest
kcat=Km. Wild-type HBG-III preferred sucrose to mal-
5
mM, maltose was the best substrate for Y227H. Double
mutant P226N-Y227H showed marked reduction of all
rates, but preferred maltose as substrate at a 5-times
higher reaction velocity than sucrose. The other dis-
accharides tested were poor substrates for the three
HBG-III mutants as observed for the wild type.
The mutations of HBG-II also affected substrate
specificity (Fig. 2). N226P showed high preference for
maltose, with 3.5-fold elevated velocity for it, together
tose. Sucrose was catalyzed with the highest k , but the
cat
Km for it was also very high. On the other hand, wild-
type HBG-II preferred maltose to sucrose with respect to
k =K , due mainly to a K as low as that for
cat
m
m
maltotriose. Wild-type HBG-II showed lower kcat and

Crucial Amino Acids for Specific Properties of GH-13 HBGs
1971
sucrose
glc fru
maltose
A
B
C
D
glc
erlose
maltotriose
G1
G2
G3
G4
fru
glc
sucrose
III-10
erlose
III-10
III-6
III-3
III-6
III-3
G5
G6
panose
S
0 15 180
Reaction (min)
theanderose
II-60
II-30
II-6
II-30
II-6
G1
G2
glc
maltose
panose maltotriose
erlose
G3
G4
maltotriose
II(PY)-60
II(PY)-30
II(PY)-6
G5
G6
II(PY)-30
II(PY)-6
2
3
4
5
2
3
4
5
6
S
0 15180 360
Reaction (min)
Retention time (min)
Retention time (min)
Fig. 3. Analyses of Transglucosylation Products.
A and B, TLC analysis of products on 150 mM sucrose (A) and maltose (B) during reactions by wild-type HBG-III. S, standards; G1–G6,
glucose and maltooligosaccharides to maltohexaose. Products are indicated at the right. C and D, HPAEC-PAD analysis of transglucosylation
products on 150 mM sucrose (C) and maltose (D). The enzymes used and reaction times are indicated at the right side of each chromatogram. III,
wild-type HBG-III; II, wild-type HBG-II; II (PY), HBG-II mutant, N226P-H227Y.
Km for all three substrates than wild-type HBG-III.
Particularly, the kcat for sucrose and the K for maltose
were 2.5- and 3.5-fold lower respectively than those for
wild-type HBG-III.
The kinetic parameters of the wild-type enzymes were
drastically changed by mutation (Table 1). Among the
HBG-III mutants, only L225V showed higher kcat for all
three substrates than the wild type, but the mutant
showed slight reductions in kcat/Km values due to the
mutant for maltose and maltotriose were very similar to
those of wild-type HBG-III. No increase in Km was
observed for the single mutant N226P. Hence, it showed
kcat=Km elevated by 5.1-fold for sucrose and 4.2-fold for
maltose.
m
Reaction at a high substrate concentration
The reactions products on 150 mM sucrose and
maltose were analyzed (Fig. 3). The TLC results clearly
indicated that wild-type HBG-III formed transglucosy-
lation products migrating as erlose and maltotriose at a
relatively early stage of the reaction on sucrose and
maltose respectively. Both products were degraded by
further incubation (Fig. 3A and B). The products were
analyzed also with HPAEC-PAD (Fig. 3C and D).
HBG-III produced exclusively ꢀ-1,4-transglucosylation
products from sucrose and maltose. On the other hand,
HBG-II formed predominantly ꢀ-1,6-transglucosylation
products, theanderose (ꢀ-Glc-(1!6)-ꢀ-Glc-(1$2)-ꢁ-
Fru) and panose, from the substrates respectively,
increase in K . Most of the other HBG-III mutants,
m
particularly those harboring the P226!N mutation,
showed lower k values for sucrose. However, they
cat
also showed lower Km values for sucrose, and the
kcat=Km values of HBG-III mutants were in a range of
3
5% (P226N) and 163% (C229F) as compared with the
wild type. P226N-H227Y exceptionally showed very
low kcat and kcat=Km values. For maltose and maltotriose,
P226N and its derivatives showed decreases in kcat=Km,
mainly due to a reduction in kcat. The mutants containing
the Y227!H mutation displayed elevated kcat=Km, 11 to
2
3)
1
4-fold for maltose and 5-fold for maltotriose, due to the
judging by a comparison with authentic substances
increase in kcat and the drastic reduction in K , except
m
in the TLC analyses (data not shown) and the HPAEC-
PAD analyses (Fig. 3C and D). Their distinctive
regioselectivity in transglucosylation was changed by
introducing mutations in the amino acids at 226 and 227.
