Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Article abstract of DOI:10.1021/jf073320b

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wildtype MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75°C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields.

Full text of DOI:10.1021/jf073320b

5628 J. Agric. Food Chem. 2008, 56, 5628–5633
Identification of the Essential Catalytic Residues and
Selectivity-Related Residues of
Maltooligosyltrehalose Trehalohydrolase from the
Thermophilic Archaeon Sulfolobus solfataricus
ATCC 35092
TSUEI-YUN FANG,*,† WEN-CHI TSENG,‡ TONG-YUAN SHIH,† AND
†
MEI-YING WANG
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, and Department of
Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan
Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the
R-1,4-glucosidic linkage next to the R-1,1-linked terminal disaccharide of maltooligosyltrehalose.
Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic
residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were
constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified
D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-
type MTHase, suggesting that these three residues are essential catalytic residues. Compared with
wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios,
which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose
formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while
W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried
out at 75 °C and wild-type and mutant MTHases were, respectively, used with isoamylase and
maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of
trehalose formation were inversely correlated to the peak trehalose yields.
KEYWORDS: Maltooligosyltrehalose trehalohydrolase; selectivity; mutation; trehalose; substrate specific-
ity; starch; Sulfolobus
INTRODUCTION
produced from starch by thermophilic maltooligosyltrehalose
synthase (MTSase) and MTHase combined with a debranching
enzyme. However, both MTSase and MTHase catalyze a side
hydrolysisreactionthatwoulddecreasetheyieldoftrehalose(3–5).
This suggests that engineering the selectivity of both MTSase
and MTHase to decrease the side hydrolysis reactions would
increase the trehalose yield. Our previous study also showed
that F405Y MTSase, which had a decreased reaction of the side
hydrolysis, had resulted in a higher yield of trehalose production
from starch when it replaced wild-type MTSase (6, 7).
MTHase from Sulfolobus solfataricus KM1 contains three
major domains (A, C, and E) and two subdomains (B and D),
of which domain A is the (ꢀ/R)8 barrel catalytic domain with
B and D subdomains as insertions into this barrel (8). According
to the information from CAZy, a Web resource on glycosyl
hydrolases at http://afmb.cnrs-mrs.fr/CAZY/, MTHase has been
previously classified in family 13 of glycosyl hydrolases. The
sequence and structural analysis suggested that domain A
contains three catalytic residues (Asp252, Glu283, and Asp377)
that are conserved in family 13 of glycosyl hydrolases (8).
Asp252 and Glu283 likely act as the catalytic base and acid,
Maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141,
MTHase, also known as glycosyltrehalose trehalohydrolase,
glycosyltrehalose-hydrolyzing enzyme, and trehalose-forming
enzyme) mainly catalyzes the release of trehalose by cleaving
the R-1,4-glucosidic linkage next to the R-1,1-linked terminal
disaccharide of maltooligosyltrehalose. Trehalose (R-D-glucopy-
ranosyl-R-D-glucopyranoside) is a nonreducing sugar formed
from two glucose (G1) units joined by an R-1,1 linkage. The
sugar has a remarkable ability to protect proteins and membranes
from dryness, heat, and osmotic changes and has been approved
as a novel food ingredient under the GRAS terms in the United
States and the European Union (1, 2). It is gaining more
applications in many different areas, such as a sweetener
component, a preservative or stabilizer for food, cosmetics,
vaccines, medicines, cells, and organs (2). Trehalose can be
* Corresponding author. Telephone: 886-2-2462-2192, ext. 5141.
Fax: 886-2-2462-2586. E-mail: tyfang@mail.ntou.edu.tw.
† National Taiwan Ocean University.
‡ National Taiwan University of Science and Technology.
