The crystal structure of Thermoactinomyces vulgaris R-47 a-amylase II TVA II complexed with transglycosylated product Masahiro Mizuno1, Takashi Tonozuka1, Akiko Uechi1, Akashi Ohtaki2, K
Trang 1The crystal structure of Thermoactinomyces vulgaris R-47 a-amylase II (TVA II) complexed with transglycosylated product
Masahiro Mizuno1, Takashi Tonozuka1, Akiko Uechi1, Akashi Ohtaki2, Kazuhiro Ichikawa1,
Shigehiro Kamitori2, Atsushi Nishikawa1and Yoshiyuki Sakano1
1
Departments of Applied Biological Science and2Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan
An a-amylase (TVA II) from Thermoactinomyces vulgaris
R-47 efficiently hydrolyzes a-1,4-glucosidic linkages of
pullulan to produce panose in addition to hydrolyzing
starch TVA II also hydrolyzes a-1,4-glucosidic linkages of
cyclodextrins and a-1,6-glucosidic linkages of isopanose To
clarify the basis for this wide substrate specificity of TVA II,
we soaked 43-a-panosylpanose (43-P2) (a pullulan
hydro-lysate composed of two panosyl units) into crystals of
D325N inactive mutated TVA II We then determined the
crystal structure of TVA II complexed with 42
-a-pano-sylpanose (42-P2), which was produced by
transglycosyla-tion from 43-P2, at 2.2-A˚ resolution The shape of the active
cleft of TVA II is unique among those of a-amylase family
enzymes due to a loop (residues 193–218) that is located at
the end of the cleft around the nonreducing region and forms
a dam-like bank Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II While Trp356
is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-li-ganded state and the other is supplying the water that is necessary for substrate hydrolysis
Keywords: a-amylase; GH family 13; 42-a-panosylpanose; substrate specificity; transglycosylation
a-Amylase (1,4-a-D-glucan-4-glucanohydrolase; EC 3.2.1.1)
hydrolyzes a-1,4-glucosidic linkages of starch to release
a-anomer products Numerous enzymatic properties of
a-amylase have been reported, due to the industrial
importance of this enzyme in food and pharmaceutical
fields According to the classification system proposed by
Henrissat et al [1–3], a-amylases are classified into
glycoside hydrolase (GH) family 13
Thermoactinomyces vulgarisR-47 produces a-amylase II
(TVA II) as an intracellular enzyme [4] TVA II hydrolyzes
a-1,4-glucosidic linkages of starch like other a-amylase
family enzymes to produce mainly maltose In addition
to a-amylase activity, TVA II hydrolyzes a-1,4-glucosidic
linkages of pullulan to produce panose [5,6] via an activity
proposed as neopullulanase activity by Kuriki et al [7] TVA II also hydrolyzes a-1,4-glucosidic linkages of cyclo-dextrins [8] and a-1,6-glucosidic linkages of isopanose [9,10] The crystal structure of TVA II has been determined at 2.3-A˚ resolution [11,12], and TVA II has been shown to form a dimeric structure (Fig 1A) Each monomeric subunit of TVA II is composed of four structural domains,
N (residues 1–121), A (residues 122–242 and 298–502),
B (residues 243–297), and C (residues 503–585) (Fig 1B) Domain A forms a (b/a)8-barrel structure that is the catalytic unit containing three catalytic residues (Asp325, Glu354 and Asp421), which is typical of a-amylase family enzymes Domain B is a small component which protrudes from the third b-strand of domain A Domain C is also highly conserved among a-amylase family enzymes, but its function is still not so clear TVA II has a notable extra domain consisting of 120 amino acid residues at the N-terminus, called domain N, which appears to be involved
in forming the dimeric structure [13] The N domains of both molecules are involved in forming each of the active clefts in cooperation with the A domains
TVA II shows broader substrate specificity than other a-amylase family enzymes: for example, it hydrolyzes a-1,4-glucosidic linkages of starch, pullulan and cyclodextrin, and a-1,6-glucosidic linkages of isopanose It is still unclear what accounts for this broad substrate specificity of TVA II We have already reported the structures of TVA II complexed with maltotetraose [14] and cyclodextrins [14,15], while the structure of the complex with an oligosaccharide based on pullulan has not been analyzed In this study, to analyze the pullulan recognition mechanism, we first developed a
Correspondence to Y Sakano, Department of Biotechnology and
Life Science, Tokyo University of Agriculture and Technology,
3-5-8 Saiwai-Cho, Fuchu, Tokyo 183-8509, Japan.
