Tài liệu Báo cáo khoa học: nsights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase doc

Insights into the reaction mechanism of glycosyl hydrolase family 49
Site-directed mutagenesis and substrate preference of isopullulanase
Hiromi Akeboshi
1
, Takashi Tonozuka
1
, Takaaki Furukawa
1
, Kazuhiro Ichikawa
1
, Hiroyoshi Aoki
1,2
,
Akiko Shimonishi
1
, Atsushi Nishikawa
1
and Yoshiyuki Sakano
1
1
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan;
2
Fuence Co., Shibuya, Tokyo, Japan
Aspergillus n iger isopullulanase (IPU) is the only pullulan-
hydrolase in glycosyl hydrolase (GH) family 49 and does not
hydrolyse d extran at all, while all o ther GH family 49
enzymes a re dextran-hydrolysing enzymes. T o i nvestigate
the common catalytic mechanism of GH family 49 enzymes,
nine mutants were prepared to replace residues conserved
among GH family 49 (four Trp, three A sp and two Glu).
Homology modelling of I PU was also carried out based o n

the structure of Penicillium minioluteum dextranase, and the
result showed that Asp353, Glu356, Asp372, Asp373 and
Trp402, whose substitutions resulted in the reduction of
activity for both pu llulan and panose, were p redicted to b e
located in the negatively numbered subsites. Three Asp-
mutated enzymes, D353N, D372N and D373N, lost their
activities, indicating that these r esidues are candidates f or the
catalytic residues of IPU. The W402F enzyme significantly
reduced IPU activity, and the K
m
value was sixfold higher
and the k
0
value was 5 00-fold lower than t hose for the wild-
type enzyme, suggesting that Trp402 is a residue participa-
ting in subsite )1. Trp31 and Glu273, whose substitutions
caused a d ecrease in the activity for pullulan but not for
panose, were predicted to be located in the interface between
N-terminal and b-helical domains. T he substrate p reference
of the negatively numbered subsites of IPU resembles that of
GH family 49 dextranases. These findings suggest that IPU
and the GH family 49 dextranases have a similar c atalytic
mechanism i n their negatively numbered s ubsites in spite of
the d ifference of their substrate s pecificities.
Keywords: d extranase; GH family 49; isopullulanase; pullu-
lan-hydrolase; site-directed mutagenesis.
Isopullulanase (IPU, EC 3.2.1.57; pullulan 4 -glucanohydro-
lase) f rom Aspergillus niger ATCC9642 hydrolyses pullula n
to produce isopanose (Glc-a-(1fi4)-Glc-a-(1fi6)-Glc) and
also hydrolyses substrates containing the panose (Glc-a-

(1fi6)-Glc-a-(1fi4)-Glc) s tructure, and cleaves the a-1,4-
glucosidic linkage in the panose motif [1,2]. Enzymes that
hydrolyse specific sites of pullulan can be classified into the
following three types (schematic action patterns of these
enzymes have been illustrated previously [2]). (a) Pullulanase
(EC 3 .2.1.41), which hyd rolyses a-1,6-glucosidic linkages t o
produce maltotriose [3]; (b) Thermoactinomyces vulgaris
R-47 a-amylase (TVA, EC 3.2.1.1) [4] and neopullulanase
(EC 3.2.1.135) [5], which hydrolyse a-1,4-glucosidic linkages
to produce panose; and (c) IPU, which h ydrolyses the other
a-1,4-glucosidic linkages to produce isopanose. Except for
IPU, these enzymes are c lassified into glycosyl h ydrolase
(GH) family 13, known as the a-amylase family (reviewed in
[6–8]). In contrast, IPU is the sole enzyme classified into GH
family 49 [2,9,10] among these pullulan-hydrolases, and no
homology between IPU a nd a-amylase family e nzymes
has been found ( />index.html).
Interestingly, IPU does not hydrolyse dextran at all, while
all o ther GH family 49 enzymes are dextran-hydrolysing
enzymes, such as endo-dextranase (EC 3.2.1.11) [11–14] and
isomaltotrio-dextranase (EC 3.2.1.95) [15]. We have repor-
ted the molecular cloning of IPU, and indicated that seven
highly co nserved regions are found among the p rimary
structures of these dextran-hydrolases and IPU [2]. The
expression systems of IPU have been constructed with
eukaryotic hosts Aspergillus oryzae and Pichia pastoris
[2,16]. Recently, a three-dimensional structure of GH family
49 dextranase (D ex49A), wh ich shows a 38% s equence
identity with IPU, has b een reported, and the catalytic
domain folds into a right-handed parallel b-helix [17].