10.1021/jf073320b CCC: $40.75  2008 American Chemical Society
Published on Web 06/19/2008

Essential Catalytic and Selectivity-Related Residues of MTHase
J. Agric. Food Chem., Vol. 56, No. 14, 2008 5629
Figure 1. Stereoview of the MTHase monomer from S. solfataricus KM1 (8), illustrating the active site. Residues W215, D252, A256, E283, Y325, F352,
R353, and D377 in S. solfataricus KM1 MTHase are equivalent to residues W218, D255, A259, E286, Y328, F355, R356, and D380 in S. solfataricus
ATCC 35092 MTHase. The figure was prepared with PyMOL (9).
albumin (BSA) were supplied by Sigma. Q-Sepharose, sephacryl S-200
HR, and protein low molecular weight standards were from Amersham
Pharmacia Biotech. Microcon centrifugal filter unit was obtained from
Millipore.
Site-Directed Mutagenesis. The MTHase gene in the previously
constructed vector pET-15b-∆H-treZ (10) was mutated by polymerase
chain reaction according to the instructions of the QuickChange XL
site-directed mutagenesis kit as previously described (6). The designed
mutations were included in the mutagenic primers which have 27-41
bases individually.
Production and Purification of MTHase. Wild-type and mutant
MTHases were produced by culturing pET-15b-∆H-treZ-transformed
E. coli BL21-CodonPlus (DE3)-RIL at 37 °C in Terrific broth medium
supplemented with 100 µg/mL of ampicillin plus 34 µg/mL of
chloramphenicol as previously described (10). The wild-type and mutant
MTHases were purified after cell disruption by using heat treatment,
streptomycin sulfate precipitation, and Q-Sepharose anion exchange
column chromatography as previously described (10). The molecular
weights of both wild-type and mutant MTHases were determined using
a BioSep-SEC-S4000 gel filtration column as previously described (10).
Protein concentration was quantitated by Bradford’s method (12) with
BSA as standards.
respectively (8). Figure 1 shows the stereoview of the MTHase
monomer from S. solfataricus KM1, illustrating the active
site (8, 9). The crystal structures of MTHase from S. solfataricus
KM1 also suggested that W215 is likely an important substrate
binding residue (8). The structure comparisons between the
MTHase from S. solfataricus KM1 and other several family 13
glycosidases also suggested that the trehalose end of maltoo-
ligosyltrehaloses binds subsites +1 and +2, which are located
near the C-terminal side of the catalytic barrel, to provide a
recognition for the trehalose moiety, and subsite +2 is the most
critical region for substrate discrimination between maltooli-
gosyltrehaloses and maltooligosaccharides (8). The residues
Y325, F352, and R353, which are located at the C-terminal side
of the catalytic barrel, might form a steric barrier at subsite +2
of the substrate binding cleft (8). In addition, when we compared
the four conserved regions in the active sites of MTHases from
five different strains of the Sulfolobus genus, we found that four
MTHases have Ala but only Sulfolobus shibatae B12 has Ser
corresponding to position 259 of MTHase of S. solfataricus
ATCC 35092, which is in the third conserved region and usually
a residue important for specificity (10, 11). However, no
mutation analysis has been carried out to identify these catalytic
and selectivity-related residues of MTHase.
Preparation of Maltooligosyltrehaloses. Maltotriosyltrehalose
(G3T), maltotetraosyltrehalose (G4T), and maltopentaosyltrehalose (G5T)
were prepared as previously described (10).
The MTHase from S. solfataricus ATCC 35092, also known
as P2, has been purified and characterized in our previous study
(10). In this study, site-directed mutagenesis was used to
construct various individual mutations in this enzyme to identify
the essential catalytic residues and selectivity-related residues.
To find the relationships between trehalose yield and specific
parameters, we not only investigated the kinetic parameters of
the wild-type and mutant enzymes but also carried out the
production of trehalose from the digestion of 10% (w/v) soluble
starch at 75 °C by using wild-type and mutant MTHases,
respectively, along with isoamylase and MTSase.
Enzyme Activity Assay. The MTHase activity was assayed at 60
°C for 10 min by using 2.8 mM G4T as substrate in 0.05 M citrate
phosphate buffer (pH 5), and the activity of the enzyme was determined
by the DNS assay in our previous study (10). One unit (U) of MTHase
activity was defined as the amount of enzyme required to produce 1
µmol of trehalose in one minute. The specific activity was defined as
the amount of enzyme activity/mg of the enzyme (U/mg). The effects
of pH and temperature on the activity and stabilities of wild-type and
mutant MTHases were also determined according to the methods
previously described (10).