Fax: + 81 42 3675705, Tel.: + 81 42 3675704,
E-mail: sakano@cc.tuat.ac.jp
Abbreviations: TVA II, Thermoactinomyces vulgaris R-47 a-amylase
II; GH, glycoside hydrolase; PEG, polyethylene glycol; MPD,
2-methyl-2,4-pentanediol; 4 3 -a-panosylpanose (4 3 -P2),
Glcp(a1 fi 6)Glcp(a1 fi 4)Glcp(a1 fi 4)Glcp(a1 fi 6)
Glcp(a1 fi 4)Glc; 4 2
-a-panosylpanose (42-P2), Glcp(a1 fi 6) Glcp(a1 fi 4)Glcp(a1 fi 4)[Glcp(a1 fi 6)] Glcp(a1 fi 4)Glc)
(Glcp(a1 fi 6)Glcp(a1 fi 4)Glcp(a1 fi 4)[Glcp(a1 fi 6)]
Glcp(a1 fi 4)Glc.
Enzyme: 1,4-a- D -glucan-4-glucanohydrolase (EC 3.2.1.1).
(Received 25 February 2004, accepted 23 April 2004)
Trang 2method to form a complex between TVA II and a pullulan
model substrate by using partial hydrolyates of pullulan and
an inactive TVA II mutant, D325N A hexasaccharide
containing two panose units, 43-a-panosylpanose (43-P2)
(Fig 2A), was thereby prepared We analyzed the crystal
structure of the complexed form at 2.2-A˚ resolution and
found that the transglycosylation product was bound in the
active cleft
Materials and methods
Gene construction for Y374A-TVA II mutant
The gene manipulation methods were based on those of
Sambrook et al [16] Site-directed mutagenesis was carried
out using plasmid pTN302-10 as described [8] according to
the method of Kunkel et al [17] To construct the Y374A
mutant, the following oligonucleotide was used as a
mutagenic primer: 5¢-GATCACACTCTCGCGAAACA
AATAATTCATCACCG-3¢ The underlined nucleotide in
the primer indicates the mismatched nucleotide creating the
alanine substitution mutation DNA sequencing confirmed
the presence of the mutation The gene construction for the
D325N mutant has already been reported [18]
Purification, crystallization and data collection
The mutated TVA II was prepared using recombinant
Escherichia coli MV1184 cells and was purified as
described [19] The crystals of D325N were grown at
20C using the hanging-drop method, in which 1.5 lL of
a 20 mgÆmL)1D325N solution in 5 mM Tris/HCl buffer (pH 7.5) was mixed with the same volume of a reservoir solution containing 1% (w/v) PEG6000, 5 mM CaCl2 in
40 mM Mes/NaOH (pH 6.1) The crystal complex of D325N with 43-P2 prepared by the same method as described in our previous paper [20] was obtained by soaking the crystal in cryo-protectant solution [20% (w/v) PEG6000, 20% (v/v) MPD, 2.5 mM CaCl2] containing
10 mM43-P2 for 10 h The diffraction data was collected
at the beam line of BL18B, PF (Photon Factory, Japan) The data was processed and scaled using the programs DPS/MOSFLM[21]
Structure refinement The structure of the D325N complex was solved by molecular replacement using the unliganded TVA II as the search model The 2Fo–Fc electron density map showed that a continuous density 1 r contoured level for all atoms of the protein is seen except for Ser276-Arg280 of both subunits After simulated annealing refinement using the program CNS [22], the different Fourier maps clearly revealed a density corresponding to
a hexasaccharide Water molecules were added automat-ically using CNS and a 3.0 r cut-off for peaks in Fo–Fc maps To avoid overfitting of the diffraction data, a free
R factor with 10% of the test set excluded from
Fig 1 Ribbon representation of the fold of TVA II in complex with 42-P2 (A) Dimeric form (B) Monomeric form MOL-1, MOL-2 and each domain are shown by different gray scales Darker hues are used for MOL-1 Names of each domain with or without the asterisk represent MOL-1
or MOL-2 Three catalytic residues are drawn in black stick and 4 2 -P2 molecules are drawn in red sticks The bound calcium ions are shown as black spheres Figures were produced with MOLSCRIPT [35] and RENDER from the R ASTER 3D package [36].
