To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 four Trp, three Asp and two Glu.. Homol
Trang 1Insights into the reaction mechanism of glycosyl hydrolase family 49
Site-directed mutagenesis and substrate preference of isopullulanase
Hiromi Akeboshi1, Takashi Tonozuka1, Takaaki Furukawa1, Kazuhiro Ichikawa1, Hiroyoshi Aoki1,2,
Akiko Shimonishi1, Atsushi Nishikawa1and Yoshiyuki Sakano1
1
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan;
2
Fuence Co., Shibuya, Tokyo, Japan
Aspergillus nigerisopullulanase (IPU) is the only
pullulan-hydrolase in glycosyl pullulan-hydrolase (GH) family 49 and does not
hydrolyse dextran at all, while all other GH family 49
enzymes are dextran-hydrolysing enzymes To investigate
the common catalytic mechanism of GH family 49 enzymes,
nine mutants were prepared to replace residues conserved
among GH family 49 (four Trp, three Asp and two Glu)
Homology modelling of IPU was also carried out based on
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 pullulan and panose, were predicted to be
located in the negatively numbered subsites Three
Asp-mutated enzymes, D353N, D372N and D373N, lost their
activities, indicating that these residues are candidates for the
catalytic residues of IPU The W402F enzyme significantly
reduced IPU activity, and the Kmvalue was sixfold higher and the k0value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participa-ting in subsite)1 Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and b-helical domains The substrate preference
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 catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities
Keywords: dextranase; GH family 49; isopullulanase; pullu-lan-hydrolase; site-directed mutagenesis
Isopullulanase (IPU, EC 3.2.1.57; pullulan
4-glucanohydro-lase) from Aspergillus niger ATCC9642 hydrolyses pullulan
to produce isopanose (Glc-a-(1fi4)-Glc-a-(1fi6)-Glc) and
also hydrolyses substrates containing the panose
(Glc-a-(1fi6)-Glc-a-(1fi4)-Glc) structure, 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 hydrolyses a-1,6-glucosidic linkages to
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 hydrolyses the other a-1,4-glucosidic linkages to produce isopanose Except for IPU, these enzymes are classified into glycosyl hydrolase (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 and a-amylase family enzymes has been found (http://afmb.cnrs-mrs.fr/cazy/CAZY/ index.html)
Interestingly, IPU does not hydrolyse dextran at all, while all other 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 indicarepor-ted that seven highly conserved regions are found among the primary 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 (Dex49A), which shows a 38% sequence identity with IPU, has been reported, and the catalytic domain folds into a right-handed parallel b-helix [17] Crystal structures of polygalacturonases and rhamnogalac-turonases, which belong to GH family 28, have been solved
by many researchers (for example [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: sakano@cc.tuat.ac.jp
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-endo-dextranase (EC 3.2.1.95);
glucoamylase (anomer-inverting enzyme; EC 3.2.1.3); a-glucosidase
(anomer-retaining enzyme; EC 3.2.1.20).