Crystal structures o f polygalacturonases a nd rhamnogalac-
turonases, which belong to GH family 28, have been solved
by many researchers (for e xample [18–20]). Although the
substrate specificities between GH family 49 and 28 are
completely different, the GH family 28 polygalacturonases
and rhamnogalacturonases consist of the similar b-helical
structures, and GH family 49 and 28 form clan GH-N
Correspondence to Y. Sakano, Department of Applied Biological
Science, Faculty of Agriculture, Tokyo University of Agriculture and
Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509 Japan.
Fax: +81 42 367 5705, E - mail: s
Abbreviations: IPU, isopullulanase; GH, glycosyl hydrolase; BMM,
buffered minimum methanol medium; YPGY, yeast peptone glycerol
medium.
Enzyme: isopullulanase (EC 3.2.1.57); pullulanase (EC 3.2.1.41); neo-
pullulanase ( EC 3 .2.1.135); R-47 a-amylase (TVA, EC 3.2.1.1); endo-
dextranase (EC 3.2.1.11); isomaltotrio-dextranase (EC 3 .2.1.95);
glucoamylase (anomer-inverting enzyme; EC 3.2.1.3); a-glucosidase
(anomer-retaining en zyme; EC 3 .2.1.20).
(Received 2 3 June 2004, revised 1 6 August 2004,
accepted 27 September 200 4)
Eur. J. Biochem. 271, 4420–4427 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04378.x
[10,17]. D espite these a dvances, little is known a bout the
unique substrate preference and the catalytic mechanism
of IPU.
To clarify the catalytic mechanism of IPU, site-directed
mutagenesis was carried out. Acidic amino acid residues,
Asp a nd/or Glu, are commonly r eported a s the catalytic
residues of GHs [6,21–26], and it is probable that IPU has
Asp a nd/or Glu as the catalytic residues. In addition, in the

chemical modification experiment IPU was inactivated by
N-bromosuccinimide, i ndicating that a Trp residue is
required f or IPU activity [27]. Therefore, several Asp and/
or Glu, and Trp residues located in the conserved regions
are predicted to be indispensable for IPU a nd the other GH
family 49 enzymes. Here we determined the residues that are
essential for the catalytic activity of IPU, and also investi-
gated some detailed properties of this enzyme. The results
indicated that the functionally important residues o f GH
family 49 enzymes are conserved in the negatively numbered
subsites, and the substrate preference of the negatively
numbered subsites of IPU also resembles that of GH family
49 dextranases.
Materials and methods
Host strains and media
Escherichia coli JM109 was used for the plasmid con-
structions. P. pastoris GS115 (Invitrogen) was used for the
heterologous expression of IPU. The Luria–Bertani
medium for E. coli, and the yeast peptone dextrose
medium and buffered minimum methanol medium
(BMM) for P. pastoris were prepared according t o t he
manufacturer’s r ecommendations. Y east peptone glycerol
medium (YPGY: 1% yeast extract, 2% peptone, and 1%
glycerol) was prepared for the propagat ion of recombinant
P. pastoris strain for t he expression of enzymes. All
cultivation was done at 37 °CforE. coli and 30 °Cfor
P. pastoris.
Mutant constructions
All c loning procedures were carried out by applying
standard molecular b iological t echniques [28]. Transforma-

tion of P. pastoris was done according to the manufacturer’s
instructions for the Pichia Expression Kit (Invitrogen),
which has been described elsewhere [16]. Plasmids coding
for the W31F, W32F, W240F, and W402F enzymes were
constructed by PCR using p IPA118(–), a plasmid DNA
coding for mature I PU. T he complementary m utagenic
primers encoding the desired mutations (Table 1) were
paired with universal primers, M13 forward and M13
reverse, respectively, and two partial DNA fragments were
amplified. Subsequently, PCR was performed with these
two amplified fragments as the tem plates and pri mers, M13
forward a nd M13 r everse. After sequence confirmation, the
EcoRI–BamHI fragment w as inserted into the e xpression
vector for P. pastoris, pHIL-S1 (Invitrogen). Plasmids
coding for E273Q, D353N, E3 56Q, D372N, and D373N
were constructed b y the method of Kunkel [ 29]. The s ites at
which the mutations were introduced are located in the
StyI–XbaI fragment. To construct reliable mutants,
mutated St yI–XbaI fragments were verified b y DNA
sequencing, and the origin al StyI–XbaI fragment of
pIPA118(–) was replaced with the mutated StyI–XbaI
fragment. The EcoRI–BamHI fragments o f these plasmids
were further subcloned into pHIL-S1.
Expression of wild-type and mutated IPU enzymes
The wild-type IPU was expressed in P. pastoris harbouring
pSig-PHO as d escribed [16] with slight modifications.
Briefly, P. pastoris harbouring pSig-PHO w as cultured in
250 mL YPGY for 2 days, and the propagated cells
collected by centrifugation (5000 g for 1 0 min) were resus-
pended a nd cultivated for 6 days in 100 mL B MM. The