FEBS 2004 Structure of T vulgaris a-amylase II complex (Eur J Biochem 271) 2531
Trang 3refinement was monitored [23] Refinement of the final structure were converged at an R factor of 0.194 (Rfree¼ 0.233), and contained 1170 amino acid residues, two cal-cium ions, two 42-P2 molecules and 399 water molecules Model quality and refinement statistics
Refinement statistics are presented in Table 1 Analysis of the Ramachandran plot [24], calculated with the program PROCHECK[25], revealed that 86.2% of residues in MOL-1 and 84.9% of residues of MOL-2 were in the most favored region, and only one residue (Thr278 of MOL-2) was found
in disallowed region
Protein Data Bank accession number The atomic coordinates and structure factors of the D325N complex (PDB code 1VB9) have been deposited in the Protein Data Bank
Kinetic study Purified enzyme (diluted to 0.01 mgÆmL)1, 120 lL) was added to 480 lL of various concentrated substrates (soluble starch was purchased from Merck, Germany; pullulan was obtained from Hayashibara Biochemical Laboratories, Japan) in 100 mMsodium phosphate buffer (pH 6.0), and the hydrolysis reaction was started at 40C, with sampling every 5 min After the reaction had stopped, the method of Somogyi-Nelson [26] was followed
Table 1 Data collection and refinement statistics.
42-P2 complex Data collection
Cell dimensions
R merge
a
0.053 (0.236)b
Structure refinement
a R merge ¼ SS|I i – <I>|/S<I> b The values for the highest
resolution shell are given in parentheses (2.34–2.20-A˚ resolution).
Fig 2 Topologies of pullulan model oligosaccharides (A) 43-a-Panosylpanose is abbreviated 43-P2 (B) 42-a-Panosylpanose is abbreviated 42-P2 (C) The F o –F c electron density map of 4 2 -P2 bound at the active site The number of glucose units is labeled from )3 (nonreducing end) to +2 (reducing end), except for +2¢, which branches from +1 with an a-1,6-glucosidic linkage in 42-P2 The contour level is 2.0 r.
Trang 4Carbohydrate in the catalytic site
The structure of the complex was determined by
molecular replacement using the structure of unliganded
TVA II (PDB code 1JI2) [12] as a search model In the
final model, there were two subunits (MOL-1 and
MOL-2) related by noncrystallographic twofold
sym-metry, in an asymmetric unit (Fig 1A) MOL-1 and
MOL-2 are homodimers The overall structure of the
complex form was essentially identical to that of
unliganded TVA II except for the induced fitting of
some regions, residues 137–170, residues 417–425 and
residues 455–481, composed the catalytic cleft The root
mean square deviation value calculated for the whole Ca
chain is 1.10 A˚
After the initial structural refinement, the difference
Fourier map indicated that the clear continuous electron
densities for the pentasaccharide consisted of the five
glucose units, labeled Glc)3, )2, )1, +1 and +2, at the
active sites of both MOL-1 and MOL-2 This
pentasaccha-ride occupied subsites)3 to +2 (The subsites are numbered
based on the nomenclature of Davies et al [27]) Also, weak
density was seen around the O6 of Glc +1 at the center of
the active site After refinement with this pentasaccharide,
the rest of the density was clearly shown This new density
was assigned as a glucose unit and labeled as Glc +2¢, and
its electron density between O1 of Glc +2¢ and O6 of Glc
+1 seemed to be connected with the a-1,6-glucosidic
linkage (Fig 2C) Therefore, the oligosaccharide bound
at the active site was determined to be 42-a-panosylpanose
(42-P2) (Fig 2B)
The most surprising thing about these results is that
the mutated enzyme was soaked in a solution of 43-P2
(Fig 2A), bu t that 42-P2 (Fig 2B) was actually bound in
the active site instead of 43-P2 In TVA II, Asp325, Glu354
and Asp421 have been identified as the catalytic residues
[18] The activity for pullulan of the D325N used in the
crystallization was less than 0.006% that of the wild-type
enzyme [18] However, D325N released a small amount of
panose from pullulan at a high enzyme concentration with a
long reaction time, as observed by thin layer
chromato-graphy [18] TVA II also carries out a transglycosylation
reaction to form both a-1,4- and a-1,6-glucosidic linkages
[20] Thus, it is possible that 42-P2 is produced by a
transglycosylation reaction
42-P2 binding
The complexed structure enables a detailed analysis of the
interactions of the active site with 42-P2 To facilitate
description of these interactions, the active cleft of
TVA II is separated into two parts called the nonreducing
region (containing subsites )1, )2 and )3) and the
reducing region (containing subsites +1, +2 and +2¢) in
this report
TVA II forms a homodimeric structure, while most of
the a-amylases form a monomeric structure Although the
active cleft of TVA II in the monomeric structure is wide
and shallow, domain N of MOL-2 contributes to the
formation of a narrow, deep cleft around the reducing