(Received 23 June 2004, revised 16 August 2004,
accepted 27 September 2004)
Trang 2[10,17] Despite these advances, little is known about 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 and/or Glu, are commonly reported as the catalytic
residues of GHs [6,21–26], and it is probable that IPU has
Asp and/or Glu as the catalytic residues In addition, in the
chemical modification experiment IPU was inactivated by
N-bromosuccinimide, indicating that a Trp residue is
required for IPU activity [27] Therefore, several Asp and/
or Glu, and Trp residues located in the conserved regions
are predicted to be indispensable for IPU and 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 of 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 to the
manufacturer’s recommendations Yeast peptone glycerol
medium (YPGY: 1% yeast extract, 2% peptone, and 1%
glycerol) was prepared for the propagation of recombinant
P pastoris strain for the expression of enzymes All
cultivation was done at 37C for E coli and 30 C for
P pastoris
Mutant constructions
All cloning procedures were carried out by applying
standard molecular biological techniques [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 pIPA118(–), a plasmid DNA
coding for mature IPU The complementary mutagenic
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 templates and primers, M13
forward and M13 reverse After sequence confirmation, the
EcoRI–BamHI fragment was inserted into the expression
vector for P pastoris, pHIL-S1 (Invitrogen) Plasmids
coding for E273Q, D353N, E356Q, D372N, and D373N
were constructed by the method of Kunkel [29] The sites at
which the mutations were introduced are located in the
StyI–XbaI fragment To construct reliable mutants,
mutated StyI–XbaI fragments were verified by DNA
sequencing, and the original StyI–XbaI fragment of pIPA118(–) was replaced with the mutated StyI–XbaI fragment The EcoRI–BamHI fragments of 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 described [16] with slight modifications Briefly, P pastoris harbouring pSig-PHO was cultured in
250 mL YPGY for 2 days, and the propagated cells collected by centrifugation (5000 g for 10 min) were resus-pended and cultivated for 6 days in 100 mL BMM The clear supernatant of cultured BMM (crude IPU) was then obtained by centrifugation (5000 g for 15 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 inducer The expression of IPU mutants was performed using the same procedure as for the wild-type IPU
Substrates Pullulan was obtained from Hayashibara, Japan Panose [30], IMTG (41-a-isomaltotriosylglucose, also called
62-a-isomaltosylmaltose; Glc-a-(1fi6)-Glc-a-(1fi6)-Glc-a-(1fi4)-Glc) [31], and MM (62-a-maltosylmaltose; 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 mM 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 case letters indicate the nucleotide mutations To facilitate the selection of mutant clones, silent mutations were made to introduce restriction enzyme recognition sites (underlined).
Pimers Nucleotide sequence (5¢fi3¢) W31F CTGACCTtcTGGCATAACACCGGtGAAATC
AgeI W32F CCTGGTtcCATAACACCGGtGAAATC
AgeI W240F GGTGCTgAGCTCAAGTGTGACTTtcGTCTAC
SacI W402F CCGGTGGTcGAcTTTGGTTtcACGCCC
SalI E273Q ACGTACTGCTgTCCGGAAAGtACtCCATGGCCGC
ScaI D353N TCCAATCCGTtAGTCTGgCCaTAGAACGCGC
MscI E356Q GGAGAATGGTgCCAGGGTACATTTcCAATCCGTCA
KpnI D372N AATACATCTTaAGGCCGTCGTtGTCGGTGTGG
AflII D373N AATACATCTTaAGGCCGTtGCGTCGGTG
AflII
Trang 3Assays of IPU activity and protein concentration
The pullulan-hydrolysing activity of IPU was evaluated as
described previously [34] The activities for panose, IMTG
and MM were measured as follows A reaction mixture
consisting of a desired substrate (32 mM) and IPU in
40 mMacetate buffer (pH 3.5) was incubated at 40C for
30 min, and the reaction was terminated by the addition
of an equal volume of 0.1M Na2CO3 The amount of
glucose produced by IPU was assayed with Glucose 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 panose,
mixtures consisting of 4 lgÆmL)1enzyme and from 43 to
216 mM substrate, and 20 lgÆmL)1 enzyme and 64 to
480 mM substrate, respectively, were used The protein
concentration was measured by the method of Lowry
et al with BSA as standard [37]
TLC
TLC was performed to analyse the hydrolysates of the
W402F enzyme The reaction mixture consisting of
pullu-lan (5%) or panose (100 mM) and the W402F enzyme
(0.1 mgÆmL)1) in 50 mM acetate buffer (pH 3.5) was
incubated at 30C for 3 days, and the hydrolysates were
developed by TLC with 1-butanol/ethanol/H2O¼ 2/2/1
(v/v/v) The spots were detected by charring with H2SO4
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 the Swiss-Model
server (http://swissmodel.expasy.org/) [38] using the first
approach mode, and a model consisting of residues 25–540,
which is based on the structure of 1OGM, was obtained To
determine the potential catalytic site, this model was
superimposed on 1OGO using the programDEEPVIEW[38],
and a glucosyl units in subsites +1 and +2 were placed in
the model The figure was generated using the programs
RASMOL[39],RASTOP(http://www.geneinfinity.org/rastop/),
MOLSCRIPT[40] andRASTER3D[41]
Polarimetric assays
Polarimetric measurements of IPU, glucoamylase from