The HBG-II mutant, N226P-H227Y, produced erlose
and maltotriose from the substrates (Fig. 3C and D). In
particular, the formation of erlose was much higher than
that of theanderose. The accumulation of the trans-
glucosylation products over a long reaction period is
summarized in Fig. 4. HBG-III and HBG-II produced ꢀ-
1,4- and ꢀ-1,6-transglucosylation products respectively,
to approximately 5–6 mM under the reaction conditions,
but the HBG-III mutants, P226N and P226N-Y227H,
catalyzed not only ꢀ-1,4-transglucosylation but also
ꢀ-1,6-transglucosylation to produce panose and thean-
for the double mutant, P226N-Y227H. The reduced Km
values of Y227H for the three substrates were even
lower than those of wild-type HBG-II. Moreover, the
kcat of the mutant for sucrose was reduced from the wild-
type HBG-III, but still higher than that of HBG-II.
Hence, Y227H and its derivatives catalyzed the three
substrates with higher kcat=Km than wild-type HBG-III
and HBG-II.
On the other hand, all the HBG-II mutants showed
increased k , by 1.6-fold (N226P for maltotriose) to
cat
3
.9-fold (H227Y for maltose). However, the Km values
of H227Y and the double mutant increased, as observed
for wild-type HBG-III, which resulted in decreases in
kcat=Km. For instance, the kcat=Km values of the double

1972
L. NGIWSARA et al.
A
B
E
C
F
D
G
H
K
I
J
L
Fig. 4. Progress Curves for Transglucosylation Products.
Products of the reaction on 150 mM maltose (A, B, C, G, H, and I) and sucrose (D, E, F, J, K, and L) were quantified by HPAEC-PAD analysis.
HBG-III derivatives (A and D, wild type; B and E, P226N; C and F, P226N-Y227H) and HBG-II derivatives (G and J, wild type; H and K,
N226P; I and L, N226P-H227Y) were used as enzymes.
maltotriose;
panose;
erlose;
theanderose.
derose. On the other hand, accumulation of ꢀ-1,4-
transglucosylation was observed in the HBG-II mutants,
N226P and N226P-H227Y. In particular, the double
mutant accumulated erlose much more than theanderose
with sucrose as substrate (Fig. 4L).
values of Y227H and its combination with I228L on
150 mM maltose were also higher than wild-type HBG-
III. The mutants containing the Y227!H mutation
exhibited very high rtg values on maltose. The values of
rtg on sucrose of the mutants were lower, but reached
66–74%, significantly higher than that of the wild type
(43%). P226N and its derivatives catalyzed with low vag.
Mutants P226N and P226N-Y227H, however, catalyzed
not only ꢀ-1,4-transglucosylation, but also ꢀ-1,6-trans-
glucosylation. In particular, P226N-Y227H catalyzed ꢀ-
1,6-transglucosylation from both substrates. In addition,
the rate of ꢀ-1,6-transglucosylation on maltose of the
mutant was even higher than that of ꢀ-1,4-transgluco-
sylation (Table 2).