region in the dimeric structure, while the nonreducing region is not affected by formation of the dimeric structure (Fig 3A) Yokota et al [13] constru cted a mutated TVA II truncated domain N and showed that domain N was necessary for the formation of the dimeric structure and enzymatic activities The average tempera-ture factors for Glc )3, )2, )1, +1 and +2 are 36.1, 27.6, 28.8, 35.1 and 42.2 A˚2, respectively The values for Glc)1 and )2 are lower than those for the other glucose units because the maltose unit bound at subsites )1 and )2 is taken up into the bottom of the active cleft and tightly bound to the enzyme by multiple hydrogen bonds Table 2 lists the hydrogen bond environments of the bound 42-P2
Non-reducing region Figure 3(B) shows the residues engaged in 42-P2 binding at the nonreducing region O2 and O3 of Glc )1 form hydrogen bonds with Asp421 at distances of 2.4 and 2.7 A˚, respectively Asp421 is one of the three catalytic residues and the pullulan-hydrolyzing activity of mutated TVA II (D421N) was drastically decreased to 0.001% of that of the wild-type enzyme [18] Asp421, which has been proposed to function as a fixer for Glc)1, causes deformation of the glucose ring, which is essential for the catalysis [28] In this complexed structure, the conformation of the ring of Glc)1 was slightly distorted from the4C1chair form Asp465 and Arg469 are involved in the binding of Glc )2 Asp465 interacts with O3 of Glc )2 at a distance of 2.6 A˚ and Arg469 also interacts with O2 and O3 of Glc)2 at distances
of 2.8 and 3.1 A˚, respectively The recognition of the maltose unit by Asp421, Asp465 and Arg469 is widely found in a-amylase family enzymes, indicating that this mode of maltose recognition is a common mechanism regardless of the diversity of the substrate specificity His202
is located at the bottom of the active cleft, and only forms a hydrogen bond with the O2 of Glc)3 at the distance of 2.8 A˚
Reducing region While many interactions between the enzyme and the substrate were identified in the nonreducing region, relat-ively few interactions with the substrate were seen in the reducing region (Fig 3C) Remarkable conformational changes of two amino acid residues around subsite +2, Trp356 and Tyr374, were observed between the unliganded and complexed structure Once 42-P2 is taken into the active site, the side chain of the Trp356 is rotated from)174.7 to 169.4 in torsion angle of Cc–Cb–Ca–C on Trp356 This conformational change makes a plane of its side chain parallel to the ring of Glc +2 and contributes to interaction with Glc +2 through a stacking effect Furthermore, this adjustment of Trp356 seems to trigger a rotational change
of Tyr374 The side chain of Tyr374 is rotated from)51.1
to 149.8 in torsion angle of Cc–Cb–Ca–C on Tyr374 without the steric barrier of Trp356, and also precisely becomes parallel to Trp356 and Glc +2
The reducing region of the active cleft is coordinately composed of domain A of MOL-1 and domain N of MOL-2 Two loops (Asp43-Glu51 and Glu104-Tyr113) of
FEBS 2004 Structure of T vulgaris a-amylase II complex (Eur J Biochem 271) 2533
Trang 5domain N appear to be strongly involved in substrate
binding O2 and O3 of Glc +2 form hydrogen bonds with
Gln112 and Arg44, both of which belong to domain N
of MOL-2, at distances of 2.9 and 3.3 A˚, respectively
Glc +2¢ occupied the center of the active site without any
interactions with MOL-1 or MOL-2, and its average
temperature factor was 48.9 A˚2 The configuration of the
a-1,6-glucosidic linkage between Glc +1 and +2¢ was
completely different from that between Glc)3 and )2 The
torsion angles of O6–C6–C5–C4 in Glc +1 and +2¢, and
Glc)3 and )2, were )168.8 and 47.2, respectively The
distances between O2 (+2¢) and O6 (+2), and O2 ()3) and
O6 ()1) were 4.2 and 6.7 A˚, respectively Phe286 of MOL-1
and Tyr45 of MOL-2, which play important roles in the
binding of cyclodextrins [29], are located at the nearest
distances of 3.8 A˚ and 4.1 A˚, and interact with Glc +2¢ via
van der Waals force In the structure of neopullulanase
complexed with maltotetraose, the electron density
corres-ponding to maltose was observed proximal to the position
of Glc +2¢, and it was proposed that maltose may be
a potential acceptor in the transglycosylation reaction [30] Thus, these findings suggest that the position around Glc +2¢ has the ability to hold a monosaccharide or small oligosaccharide A summary of the intermole-cular hydrogen-bonding interactions that can be inferred for the complex between TVA II and 42-P2 is presented in Fig 4
Discussion
Four loops in the nonreducing region The nonreducing region of the active cleft, containing subsites )1, )2 and )3, consists of four loops, loop I (residues 136–171), loop II (residues 193–218), loop III (residues 257–302), and loop IV (residues 454–482) (Fig 5A) Loops III and IV are located at each side of the cleft to form a cleft, and the width of this cleft is about 10 A˚
Fig 3 Stereo-view of the active site with
42-P2 (A) The whole shape of the active cleft formed collaboratively with domain N of MOL-2 (green surface model) is shown in the molecular surface model The surface model was produced using PYMOL (http://www pymol.org) (B) Unliganded TVA II (green) superimposed into the complex structure (magenta) around the nonreducing region.