clear supernatant of cultured BMM (crude IPU) was then
obtained by c entrifugation ( 5000 g for 1 5 min). During the
cultivation in BMM, 500 lL of methanol was added every
24 h for the maintenance of 0.5% (v/v) methanol as the
carbon source and i nducer. The expressi on of IPU mutants
was performed using the same procedure as for the wild-
type IPU.
Substrates
Pullulan w as obtained from H ayashibara, Japan. Panose
[30], IMTG (4
1
-a-isomaltotriosylglucose, also called
6
2
-a-isomaltosylmaltose; Glc-a-(1fi6)-Glc-a-(1fi6)-Glc-a-
(1fi4)-Glc) [ 31], and MM (6
2
-a-malto sylmaltose; Glc-a-
(1fi4)-Glc-a-(1fi6)-Glc-a-(1fi4)-Glc) [32] were prepared
as described. Dextran T-2000 was from Amersham.
Concentrations of the substrates dissolved in 50 m
M
acetate buffer (pH 3.5) were measured by a modified
phenol/sulfuric acid method [33], using glucose as the
standard.
Table 1. Primers used for the mutant constructions. For PCR muta-
genesis (W21F, W32F, W240F and W402F), the same sequences on the
opposite strand were also used as described in Materials and methods.
Lower c ase letters indicate the nucleotide m utations. To facilitat e the
selection of mutant clones, silent mutations were made to introduce

restriction enzyme reco gnition sites ( u nderlined).
Pimers Nucleotide sequence (5¢fi3¢)
W31F CTGACCTtcTGGCATAAC
ACCGGtGAAATC
AgeI
W32F CCTGGTtcCATAAC
ACCGGtGAAATC
AgeI
W240F GGTGCT
gAGCTCAAGTGTGACTTtcGTCTAC
SacI
W402F CCGGTG
GTcGAcTTTGGTTtcACGCCC
SalI
E273Q ACGTACTGCTgTCCGGAA
AGtACtCCATGGCCGC
ScaI
D353N TCCAATCCGTtAGTC
TGgCCaTAGAACGCGC
MscI
E356Q GGAGAAT
GGTgCCAGGGTACATTTcCAATCCGTCA
KpnI
D372N AATACAT
CTTaAGGCCGTCGTtGTCGGTGTGG
AflII
D373N AATACAT
CTTaAGGCCGTtGCGTCGGTG
AflII
Ó FEBS 2004 Reaction mechanism of isopullulanase (Eur. J. Biochem. 271) 4421

Assays of IPU activity and protein concentration
The p ullulan-hydrolysing activity of IPU was evaluated as
described previously [34]. The activities for panose, IMTG
and MM were measured as follows. A re action mixture
consisting of a desired substrate (32 m
M
)andIPUin
40 m
M
acetate buffer (pH 3.5) was incubated at 40 °Cfor
30 min, and the reaction was terminated by t he addition
of an equal v olume o f 0.1
M
Na
2
CO
3
. T he amount of
glucose p roduced by IPU was assayed with G lucose CII
(Wako Pure Chemical Co., Osaka, Japan) [35,36] using a
Bio-Rad550 Microplate reader. To determine the kinetic
parameters of wild-type and W402F IPU for p anose,
mixtures consisting of 4 lgÆmL
)1
enzyme and from 43 to
216 m
M
substrate, and 20 lgÆmL
)1
enzyme and 64 to

480 m
M
substrate, respectively, were used. The protein
concentration was measured by the method of Lowry
et al. with B SA as standard [37].
TLC
TLC was p erformed to analyse t he hydrolysates of the
W402F enzyme. The reaction mixture consisting of pullu-
lan ( 5%) o r p anose ( 100 m
M
) a nd the W402F e nzyme
(0.1 mg ÆmL
)1
)in50m
M
acetate buffer (pH 3.5) was
incubated at 30 °C for 3 days, and the hydrolysates were
developed by TLC with 1-butanol/ethanol/H
2
O ¼ 2/2/1
(v/v/v). The spots were detected by charring with H
2
SO
4
.
Homology modelling
The primary structure of IPU is homologous to that of
Penicillium minioluteum dextranase (38% identity), whose
three-dimensional structures of unliganded form and com-
plex form with a product, isomaltose, have been reported