Enhancement of the reaction velocities of all three
HBG-II mutants was observed not only in the release of
glucose, but also in aglycone release (Table 2). The
values of vag increased by 9.3-fold at maximum. All
three mutants catalyzed ꢀ-1,4-transglucosylation from
sucrose as substrate, while wild-type HBG-II catalyzed
predominantly ꢀ-1,6-transglucosylation. The N226!P
mutation particularly altered regioselectivity, even on
maltose. Single mutant N226P increased rtg (41% on
By measuring those products with HPAEC-PAD, the
initial reaction rates of hydrolysis (vh), transglucosyla-
tion (v ), and aglycone release (v ¼ v þ v ) at very
tg
ag
h
tg
early stages of the reaction, when substrate consumption
was 0.35% or less, were determined (Table 2). HBG-III
produced exclusively ꢀ-1,4-transglucosylation products
from both substrates, and HBG-II mainly catalyzed ꢀ-
1
,6-transglucosylation, and produced theanderose and
panose from sucrose and maltose respectively, even
judging from the initial velocities. Wild-type HBG-III
showed much higher vag than HBG-II. The transgluco-
sylation ratios (rtg) of HBG-III were 43% and 67% for
sucrose and maltose respectively, higher than those of
HBG-II (22% and 59%).
A variety of reaction velocities and transglucosylation
ratios were observed in the reactions of the HBG-III
mutants on 150 mM substrates (Table 2). L225V showed
higher vag than the wild type for both substrates. The vag

Crucial Amino Acids for Specific Properties of GH-13 HBGs
1973
Table 2. Reaction Velocities on 150 mM Substrates and Transglucosylation Ratios
Sucrose
Maltose
Enzyme
vag
v4tga
v6tgb
vag
v4tga
v6tgb
rtg
rtg
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
)
(
mmol min mg
)
(%)
(mmol min mg
(%)
HBG-III
Wild type
L225V
L225I
198
256
196
85.5
86.2
82.1
5.7
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.8
nd
nd
43
34
42
29
69
33
7
45.0
30.4
86.0
58.1
1.8
nd
nd
nd
1.2
nd
nd
nd
nd
nd
0.5
nd
nd
68
69
126
81.0
4.7
72
63
P226N
Y227H
19.3
97.2
114
87.3
213
67.0
37.7
5.7
63.4
22.6
67.5
59.7
23.2
0.8
63.2
7.3
100
32
I228L
I228M
C229F
29.0
26.9
21.5
0.3
43
45
45.5
28.0
1.3
21
33
74
66
73
L225I-P226N
P226N-Y227H
Y227H-I228L
Y227H-I228M
83.5
2.8
93
97
72.1
81.7
47.8
59.5
95.0
34.3
95.0
26.0
100
76
HBG-II
Wild type
N226P
H227Y
41.2
nd
8.9
17.6
10.0
1.7
22
41
9
8.2
35.9
76.8
59.2
nd
4.9
21.4
28.6
9.1
59
72
37
31
105
29.4
6.7
4.3
nd
152
143
N226P-H227Y
31.0
30
9.3
a
Velocity of ꢀ-1,4-transglucosylation yielding erlose and maltotriose from sucrose and maltose respectively.
Velocity of ꢀ-1,6-transglucosylation yielding theanderose and panose from the substrates respectively.
nd, Not determined due to velocities too low.
b
sucrose, 72% on maltose), while the two others
containing H227!Y mutation decreased in these ratios
significantly.
The crucial amino acids were also effective as HBG-I
determinants. Two mutants, Y227H of HBG-III and
N226P of HBG-II, which shared Pro-His at the positions
1
2)
10)
with HBG-I and JBG-I indicated in Fig. 1, exhib-
7
,8)
Discussion
ited enzymatic properties similar to HBG-I.
Both
mutants showed high activity particularly on maltooli-
gosaccharides (Table 1), and the substrate specificity
toward maltose was very high (Fig. 2). The ꢀ-1,6-
regioselectivity of HBG-II was changed to ꢀ-1,4 by the
N226!P mutation (Fig. 4 and Table 2). In addition,
drastic increases in rtg were observed in both mutant
enzymes. All of the features the mutants acquired are
ꢀ
-Glucosidase isozymes from the European honeybee
exhibit many differences in substrate specificity, trans-
glucosylation, and other kinetic behaviors. HBG-II,
purified from honeybees, has broad substrate specificity
with activity lower than HBG-I and HBG-III.9) In
contrast, HBG-III acts specifically on sucrose, maltose,
and maltotriose with high kcat and Km. Recombinant
wild-type HBG-II and HBG-III, investigated in this
study, exhibited characteristics similar to those of the
native enzymes. The kinetic parameters, determined at
low substrate concentrations, were in good agreement
with those previously reported. In this study, rate
determination at a high substrate concentration, 150 mM,
revealed the high transglucosylation ability of HBG-III
and the regioselectivity of the enzymes in transgluco-
sylation.