Enzyme Kinetics. The initial rates for hydrolysis of G3T-G5T and
G5-G7 were determined at 60 °C in 50 mM citrate phosphate buffer
at pH 5 by using 8-10 substrate concentrations ranging from 2 to 50
mM as previously described (9). The values of kcat, KM, and kcat/KM
were calculated by fitting the initial rates as a function of substrate
concentration to the Michaelis-Menten equation using Enzfitter
software (Elsevier-Biosoft). The standard errors of these parameters
were obtained from the fitting results. The change of transition-state
MATERIALS AND METHODS
Materials. Escherichia coli Rosetta (DE3) was from Novagen.
QuickChange XL site-directed mutagenesis kit was purchased from
Stratagene. G1, maltopentaose (G5), maltohexaose (G6), maltoheptaose
(G7), glucoamylase, 3,5-dinitrosalicyclic acid (DNS), and bovine serum

5630 J. Agric. Food Chem., Vol. 56, No. 14, 2008
Fang et al.
Table 1. Specific Activities of the Wild-Type and Mutant MTHases for
Hydrolysis of G4T at 60 °C in 50 mM Citrate Phosphate Buffer, pH 5a
MTHase form
specific activity (U/mg)
wild-type
W218A
W218F
D255A
A259S
E286A
Y328F
F355Y
R356K
D380A
814 ( 20
5.31 ( 0.11
52.0( 3.3
1.23 ( 0.11
787 ( 18
0.74 ( 0.04
492 ( 20
597 ( 16
472 ( 8
0.10 ( 0.01
a One unit (U) of MTHase activity was defined as the amount of enzyme required
to produce 1 µmol of trehalose in one minute. The specific activity was defined as
the amount of enzyme activity/mg of the enzyme (U/mg). Data represent the mean
( SD from triplicate experiments.
Figure 2. SDS-PAGE of the purified wild-type and mutant MTSases. A
10% minigel was stained with Coomassic Brilliant Blue R-250. Lane M:
molecular weight standards; lanes 1-10: wild-type, D255A, E286A, D380A,
A259S, Y328F, W218A, W218F, F355Y, and R356K MTHases, respectively.
activities of D255A, E286A, and D380A MTHases were only
0.15, 0.09, and 0.01%, respectively, of that of wild-type. The
severe losses in activity indicate that D255, E286, and D380
are catalytic residues.
Wild-type and W218A, W218F, A259S, Y328F, F355Y, and
R356K MTHases had optimal activities at pH 5 and 85 °C (data
not shown). After a two-hour incubation at 80 °C, wild-type
and those six mutant MTSases retained virtually all of their
activities (data not shown). The good thermostability of these
enzymes suggested the purified mutant MTHases were well
folded without any long-range conformational changes that may
be a consequence of mutation.
Enzyme Kinetics. Kinetic parameters (kcat and KM) for the
hydrolysis of G3T-G5T and G5-G7 at 60 °C and pH 5 are given
in Table 2. The catalytic efficiencies (kcat/KM) for G4T and G6
hydrolysis of W218A MTHase were 0.97 and 0.62%, respec-
tively, of those of wild-type MTHase, while those of W218F
MTHase were 4.1 and 0.62%, respectively, of those of wild-
type MTHase. The low specific activities (Table 1) and catalytic
efficiencies (Table 2) of these two mutant enzymes might
prevent their applications in an industrial process to produce
trehalose from starch, and therefore we did not carry out further
the kinetic studies by using other forms of substrates and the
trehalose production from starch.
By comparison with wild-type MTHase, A259S MTHase had
slightly lower catalytic efficiencies for hydrolysis of G3T-G5T
and similar catalytic efficiencies for hydrolysis of G5 and G6,
while a slightly higher catalytic efficiency for hydrolysis of G7.