(PDB IDs, 1OGM and 1OGO, respectively) [17]. The
primary structure of mature IPU (residues 20– 564) was
submitted for automatic modelling on t he Swiss-Model
server ( asy.org/) [ 38] u sing the first
approach mode, a nd a m odel consisting of residues 2 5–540,
which is based on the structure of 1OGM, was obtained. To
determine the potential catalytic site, this mo del was
superimposed on 1OGO using the program
DEEPVIEW
[38],
and a glucosyl units in subsites +1 and +2 w ere placed i n
the m odel. The figure was generated using the programs
RASMOL
[39],
RASTOP
(einfinity.org/rastop/),
MOLSCRIPT
[40] and
RASTER
3
D
[41].
Polarimetric assays
Polarimetric measurements of IPU, glucoamylase from
Rhizopus niveus (Seikagaku K ogyo, Japan; a nomer-invert-
ing enzyme; EC 3.2.1.3), and a-glucosidase from Bacillus sp.
(Toyobo, Japan; an omer-retaining enzyme; EC 3 .2.1.20)
were compared. An enzyme solution (equivalent to
1.2 UÆmL
)1

), and 1.6–1.8% of panose (for IPU) or malto-
tetraose ( for R. niveus glucoamylase and Bacillus a-glucosi-
dase) were d issolved in 50 m
M
acetate buffer (pH 4.5), and
the optical r otations were measured at 1-min intervals at
589 nm using a JASCO DIP-360 polarim eter. After 1 0 min
(IPU) o r 2 0 min ( R. niveus glucoamylase and Bacillus
a-glucosidase), 20 lLof15
M
ammonium hydroxide w as
added to the reaction mixture t o raise mutarotation and the
anomeric form of the product was determined [42,43].
Results and Discussion
Purification of wild-type and mutated IPU
Purification of I PU using HiTrap Con A Sepharose HP
column (Amersham Biosciences) has been described [ 16].
However, because the recovery of IPU by this method was
low (13%), several other purification methods were tested.
When a hydrophobic column, TOYOPEARL Hexyl-650C
(Tosoh, Japan) was used, the recovery increased to 50%.
Ammonium sulfate was added directly to the dialysed crude
enzyme to adjust it to 70% s aturation and the supernatant
was loaded on to the column. The specific activity of
purified IPU for pullulan using this method was 40 UÆmg
)1
,
while the previous method gave only 25 UÆmg
)1
.This

specific activity was also h igher t han t hose o f IPU from
original A. niger and heterologously expressed I PU fr om
A. oryzae (27 a nd 38ÆUmg
)1
, r espectively) [7,27]. The
mutatedIPUswerepurifiedwiththesameprocedureas
wild-type IPU.
Properties of Trp-mutated enzymes
Previous experiments indicated that some T rp residues are
essential f or IPU a ctivity [27]. Four Trp residues, conserved
in the seven regions of GH family 49 (Fig. 1), are replaced
by Phe (W31F, W32F, W240F and W402F). The relative
activity of mutated enzymes towards pullulan and panose
are shown in Table 2. The W 402F enzyme lost the activity
for pullulan ( 0.4% of the wild-type IPU) and the activity
for p anose w as almost undetectable ( 0.1%) under the
given conditions. The W31F enzyme had only 38% activity
for pullulan, but the a ctivity for panose w as 1.4-fold higher
than that of wild-type enzymes. The activities of W32F
and W 240F were similar (90–160%) to that o f wild-type
enzyme.
As t he a ctivity o f W402F drastically decr eased, it s action
pattern was investigated using TLC. T he W402F enzyme
liberated isopanose from pullulan, and isomaltose and
glucose from panose, and the action patterns of the wild-
type and the W402F enzymes were almost identical (Fig. 2).
The kinetic study for W402F towards panose showed that
the K
m
value was sixfold higher and the k