The distinctive differences between HBG-II and
HBG-III in substrate specificity, kinetic parameters,
and transglucosylation were drastically changed by
introducing single and double mutations in conserved
region II, particularly at Pro226-Tyr227 of HBG-III and
the equivalent. By replacement of the corresponding
Asn226-His227 of HBG-II by Pro-Tyr, the lower kcat
and Km of HBG-II were elevated to the level of HBG-III
or even higher. The kcat=Km values of the resulting
double mutant on maltose and maltotriose were in a
good agreement with those of HBG-III (Table 1). The
regioselectivities of HBG-III and HBG-II were altered
toward the other by exchanging residues (Fig. 4 and
Table 2). Therefore, it can be concluded that Pro226-
Tyr227 of HBG-III and the equivalents of HBG-II are a
determinant distinguishing the two enzymes.
5
)
7,8)
similar to those of HBG-I.
The roles of the amino acids in enzymatic function
can be concluded to be as follows: His227 and Tyr227
are involved in substrate preferences for maltose and
sucrose respectively. The residues at position 226 are a
determinant of regioselectivity in transglucosylation.
Pro226 and Asn226 respectively are associated with ꢀ-
1,4- and ꢀ-1,6-transglucosylation. A high transglucosy-
lation ratio can be achieved in mutants harboring
Pro226-His227, which resemble HBG-I in high trans-
glucosylation ratios and amino acids at the correspond-
1
2)
8
,12)
ing positions.
The residues corresponding to Pro226-Tyr227 in
HBG-III are in conserved sequence region II of the
1
1–14)
GH-13 enzymes.
Conserved regions I–IV include
seven invariant amino acids essential to activity. There
are variations in the other amino acids in these regions,
and some of them are responsible for the specific
1
4)
properties of enzymes.
The residue C-terminally
adjacent to the catalytic Asp in region II, corresponding
to Ala224 of HBG-III and HBG-II, is a determinant of
1
6,18)
the substrate specificity of GH-13 enzymes.
The
two following amino acid residues, equivalent to
Pro226-Tyr227 of HBG-III, have been pointed out as
1
4)
the characteristic signature of GH-13 enzymes. Ac-
cording to a recent subfamily classification of GH-13

1974
L. NGIWSARA et al.
enzymes,24) the sequence motif DAxxH (corresponding
Asp223-Tyr227 of HBG-III as shown in Fig. 1) is often
found in subfamilies 1, 2, 5, 15, 17, 19, 24, 27, 28, 31,
4) Takewaki S, Chiba S, Kimura A, Matsui H, and Koike Y, Agric.
Biol. Chem., 44, 731–740 (1980).
5
)
Nishimoto M, Kubota M, Tsuji M, Mori H, Kimura A, Matsui
H, and Chiba S, Biosci. Biotechnol. Biochem., 65, 1610–1616
3
2, and 36, which include ꢀ-1,4-linkage-specific en-
(
2001).
zymes such as ꢀ-amylases, cyclomaltodextrin glucano-
transferases, and ꢀ-glucosidases. Most ꢀ-amylases and
cyclomaltodextrin glucanotransferases have dipeptide
Lys-His at the C-terminal end of the motif. The
corresponding His210 of Taka-amylase makes a hydro-
gen bond with 2OH of the 6-deoxyglucose moiety of an
acarbose derivative at subsite þ1 in a complex struc-
ture.2 The equivalent His180 of Bacillus subtilis ꢀ-
amylase is also involved in the hydrogen bond at the
same position.2 Our biochemical data clearly indicate
the involvement of the corresponding His227 of HBG-II
in the binding of maltooligosaccharides. The preference
of HBG-II for maltooligosaccharides is most likely due
to the hydrogen-bond formation of His227 with a
glucose residue of maltooligosaccharides at subsite þ1.