Y328F MTHase had 40-73 and 76-84% catalytic efficiencies
of those of wild-type for hydrolysis of G3T-G5T and G5-G7,
respectively. F355Y MTHase had somewhat lower catalytic
efficiencies for hydrolysis of G3T-G5T and slightly lower
catalytic efficiencies for hydrolysis of G5 and G7, while a slightly
higher catalytic efficiency for hydrolysis of G6. R356K MTHase
had 45-59 and 101-130% catalytic efficiencies of those of
wild-type for hydrolysis of G3T-G5T and G5-G7,
respectively.
Production of Trehalose from Starch. The digestion of 10%
(w/v) soluble starch was used to study the yield of trehalose
production by wild-type, A259S, Y328F, F355Y, and R356K
MTHases along with isoamylase and MTSase at both a high
reaction temperature and a high substrate concentration. All of
the reactions reached more than 80% trehalose yield in 6 h,
and the yield almost reached equilibrium in 12 h (data not
shown). The G1 formation in the starch digestion was also
determined because of the side reactions to release G1 from
maltooligosaccharides also catalyzed by both MTHase and
binding energy [∆(∆G)] for substrate hydrolysis caused by the mutation
was used to estimate the binding strength of the substrate in the
transition-state complex and was calculated by the equation ∆(∆G) )
-RT ln[(kcat/KM)mut/(kcat/KM)wt], where the subscripts mut and wt denote
mutant and wild-type enzymes, respectively (13). The selectivity ratio
was calculated as [kcat/KM(Gn)]/[kcat/KM(G(n-2)T)].
Production of Trehalose from Starch. In an effort to produce
trehalose from starch, purified thermophilic isoamylase, MTSase, and
MTHase were used concurrently. The purified thermophilic isoamylase
and MTSase were prepared according to the method described in our
previous studies (6, 14). The reaction was carried out at 75 °C with
10% (w/v) soluble starch in a 50 mM citrate phosphate buffer of pH 5.
The enzyme concentration was 0.063 µM for both wild-type and mutant
MTHases. Aliquots of 800 U/g-starch of isoamylase and 40 U/g-starch
of MTSase were also added to the reaction mixture. The samples were
taken at 10, 20, 30, 40, and 50 min to determine the initial rates and
were also taken at 2, 4, 6, 8, 12, 18, and 24 h to determine the trehalose
yield. The formations of G1 were determined by a glucose oxidase
method as previously described (7). For the detection of trehalose,
glucoamylase was added to hydrolyze the residual starch, maltooli-
gosaccharides and maltooligosyltrehalose, and then the reaction mixtures
were filtered and analyzed by HPLC as previously described (7). The
initial rates of trehalose and G1 formation were calculated by fitting
experimental data obtained within 1 h to the equation c ) At + B,
where c is the product concentration, t is the time and A (the initial
rate) and B are constants obtained by linear regression as previously
described (7). Peak trehalose yield was the average of the three highest
consecutive trehalose yield measurements, which were sampled at 12,
18, and 24 h in this study.
RESULTS
Purity and Characteristics of Wild-Type and Mutant
MTHases. The purity of the enzyme was analyzed by sodium
dodecyl sulfate-polyacrylamide gel (SDS-PAGE; 10% minigel)
according to the method of Laemmli (15). The purity of wild-
type and mutant MTHases was very good, as indicated by SDS-
PAGE (Figure 2). Since the wild-type MTHase is a homodimer
(10), the molecular masses of the purified wild-type and mutant
MTHases under nondenaturing conditions were analyzed by
HPLC using a BioSep-S4000 gel filtration column as previously
described (10). The molecular masses of HPLC analysis showed
that all mutant MTHases are homodimers and have a molecular
weight around 129 kDa.