0
value was 500-
fold lower than those for the wild-type enzyme (Table 3).
Properties of Asp- and Glu-mutated enzymes
Three Asp residues (353, 372 and 373) and three Glu
residues (157, 273 and 356) are found in the seven conserved
regions of all the GH family 49 enzymes (Fig. 1). To
determine the catalytic r esidues of I PU, these Asp a nd Glu
residues are replaced by Asn and Gln, respectively. Five of
these enzymes (E273Q, D353N, E356Q, D372N and
D373N) were obtained in soluble form, and were purified.
Their activities for pullulan and panose were compared
with those of wild-type e nzyme (Table 2). All three of the
Asp-mutated enzymes, D353N, D372N and D373N,
virtually lost their activities. The activities of mutated
enzymes, E273Q and E356Q, were also decreased but less
4422 H. Akeboshi et al. (Eur. J. Biochem. 271) Ó FEBS 2004
significantly. The mutant E356Q had 38% and 50% of the
activities for pullulan and panose, respectively. In contrast,
the activity of E273Q for p ullulan was 45%, w hile that for
panose remained at 74%. The sixth mutant E157Q is not
obtained in the P. pastoris expression system, but h as been
expressed by u sing A. nidulans as a host and shown to have
detectable activity to both pullulan and panose (data not
shown).
Prediction of the positions of amino acid residues whose
substitutions resulted in the reduction of the IPU activity
While the study of site-directed mutagenesis described
above was carried out, a crystal structure of Penicillium
minioluteum dextranase, Dex49A, complexed with a prod-

uct, isomaltose, has been reported (PDB ID, 1OGO) [17].
The identity between the primary structures of Dex49A a nd
IPU is 38%, and a t hree-dimensional structure of IPU was
modelled based on the structure of Dex49A using the Swiss-
Model server. IPU consists of a signal sequence (residues
1–19) and a mature part (residues 20–564), and the model is
composed of residues 25–540. The overall structure of the
model of IPU and the mutated residues in this study are
shown in Fig. 3. To elucidate the mechanism of the
substrate recognition of IPU, two glucosyl units (Glc +1
and +2, respectively) were forced to be placed based on the
position o f isomaltose bound in the subsites +1 and + 2 of
Dex49A, although IPU does not produce isomaltose.
IPU was predicted to consist of two domains, N-terminal
domain (residues 25–189) and b-h elical domain ( residues
190–540). Asp353, Glu356, Asp372, Asp373, and Trp402,
whose substitutions resulted in the reduction of the activity
for both pullulan an d panose, were predicted to be l ocated
in potential subsites )1and)2 ( a d etailed description is
given i n the next section). T rp31 and Glu273, whose
Fig. 1. Conserved regions of GH family 49 e nzymes. Identical amino acid residues are shown in white on black, and conserved Trp, Asp and Glu
residues are indicated b y asterisks. PMDEX, Penicillium minioluteum dextranase [12]; DEX49A, Penicillium minioluteum dextranase isoform [13];
PFDEXA, Penicillium funiculosum dextranase (DDBJ/EMBL/GenBank No. AJ272066); AGTDEX1 and 2, Arthrobacter globiformis T-3044
endodextranase 1 an d 2 [1 4]; AGCDEX, Arthrobacter sp. CB-8 d extranase [11]; I MTD, Brevibacterium fuscum var. dextranlyticum isomaltotrio-
dextranase [15].
Table 2. Relative activities of wild-type and mutant IPUs for pullulan
and panose. Activities for 0.4% (w/v) pullulan and 32 m
M
panose were
measured. ND, Not d etected.

Pullulan Panose
Wild-type 100 100
W31F 38 140
W32F 90 120
W240F 130 160
W402F 0.4 0.1
E273Q 45 74
D353N ND ND
E356Q 38 50
D372N ND ND
D373N ND ND
AB
Fig. 2. Patterns of hydrolysis for pullulan (A) and panose (B) by W402F
IPU. Reaction mixtures of 5% (w/v) pullulan or 100 m
M
panose with
0.1 m gÆmL
)1
of wild-type or W402F IPU were incubated at 30 °Cfor
1 day (wild-type) or 3 days (W402F). The hydrolysates were analysed
by TLC using the conditions described in Materials and methods.
M, Maltooligosaccharide marker; G1–G7, maltooligosaccharides
glucose to maltoheptaose; Pu, pullulan; Pa, panose; W, wild-type IPU
added; W402F, W402F IPU added. The numbers indicate the time of
reaction in days; 0 , no enzyme added. I P, isopanose; IM, isomaltose.
Table 3. Kinetic parameters of wi ld-type and W402F IPUs for panose.
K
m
(m
M