Rhizopus niveus(Seikagaku Kogyo, Japan;
anomer-invert-ing enzyme; EC 3.2.1.3), and a-glucosidase from Bacillus sp
(Toyobo, Japan; anomer-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 dissolved in 50 mMacetate buffer (pH 4.5), and
the optical rotations were measured at 1-min intervals at
589 nm using a JASCO DIP-360 polarimeter After 10 min
(IPU) or 20 min (R niveus glucoamylase and Bacillus
a-glucosidase), 20 lL of 15 ammonium hydroxide was
added to the reaction mixture to 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 IPU 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% saturation 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 higher than those of IPU from original A niger and heterologously expressed IPU from
A oryzae (27 and 38ÆU mg)1, respectively) [7,27] The mutated IPUs were purified with the same procedure as wild-type IPU
Properties of Trp-mutated enzymes Previous experiments indicated that some Trp residues are essential for IPU activity [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 W402F enzyme lost the activity for pullulan ( 0.4% of the wild-type IPU) and the activity for panose was almost undetectable ( 0.1%) under the given conditions The W31F enzyme had only 38% activity for pullulan, but the activity for panose was 1.4-fold higher than that of wild-type enzymes The activities of W32F and W240F were similar (90–160%) to that of wild-type enzyme
As the activity of W402F drastically decreased, its action pattern was investigated using TLC The 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 Kmvalue was sixfold higher and the k0value 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 residues of IPU, these Asp and 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 enzyme (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
Trang 4significantly The mutant E356Q had 38% and 50% of the activities for pullulan and panose, respectively In contrast, the activity of E273Q for pullulan was 45%, while that for panose remained at 74% The sixth mutant E157Q is not obtained in the P pastoris expression system, but has been expressed by using 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 minioluteumdextranase, Dex49A, complexed with a prod-uct, isomaltose, has been reported (PDB ID, 1OGO) [17] The identity between the primary structures of Dex49A and IPU is 38%, and a three-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 of 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-helical domain (residues 190–540) Asp353, Glu356, Asp372, Asp373, and Trp402, whose substitutions resulted in the reduction of the activity for both pullulan and panose, were predicted to be located
in potential subsites )1 and )2 (a detailed description is given in the next section) Trp31 and Glu273, whose
Fig 1 Conserved regions of GH family 49 enzymes Identical amino acid residues are shown in white on black, and conserved Trp, Asp and Glu residues are indicated by 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 and 2 [14]; AGCDEX, Arthrobacter sp CB-8 dextranase [11]; IMTD, 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 detected.
Pullulan Panose
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 mgÆmL)1of wild-type or W402F IPU were incubated at 30 C for
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 IP, isopanose; IM, isomaltose.
Table 3 Kinetic parameters of wild-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
Trang 5substitutions caused a decrease in the activity for pullulan
(38 and 45%, respectively) but not significant for panose
(140 and 74%, respectively), are located relatively far from
the potential catalytic site, and the side chains were
predicted to orient to the interface between 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] Numerous 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 interface between N-terminal and b-helical
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 the 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 close 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 residues of IPU 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 in 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 residues that form potential subsites )1 and )2 are reportedly more conserved in GH family 49 than the residues in subsites +1 and +2 [17] Comparison of the active sites of the model of IPU and Dex49A clearly shows that residues located in the negatively numbered subsites are highly conserved between IPU and Dex49A (Fig 4) In addition to the residues 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 subsites are relatively not conserved The report of 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 of which interact with Glc +1, are conserved between IPU and Dex49A The position equivalent to Lys315 of Dex49A
is identified as Gly290 of IPU, which may enable IPU to incorporate the a-(1fi4)-linked glucose units In addtion, in Dex49A, Phe373 protrudes to the active cleft, which appears