The specific activities of mutant MTHases were in general
lower than that of wild-type MTHase (Table 1). The specific

Essential Catalytic and Selectivity-Related Residues of MTHase
J. Agric. Food Chem., Vol. 56, No. 14, 2008 5631
Table 2. Kinetic Parameters of the Wild-Type and Mutant MTHases for Hydrolysis of G3T-G5T and G5-G7 at 60 °C in 50 mM Citrate Phosphate Buffer, pH
5a
trehalose formation
G4T
G1 formation
G6
TFE form
Wild-Type
kcat (s-1
KM (mM)
cat/KM (s-1 mM-1
G3T
G5T
G5
G7
)
1030 ( 28b
7.22 ( 0.48
143 ( 6
2.12
1192 ( 34
5.66 ( 0.37
210 ( 8
1.54
1226 ( 36
5.89 ( 0.45
198 ( 9
1.16
17.7 ( 0.5
5.86 ( 0.40
3.02 ( 0.13
18.2 ( 0.7
5.63 ( 0.51
3.24 ( 0.22
6.01 ( 0.13
2.63 ( 0.20
2.29 ( 0.13
k
)
selectivity ratioc (×102)
W218A
kcat (s-1
)
16.0 ( 0.5
7.91 ( 0.61
2.03 ( 0.09
0.99
0.36 ( 0.01
20.5 ( 1.7
0.02 ( 0.00
KM (mM)
cat/KM (s-1 mM-1
k
)
selectivity ratio (×102)
∆(∆G)d (kJ/mol)
W218F
12.8
14.4
kcat (s-1
KM (mM)
cat/KM (s-1 mM-1
)
58.4 ( 2.4
6.79 ( 0.54
8.59 ( 0.64
0.23
0.54 ( 0.02
23.2 ( 1.6
0.02 ( 0.00
k
)
selectivity ratio (×102)
∆(∆G) (kJ/mol)
A259S
8.86
13.7
kcat (s-1
KM (mM)
cat/KM (s-1 mM-1
)
1002 ( 29
8.55 ( 0.65
117 ( 5
2.76
1106 ( 60
6.79 ( 0.52
163 ( 14
1.77
1282 ( 31
7.15 ( 0.42
179 ( 7
1.60
17.4 ( 0.6
5.37 ( 0.48
3.24 ( 0.18
13.5 ( 0.6
4.69 ( 0.44
2.89 ( 0.16
6.49 ( 0.24
2.19 ( 0.25
2.97 ( 0.25
k
)
selectivity ratio (×102)
∆(∆G) (kJ/mol)
Y328F
0.55
0.71
0.27
-0.19
0.32
-0.63
kcat (s-1
KM (mM)
cat/KM (s-1 mM-1
)
634 ( 26
10.9 ( 0.9
57.8 ( 2.6
4.27
654 ( 22
5.03 ( 0.45
130 ( 8
2.10
760 ( 34
5.26 ( 0.42
144 ( 11
1.20
14.4 ( 0.6
5.82 ( 0.57
2.47 ( 0.14
8.23 ( 0.28
3.01 ( 0.28
2.73 ( 0.17
5.00 ( 0.13
2.88 ( 0.20
1.73 ( 0.08
k
)
selectivity ratio (×102)
∆(∆G) (kJ/mol)
F355Y
2.50
1.33
0.87
0.56
0.47
0.77
kcat (s-1
KM (mM)
cat/KM (s-1 mM-1
)
734 ( 25
13.6 ( 1.0
54.1 ( 2.4
4.57
721 ( 30
6.94 ( 0.64
104 ( 5
4.24
741 ( 29
5.81 ( 0.44
127 ( 10
1.53
19.1 ( 0.7
7.74 ( 0.58
2.47 ( 0.10
9.50 ( 0.28
2.16 ( 0.20
4.39 ( 0.30
5.49 ( 0.17
2.81 ( 0.25
1.95 ( 0.12
k
)
selectivity ratio (×102)
∆(∆G) (kJ/mol)
R356K
2.69
1.96
1.22
0.56
-0.85
0.44
kcat (s-1
KM (mM)
)
572 ( 23
8.90 ( 0.74
64.2 ( 3.8
4.77
627 ( 24
6.73 ( 0.65
93.7 ( 5.8
3.93
675 ( 26
5.75 ( 0.45
117 ( 9
2.53
15.5 ( 0.6
5.08 ( 0.43
3.06 ( 0.16
12.6 ( 0.3
3.42 ( 0.22
3.68 ( 0.15
6.49 ( 0.24
2.19 ( 0.25
2.97 ( 0.25
k
cat/KM (s-1 mM-1
)
selectivity ratio (×102)
∆(∆G) (kJ/mol)
2.21
1.53
1.44
-0.037
-0.36
-0.72
a The degrees of polymerization of G3T, G4T, and G5T were equal to those of G5, G6, and G7, which were 5, 6, and 7, respectively. b Standard error. c Selectivity ratio:
[kcat/KM(Gn)]/[kcat/KM(G(n-2)T)]. d Change of transition-state energy: ∆(∆G) ) -RT ln[(kcat/KM)mut/(kcat/KM)wt] (13).