) k
0
(s
)1
) k
0
/K
m
(m
M
)1
Æs
)1
)
Wild-type 160 ± 3.8 180 ± 2.2 1.13 ± 0.04
W402F 920 ± 140 0.36 ± 0.036 (3.9 ± 1.0) · 10
)4
Ó FEBS 2004 Reaction mechanism of isopullulanase (Eur. J. Biochem. 271) 4423
substitutions caused a decrease in the activity for pullulan
(38 and 45%, r espectively) but not significant for panose
(140 and 74%, respectively), are located r elatively far from
the potential catalytic site, and the side chains were
predicted t o o rient t o the interface b etween N-terminal
and b-helical domains. A structural homology search for the
N-terminal domain (residues 25–189) was also carried out
using the Dali server [44]. N umerous proteins containing an
immunoglobulin-like fold were listed, and among glycosyl
hydrolases, domain N of a pullulan-hydrolysing enzyme
from Thermoactinomyces vulgaris, TVA II (PDB ID, 1BVZ;
Z score of 3.2) [45] was a solution in the Dali result. It is

likely that the inte rface between N-terminal and b-he lical
domains participate in binding of the polysaccharide,
pullulan.
Comparison of the active sites of the model of IPU
and Dex49A
The active site structures of t he model of IPU (Fig. 4A) and
Dex49A (Fig. 4B) were compared. The three Asp residues,
Asp353, Asp372 and Asp373, mutation of which causes
nearly complete loss of the enzymatic activity, were
positioned c lose to the O4 hydroxyl group of Glc +1
residue (Fig. 4A). Larsson et al. reported that, in Dex49A,
the corresponding aspartyl residues , Asp376, 395 and
Asp396, are conserved within GH family 49, and Asp376
and 396 are positioned in a potential )1 subsite [17]
(Fig. 4B). These findings show that Asp353, 372, and 373
are the potential catalytic r esidues of I PU. Also, Trp425 of
Dex49A, which is the corresponding residue of Trp402 of
IPU, is reportedly located in the vicinity of the active site
and could form a binding site for a glucosyl unit i n subsite
)1 (Fig. 4B). Together with the observations from the
site-directed mutagenesis, Trp402 of IPU appears to be a
residue participating in subsite )1.
The r esidues that form potential subsites )1and)2
are reportedly more conserved in GH family 49 than the
residues in s ubsites +1 and +2 [17]. Comparison of the
active sites o f t he model of IPU and D ex49A c learly shows
that residues located in the negatively numbered subsites are
highly conserved between IPU and Dex49A (Fig. 4). In
addition t o the r esidues Asp353(IPU)-376(Dex49A),
Asp373–396, Glu356–379, and Trp402–425, numerous

aromatic and charged residues Arg297–322, Asn323–348,
Asp326–351, Tyr358–381, Lys376–399, Tyr378–401,
Tyr379–402, and Tyr440–463, are conserved in the negat-
ively numbered subsites. On the other hand, residues located
in the positively numbered s ubsites are relatively not
conserved. The report o f Dex49A shows an illustration
where seven amino acid residues, Asp86, Tyr303, Lys315,
Asp395, Asn417, Lys447, and Glu449, interact with Glc +1
and +2 [17] (Fig. 4B). Only two of these residues,
Tyr278(IPU)-303(Dex49A), and Asp372–395, both o f
which interact with Glc +1, are conserved between IPU
and Dex49A. The position equivalent to Lys315 o f Dex49A
is identified as Gly290 of IPU, which may enable IPU to
incorporate the a-(1fi4)-linked glucose un its. In addtion, in
Dex49A, Phe373 protrudes to the active cleft, which appears
to restrict the c onformation of the s ubstrate and accom-
modate only the a-(1fi6)-linked glucose units. In IPU, the
position e quivalent to Phe373 of Dex49A is identified as
Gly350, which allows IPU to have a relatively wide cleft,
thus it seems to b e possible that both a-(1fi4)-linked and a-
(1fi6)-linked glucose units enter the active cleft of I PU.
However, residues corresponding to Asn417 and Glu449 o f
Dex49A are v irtually lack ing in I PU because positions
equivalent to Asn417 and Glu449 of Dex49A are ident ified
as Val39 4 and Gly426 of IPU, respectively. Therefore, even
if the a-( 1fi6)-linked glucose units enter to the active cleft of
IPU as shown in Fig. 4 A, it would be impossible for the
substrate to be retained in the cleft. In the model of IPU,
several aromatic and charged residues, Trp277, Tyr349, and
Asp371, are p resent in t he v icinity of G ly350, and c ould b e