We found that the Tyr residue situated at position 227
in HBG-III is involved in sucrose preference with drastic
increases in both kcat and Km. The sequence motif
DAxxY/F (corresponding to Asp223-Tyr227 of HBG-
III) frequently occurs in GH-13 enzymes belonging to
subfamilies 4 (sucrose hydrolases), 16 (trehalose syn-
thases), 17 (ꢀ-glucosidases), 18 (sucrose phosphory-
lases), and 23 (ꢀ-glucosidases). The protein structures of
Tyr-harboring enzymes, sucrose hydrolase derived from
6
)
Kubota M, Tsuji M, Nishimoto M, Wongchawalit J, Okuyama
M, Mori H, Matsui H, Surarit R, Svasti J, Kimura A, and Chiba
S, Biosci. Biotechnol. Biochem., 68, 2346–2352 (2004).
Kimura A, Takewaki S, Matsui H, Kubota M, and Chiba S,
J. Biochem., 107, 762–768 (1990).
7)
8)
9)
Kimura A, Yoshida-Kitahara F, and Chiba S, Agric. Biol.
Chem., 51, 1859–1864 (1987).
Takewaki S, Kimura A, Kubota M, and Chiba S, Biosci.
Biotechnol. Biochem., 57, 1508–1513 (1993).
5)
10) Wongchawalit J, Yamamoto T, Nakai H, Kim YM, Sato N,
Nishimoto M, Okuyama M, Mori H, Saji O, Chanchao C,
Wongsiri S, Surarit R, Svasti J, Chiba S, and Kimura A, Biosci.
Biotechnol. Biochem., 70, 2889–2898 (2006).
6)
1
1) Ohashi K, Sawata M, Takeuchi H, Natori S, and Kubo T,
Biochem. Biophys. Res. Commun., 221, 380–385 (1996).
1
2) Nishimoto M, Mori H, Moteki T, Takamura Y, Iwai G,
Miyaguchi Y, Okuyama M, Wongchawalit J, Surarit R, Svasti J,
Kimura A, and Chiba S, Biosci. Biotechnol. Biochem., 71,
1
3) Svensson B, Plant Mol. Biol., 25, 141–157 (1994).
4) MacGregor EA, Jane cˇ ek S, and Svensson B, Biochim. Biophys.
Acta, 1546, 1–20 (2001).
703–1716 (2007).
1
1
ˇ
1
5) Inohara-Ochiai M, Nakayama T, Goto R, Nakao M, Ueda T, and
Shibano Y, J. Biol. Chem., 272, 1601–1607 (1997).
16) Yamamoto K, Nakayama A, Yamamoto Y, and Tabata S, Eur.
J. Biochem., 271, 3414–3420 (2004).
1
7) Saburi W, Mori H, Saito S, Okuyama M, and Kimura A,
Biochim. Biophys. Acta, 1764, 688–698 (2006).
2
7)
Xanthomonas campestris and sucrose phosphorylase
2
8)
18) Tsujimoto Y, Tanaka H, Takemura R, Yokogawa T, Shimonaka
A, Matsui H, Kashiwabara S, Watanabe K, and Suzuki Y,
J. Biochem., 142, 87–93 (2007).
from Bifidobacterium adolescentis, indicate that the
side chain of Tyr has no direct contact with a fructose
moiety of sucrose. It is accommodated on the far side of
1
9) Lin-Cereghino J, Wong WW, Xiong S, Giang W, Luong LT, Vu
J, Johnson SD, and Lin-Cereghino GP, BioTechniques, 38, 44–
48 (2005).
2
7)
the pocket from the equivalent residues, as observed
for the isomaltotriose-binding mode of dextran glucosi-
dase.29) In contrast, maltooligosaccharides are expected
20) Laemmli UK, Nature, 227, 680–685 (1970).
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2
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bulky Tyr residue at this position probably causes steric
hindrance in the binding of substrates, and substrate-
binding in a distorted manner might increase the energy
state of the Michaelis complex, resulting in increases in
Km and kcat.
2
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2
2
2
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1
Acknowledgment
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We thank Mr. Tomohiro Hirose of Instrumental
Analysis Division, the Equipment Management Center,
Creative Research Institution, Hokkaido University for
amino acid analysis.
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