to restrict the conformation of the substrate and accom-modate only the a-(1fi6)-linked glucose units In IPU, the position equivalent to Phe373 of Dex49A is identified as Gly350, which allows IPU to have a relatively wide cleft, thus it seems to be possible that both (1fi4)-linked and a-(1fi6)-linked glucose units enter the active cleft of IPU However, residues corresponding to Asn417 and Glu449 of Dex49A are virtually lacking in IPU because positions equivalent to Asn417 and Glu449 of Dex49A are identified
as Val394 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 4A, 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 present in the vicinity of Gly350, and could be favourable for 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 NMR spectroscopy [17]
To compare the mechanism of hydrolysis of IPU and other
GH family 49 dextranases, the anomer configuration of the hydrolysate of IPU was determined A polarimetric assay was carried out using panose as the substrate The anomeric forms of the 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 The amino acid
residues whose substitutions resulted 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 Glucosyl units in subsites +1 and
+2 (Glc +1 and +2, respectively), based on the position of isomaltose
in the Dex49A structure, are shown in yellow The figure was generated
using RASTOP
Trang 6(inverting enzyme), respectively [43] The optical rotation
decreased with the addition of ammonium hydroxide 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 proton) and a general base (activating the
nucleophilic water molecule) 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, Asp353, Asp372, and Asp373, are the potential catalytic residues of IPU The 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 the catalytic centre of
GH family 28 polygalacturonases and rhamnogalacturon-ases [18–20], another family of inverting enzymes forming the clan GH-N with GH family 49 enzymes van Santen
et al [19] and Shimizu et al [20] reported that an Asp
Fig 4 Comparison of the active site structures of the model of IPU (A) and Dex49A (B) Conserved residues between IPU and Dex49A are shown in red (mutated residues in this study) or orange Residues that are uniquely 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 are as in Fig 3 The figures were generated using MOLSCRIPT [40] and RASTER 3 D [41].
Fig 5 Enzymatic properties of IPU (A) Optical rotation during the hydrolysis of substrates by a-glucosidase (top), glucoamylase (middle), and IPU (bottom) was observed Reaction mixtures 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 lL of 15 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;c, a-1,6-glucosidic linkage; –; a-1,4-glucosidic linkage; triangle, cleaving site Grey circles indicate the residue
exterior to the panose motif Activity is defined as the amount of product (mmol) released by 1 mg IPUÆmin)1.
Trang 7residue of the polygalacturonases (position equivalent to
Asp372 of IPU) has been proposed to act as the acid
(proton donor), while there are two candidates for a general
base, two Asp residues 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 such as that in
IPU Glucoamylase and IPU are classified into different
GH families of 15 and 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 to function as a
general acid and general base, respectively [46] Sierks et al
suggested that Glu179 is the general acid catalyst of pKa5.9,
and that the adjacent Glu180 is negatively charged, raising
the pKa of the general acid catalyst [23] It is likely that
catalytic residues of IPU adopt a similar catalytic
mechan-ism to those of glucoamylase
Enzymatic properties of wild-type IPU
The modelling study of the IPU structure indicated that the
conserved and functionally important residues of both
Dex49A and IPU 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 subsites of IPU also
resemble that of GH family 49 dextranases even though IPU
does not hydrolyse dextran at all? IPU not only hydrolyses
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],
42-a-isomaltosylisomaltose
(Glc-a-(1fi6)-Glc-a-(1fi4)-Glc-a-(1fi6)-Glc) [16], and 63-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 of these substrates (Fig 5B) The activities of IPU
for 32 mMof IMTG, panose and MM were assayed, and
1 mg of the enzyme liberated 18.5, 14.0 and 7.37 lmolÆ
min)1of glucose, respectively Although IPU was 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 dextran (Glc-a-(1fi6)-Glc-a-(1fi6)-Glc), was
the best substrate These findings suggest that both IPU and
the GH family 49 dextranase have a similar catalytic
mechanism in their negatively numbered subsites in spite of
the difference of their substrate specificities
IPU has been originally reported as the
pullulan-hydro-lase [1], but the principal substrate is still not clear because of
the low affinities for pullulan and panose (Km¼ 5.7% [47]
and 160 mM, 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, amylase synthesis is
induced at an extremely low concentration ( 3 lM) of isomaltose Kato et al reported that two 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 synthesis 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 the extremely low concentra-tion of isomaltose is effective for the inducconcentra-tion 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 Japan Research Fund.
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