favourable fo r binding of Glc +2 of the a-(1fi4)-linked
glucose units of pullulan (Fig. 4A).
IPU is an anomer-inverting enzyme
In GH family 49 enzymes, Dex49A has been identified as an
anomer-inverting enzyme by using N MR spectroscopy [17].
To compar e the mechanism of h ydrolysis of IPU and o ther
GH family 49 dextranases, the a nomer configuration of the
hydrolysate of IPU was determined. A polarimetric assay
was carried out using panose as the substrate. The anomeric
forms of t he hydrolysate are equilibrated immediately by
the addition of ammonium hydroxide, and the change in the
optical rotation was compared with those of bacterial
a-glucosidase (retaining enzyme) and fungal glucoamylase
Fig. 3. Overall structure of the model of IPU. N-terminal and b-helical
domains are shown in light pink and grey, respectively.Theaminoacid
residues who se s ubstitu tions re sulted in the reduction of the activity for
both pullulan and panose (red), and substitutions that caused a
decrease in the activity for pullulan (blue), are shown. Other mutated
residues are indicated in light brown. G lucosyl units in subsites +1 an d
+2 (Glc +1 an d +2, respect ively), based on the position of isomaltose
in the Dex49A structu re, are shown i n yellow. The figure was generated
using
RASTOP
.
4424 H. Akeboshi et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(inverting enzyme), respectively [43]. The optical rotation
decreased with t he addition of ammonium hyd roxide for
a-glucosidase, while it increased for glucoamylase and also
IPU. The results indicated that IPU is an anomer-inverting
enzyme (Fig. 5A).

In the case of inverting enzymes, a single displacement
mechanism has been proposed [6,17,19–21]. In this mech-
anism, two catalytic residues function as a general acid
(donating a p roton) and a general base ( activating the
nucleophilic water m olecule) in the first step of the reaction.
A carbonium ion intermediate subsequently forms, and is
further attacked by the water molecule. The study of
site-directed mutagenesis, however, indicated that the three
Asp residues, A sp353, Asp372, and Asp373, are the
potential catalytic r esidues of IPU. T he report of the crystal
structure of Dex49A also mentioned that either Asp376 or
Asp396, the residues corresponding to Asp353 and Asp373
of IPU, respectively, appears to be properly positioned to
act as a base in the hydrolytic reaction [17]. Three Asp
residues are also strictly conserved in t he catalytic centre of
GH family 28 polygalacturonases and rhamnogalacturon-
ases [18–20], another family of inverting enzymes forming
the clan GH-N w ith GH family 49 enzymes. van San ten
et al. [19] and Shimizu et al. [20] reported that a n Asp
Fig. 4. Comparison of the active site structures of the model of IPU (A) and Dex49A (B). Conserved residues between IPU and De x49A are shown in
red (mutated res idues i n this study) o r orange. Residu es that are u niqu ely found in IPU and may interact with the substrate (see Results and
Discussion), are shown in cyan. Residues that are uniquely found in Dex49A and interact with Glc +1 and +2 are shown in green. Other colour
representations a re as in Fig. 3. The figures w ere generated using
MOLSCRIPT
[40] and
RASTER
3
D
[41].
Fig. 5. Enzymatic properties of I PU. (A) Optical ro tation during the h ydrolysis of s ubstrates by a-glucosidase (top), g lu coamylase (middle), and

IPU (bott om) was observed. Reaction mixtu r es consist of 1.6% maltotetraose in 50 m
M
acetate buffer (pH 4.5) with a-glucosidase or glucoamylase,
and 1.8% panose with IPU in 50 m
M
acetate buffer (pH 3.5). The mutarotation was achieved by adding 20 lLof15
M
ammonium hydroxide at the
point indicated by arrows. (B) Hydrolysis of IPU for panose, IMTG and MM were compared. Symbols: Circle, glucose residue; Circle with line,
glucose residue of the reducing end; ¾
c
, a-1,6-glucosidic linkage; – ; a-1,4-glucosidic linkage; triangle, cleaving site. G rey circles indicate the residue
exterior to t he panose motif. A ctivity is defined a s the amount o f product (mmol) r ele ased by 1 mg IPUÆmin
)1
.
Ó FEBS 2004 Reaction mechanism of isopullulanase (Eur. J. Biochem. 271) 4425
residue of the polygalacturonases (position equivalent to
Asp372 of IPU) has been proposed to act as t he acid
(proton donor), while there are two candidates f or a general
base, t wo Asp r esidues of the polygalacturonases ( positions
equivalent to Asp353 and Asp373 of IPU).
Although the overall structure is completely different,
glucoamylase is known as an inverting enzyme and
hydrolyses a-(1fi4)-glucosidic linkages s uch as that in
IPU. Glucoamylase and IPU are c lassified into d ifferent
GH families o f 1 5 a nd 49, respectively, but three conserved
acidic residues are found in each of the GH families, and
two of them are consecutively numbered residues. In the
glucoamylase from Aspergillus awamori, the corresponding
acidic residues are Glu179, Glu180, and Glu400, and