MTSase. The initial rates of trehalose and G1 formation in the
starch digestion and their ratios were also obtained. Ratios of
the initial rates of G1 formation to trehalose formation were
calculated to evaluate the selectivity for G1 formation versus
that of trehalose formation. Figure 3 shows the relationship
between peak trehalose yields (the average of the three highest
consecutive trehalose yield measurements that were sampled
at 12, 18, and 24 h in this study) and the ratios of initial rates
of G1 formation to those of trehalose formation. All tested
mutant MTHases, including A259S, Y328F, F355Y, and R356K
MTHases, had higher ratios of initial rates of G1 formation to
trehalose formation compared with that of wild-type MTHase
(Figure 3). Peak trehalose yields and ratios of initial rates of
G1 formation to those of trehalose formation were inversely
correlated (Figure 3). Therefore, the lower ratios of the initial
rate of G1 formation to that of trehalose formation from the
starch digestion will result in the higher trehalose yield.
MTHase Selectivity toward G5-G7 Hydrolysis versus
G3T-G5T Hydrolysis. Selectivity ratios were calculated from
the kinetic parameters of wild-type and mutant MTHases for
hydrolysis of G3T-G5T and G5-G7 as given in Table 2. The
selectivity ratio is expressed as the ratio of catalytic efficiency
of Gn hydrolysis over that of G(n-2)T hydrolysis. W218A and
W218F MTHases had decreased selectivity ratios compared to
that of wild-type MTHase, whereas A259S, Y328F, F355Y, and
R356K MTHases had increased selectivity ratios.
Figure 4 depicts the relationships between peak trehalose
yield from starch digestion and the selectivity ratios from kinetic
studies for wild-type and A259S, Y328F, F355Y, and R356K
MTHases. Only the selectivity ratios obtained from the hy-
drolysis of G3T and G5 were inversely correlated to peak
trehalose yields. When Y328F MTHase was not included, the
selectivity ratios obtained from the hydrolysis of G4T and G6
were also inversely correlated to peak trehalose yields.
DISCUSSION
In a previous study, we had investigated the effects of
MTSase mutations on trehalose yield by using 10% (w/v)
soluble starch as substrate and by combining isoamylase and
MTHase in the reaction mixture. We found that the ratios of
the initial rate of G1 formation to that of trehalose formation
from the starch digestion provide a better prediction for high
trehalose yield. In addition, mutant MTSases with decreased

5632 J. Agric. Food Chem., Vol. 56, No. 14, 2008
Fang et al.
ratios obtained from the hydrolysis of G3T and G5 had a higher
peak trehalose yield.
Residues D255, E286, and D380, which are equivalent to
the proposed catalytic residues D252, E283, and D377 of
MTHase from S. solfataricus KM1, respectively (8), were
mutated to alanine to identify their roles as catalytic residues.
The almost complete loss of specific activities confirmed that
they are catalytic residues. Residue W218, which is equivalent
to residue W215 of MTHase from S. solfataricus KM1, was
mutated to Ala and Phe to identify its importance in substrate
binding. Tryptophan is the most common residue involved in
hydrophobic interactions in protein-carbohydrate complexes,
and W215 of MTHase from S. solfataricus KM1 was proposed
to make substantial contacts with bound substrate (8). We
calculated the transition-state energy to estimate the binding
strength of the enzyme-substrate complex in the transition state.
As some earlier studies pointed out, the change of transition-
state energy, ∆(∆G), associated with the loss of a hydrogen
bond between uncharged groups on the substrate and enzyme
is between 2.1 and 6.3 kJ/mol, while that for the loss of a
hydrogen bond between an uncharged group on the substrate
and a charged group on the enzyme is between 14.6 and 18.8
kJ/mol (7, 16). The large changes in ∆(∆G) for G4T and G6
hydrolysis by W218A MTHase, 12.8 and 14.4 kJ/mol, respec-
tively, and those by W218F MTHase, 8.86 and 13.7 kJ/mol,
respectively (Table 2), are approximate to the loss of two
uncharged hydrogen bonds between enzyme and the substrate
in the transition state. In addition, the atom NE1 of Trp is both
a hydrogen acceptor and hydrogen donor, and, therefore, Trp
residue has the ability to form two hydrogen bonds with the
substrate. These results suggested that there might be two
hydrogen bonds between substrate and residue W218 on the
wild-type MTHase of S. solfataricus ATCC 35092. The G4T
can occupy the subsites +2, +1, -1, -2, -3, and -4, and G6
can occupy the subsites +1, -2, -3, -4, and -5. Therefore,
the losses of hydrogen bond caused by mutations on W218
presumably occurred between subsites +1 to -4 but not on
subsite +2. Residue W218 in S. solfataricus ATCC 35092
MTHase is equivalent to residue W241 in Deinococcus radio-
durans MTHase (DrMTHase). The crystal structure of the
DrMTHase-trehalose complex also suggested that W241 can
hydrogen bond directly to the sugar ring of the substrate (17).
In addition, the hydrophobic interactions also play a role in the
substrate binding when we compared kinetic parameters and
∆(∆G) values for G4T hydrolysis obtained from W218A and
W218F MTHases.
Figure 3. Relationship between peak trehalose yields and (initial rate of
G1 formation/initial rate of trehalose formation) during the digestion of
10% (w/v) soluble starch at 75 °C in 50 mM citrate phosphate buffer (pH
5) using the wild-type and mutant MTHases along with isoamylase and
MTSase. The peak trehalose yield was represented by mean ( SD from
three highest consecutive trehalose yield measurements, which were
sampled at 12, 18, and 24 h in this study.
Residue A259 in S. solfataricus ATCC 35092 MTHase is
equivalent to residue S257 in S. shibatae B12 (10). This residue
is located in the third highly conserved region and usually
important for specificity. Mutation A259S was designed to create
a decreased hydrophobic interaction, possibly forming hydrogen
bonds between the enzyme and substrate. A259S MTHase had
slightly increased selectivity ratios compared to those of wild-
type MTHase. However, the small change of ∆(∆G) values,
from -0.63 to 0.71 KJ/mol, indicated that this mutation had
only minor effects on the transition-state substrate binding.
Figure 4. Relationships between the peak trehalose yields from starch
digestion and the selectivity ratios from kinetic studies by the wild-type
and mutant MTHases. (A) Selectivity ratios for G5 hydrolysis over G3T
hydrolysis. (B) Selectivity ratios for G6 hydrolysis over G4T hydrolysis.
(C) Selectivity ratios for G7 hydrolysis over G5T hydrolysis. The peak
trehalose yield was represented by mean ( SD as in Figure 3.
selectivity ratios for hydrolysis over transglycosylation had a
higher peak trehalose yield except for one mutant MTSase. In
this study, we investigated the effects of MTHase mutations
on trehalose yield also by using 10% (w/v) soluble starch as
substrate and by combining isoamylase and MTSase in the
reaction mixture. Since the peak trehalose yields and ratios of
initial rates of G1 formation to those of trehalose formation were
inversely correlated (Figure 3), we confirmed that the lower
ratios of the initial rate of G1 formation to that of trehalose
formation from the starch digestion could predict the higher
trehalose yield. In addition, the MTHases with lower selectivity
Residues Y328, F355, and R356 in S. solfataricus ATCC
35092 MTHases are equivalent to residues Y325, F352, and
R353, respectively, in S. solfataricus KM1 MTHase (Figure
1). Those residues probably form a steric barrier at subsite +2
of the substrate binding cleft (8). Mutations Y328F, F355Y,
and R356K were designed to create minor changes in this steric
barrier. The small change of ∆(∆G) values, from -0.85 to 2.69
KJ/mol, indicated that these mutations had only minor effects

Essential Catalytic and Selectivity-Related Residues of MTHase
J. Agric. Food Chem., Vol. 56, No. 14, 2008 5633
Sulfolobus solfataricus maltooligosyltrehalose synthase. J. Agric.