Glu179 and Glu400 have been reported t o function as a
general a cid a nd g eneral b ase, res pectively [4 6]. S ierks et al.
suggested that Glu179 is the general acid catalyst of pK
a
5.9,
and that the adjacent Glu180 is negatively charged, raising
the p K
a
of the general acid catalyst [23]. I t is likely t hat
catalytic residues of IPU adopt a similar catalytic mechan-
ism to those of glucoamylase.
Enzymatic properties of wild-type IPU
The modelling s tudy of the IPU structure i ndicated that the
conserved and functionally important residues of both
Dex49A and I PU are found in the negatively numbered
subsites. The anomeric configuration of products of both
Dex49A and IPU are identical, as well. Does the substrate
preference of the negatively numbered s ubsites of IPU also
resemble that of GH family 49 dextranases even though IPU
does not hydrolyse dextran at all? IPU not only h ydrolyses
panose and a polymer of panose, pullulan [1,30], but also the
oligosaccharides containing the panose structure such as
IMTG (Glc-a-(1fi6)-Glc-a-(1fi6)-Glc-a-(1fi4)-Glc) [16],
MM (Glc -a-(1fi4)-Glc-a-(1fi6) -Glc-a-(1fi4)-Glc) [1,16],
4
2
-a-isomaltosylisomaltose (Glc-a-(1fi6)-Glc-a-( 1fi4)-
Glc-a-(1fi6)-Glc) [16], and 6
3
-a-glucosylmaltotriose

(Glc-a-(1fi6)-Glc-a-(1fi4)-Glc-a-(1fi4)-Glc) [1,16]. We
measured the activities of IPU for panose, IMTG and
MM, because IPU releases only glucose from the reducing
end side o f t hese substrates (Fig. 5B). The activities of IPU
for 32 m
M
of IMTG, panose a nd MM were assayed, and
1 mg of the enzyme liberated 18.5, 14.0 and 7.37 lmolÆ
min
)1
of glucose, respectively. Although IPU w as originally
reported as a pullulan-hydrolase [1], MM, part of the
structure of pullulan, was the poorest substrate among these
three oligosaccharides, while IMTG, whose portion bound
to the negatively numbered subsites is composed of the
structure of d extran (Glc-a-(1fi6)-Glc-a-(1 fi6)-Glc), was
the best substrate. These findings suggest that both IPU and
the GH family 49 dextranase have a s imilar catalytic
mechanism i n their negatively numbered s ubsites in spite of
the difference o f their substrate s pecificities.
IPU h as been originally reported as the pullulan-hydro-
lase [1], but the principal substrate is still not clear because of
the low affinities f or pullulan a nd panose (K
m
¼ 5 .7% [4 7]
and 160 m
M
, respectively). What might be the physiological
role of this enzyme in A. niger? In several Aspergillus
species, A. oryzae [48] and A. nidulans [49], isomaltose and

panose are known as effective inducers for amylase
synthesis. In the case of A. nidulans, a mylase synthes is is
induced at an extremely low concentration ( 3 l
M
)of
isomaltose. Kato et al. reported that t wo a-glucosidases
from A. nidulans, AgdA and AgdB, showed strong trans-
glycosylation activity to produce isomaltose from maltose,
and they are suggested to participate in the maltose-
dependent induction of amylase s ynthesis along with other
undetected isomaltose-forming enzymes [49,50]. It is likely
that A. niger has a mechanism similar to such amylase
synthesis, and IPU may collaborate to produce isomaltose
from panose and other branched oligosaccharides with
some transglycosidases. Since t he extremely l ow concentra-
tion of isomaltose is effective for the induction of amylase
synthesis, the low activity of IPU may be suitable for control
of this regulation.
Acknowledgements
We thank Mr Masahiro Mizuno for his useful advice and discussions.
This study was supported in part by the Novozymes J apan R ese arch
Fund.
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Ó FEBS 2004 Reaction mechanism of isopullulanase (Eur. J. Biochem. 271) 4427