Food Chem. 2006, 54, 3585–3590.
(7) Fang, T. Y.; Tseng, W. C.; Pan, C. H.; Chung, Y. T.; Wang, M. Y.
Protein engineering of Sulfolobus solfataricus maltooligosyltre-
halose synthase to alter its selectivity. J. Agric. Food Chem. 2007,
55, 5588–5594.
(8) Feese, M. D.; Kato, Y.; Tamada, T.; Kato, M.; Komeda, T.; Miura,
Y.; Hirose, M.; Hondo, K.; Kobayashi, K.; Kuroki, R. Crystal
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on transition-state substrate binding. However, Y328F, F355Y,
and R356K MTHases had moderately increased selectivity ratios
compared to those of wild-type MTHase. These minor changes
near the subsite +2 substantially changed the selectivity of
MTHase especially when the degree of polymerization of
substrate was decreased.
In conclusion, in this study we confirmed the catalytic roles
of D255, E286, and D380 and suggested the existence of two
hydrogen bonds between substrate and the enzyme at position
W218. In addition, the minor changes in the substrate binding
strength at residues A259, Y328, F355, and R356 can alter the
selectivity of MTHase. The lower ratios of the initial rate of
G1 formation to that of trehalose formation from the digestion
of soluble starch predict high trehalose yield.
ABBREVIATIONS USED
(11) MacGregor, E. A.; Janecek, S.; Svensson, B. Relationship of
sequence and structure to specificity in the R-amylase family of
enzymes. Biochim. Biophys. Acta 2001, 1546, 1–20.
(12) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248–254.
(13) Wilkinson, A. J.; Fersht, A. R.; Blow, D. M.; Winter, G. Site-
directed mutagenesis as a probe of enzyme structure and catalysis:
Tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation.
Biochemistry 1983, 22, 3581–3586.
MTSase, maltooligosyltrehalose synthase; MTHase, mal-
tooligosyltrehalose trehalohydrolase; S. solfataricus, Sulfolo-
bus solfataricus; S. shibatae, Sulfolobus shibatae; G1,
glucose; G5, maltopentaose; G6, maltohexaose; G7, malto-
heptaose; G3T, maltotriosyltrehalose; G4T, maltotetraosyl-
trehalose; G5T, maltopentaosyltrehalose; DNS, 3,5-dinitro-
salicylic acid; BSA, bovine serum albumin; ∆(∆G), change
of transition-state energy; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis.
(14) Fang, T. Y.; Tseng, W. C.; Yu, C. J.; Shih, T. Y. Characterization
of the thermophilic isoamylase from the thermophilic archaeon
Sulfolobus solfataricus ATCC 35092. J. Mol. Catal. B: Enzym.
2005, 33, 99–107.
(15) Laemmli, U. K. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 1970, 227, 680–
685.
(16) Fersht, A. R.; Shi, J. P.; Knill-Jones, J.; Lowe, D. M.; Wilkinson,
A. J.; Blow, D. M.; Brick, P.; Carter, P.; Waye, M. M.; Winter,
G. Hydrogen bonding and biological specificity analysed by
protein engineering. Nature 1985, 314, 235–238.
(17) Timmins, J.; Leiros, H. K.; Leonard, G.; Leiros, I.; McSweeney,
S. Crystal structure of maltooligosyltrehalose trehalohydrolase
from Deinococcus radiodurans in complex with disaccharides.
J. Mol. Biol. 2005, 347, 949–963.
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Received for review November 13, 2007. Revised manuscript received
March 10, 2008. Accepted April 10, 2008. This work was supported by
the Center for Marine Bioscience and Biotechnology at the National
Taiwan Ocean University and Grant NSC-95-2313-B-019-021-MY2
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