42-P2, separated between )1 and +1, is dis-played as dark gray sticks The residues with asterisks are located in domain N of the MOL-2 molecule (C) Reducing region The explanation is the same as for (B).
Trang 6at its narrowest Loop I is also a component of the active
cleft, but loop I does not directly interact with 42-P2 Loop
II is located in the end of the cleft composed of loops III and
IV, and seems to act as a dam of the cleft
These four loops of TVA II are superimposed on those of
a-amylase (Taka-amylase A) from Aspergillus oryzae (PDB
code, 7TAA) [31] and cyclodextrin glucanotransferase
(CGTase) from Bacillus circulans strain 251 (PDB code,
1CDG) [32] (Fig 5A) The Ca backbones of loop IV,
where several highly conserved residues, such as Asp465 and
Arg469 of TVA II, are also located, are similar in these
three enzymes It appears that the shape of loop IV is
necessary for the recognition of the maltose unit bound Glc
)1 and )2 in a-amylase family enzymes In contrast, loop
III of TVA II adopts a different conformation from that of
other a-amylase family enzymes In TVA II, this loop is
shorter than in these other enzymes, but the C-terminus of
the loop is connected with domain B Loop II is located at
the end of the active cleft and forms a dam-like bank In
TAA and CGTase, loop II is 10 and 14 residues longer than
that in TVA II, and protrudes more markedly into the
active cleft compared to Loop II in TVA II (Fig 5B) In
most a-amylases, loop II makes a large bank in the active
cleft, as in CGTase and TAA In contrast, loop II of TVA II
is short and the bank is small, which allows an open cleft This distinctive shape of the cleft of TVA II enables TVA II
to incorporate various substrates, including pullulan, into the active cleft
We previously analyzed the structure of TVA II com-plexed with maltohexaose, but found that Glc )3 was disordered [14] We estimated the position of Glc)3 of the a-1,4-glucan using the structure of TAA complexed with acarbose (Fig 5C) The positions of the maltose unit, Glc )1 and )2, are almost the same in the two enzymes However, the position of Glc )3 of 42-P2 is completely different from that of acarbose In the case of 42-P2, Glc)3 extends toward the space between loops II and III The length of loop II of TVA II is very short and the cleft is open, which enables TVA II to bind pullulan efficiently Loop II of TAA occupies the end of the active cleft round the nonreducing region, which seems to restrain the uptake
of pullulan into the active site Tyr75, located at Loop II of TAA, also seems to be a steric barrier to the uptake of Glc )3 in TAA On the other hand, in acarbose, Glc )3 extends toward the space between loops I and II Although Glu35, located at loop I of TAA, is engaged in the binding of Glc )3, His164 of TVA II, located at the position corresponding
to Tyr75 of TAA, is too close to Glc)3 in this model The activity of TVA II for starch and its derivatives is almost equal to that for pullulan Thus, the hydrolysis of pullulan
by TVA II appears to be the result of effective binding due
to the shape of the active cleft around the nonreducing region
The substrate recognition of TVA II at the nonreducing region of the active cleft is different from those of other a-amylase family enzymes These differences are also due to the individual amino acid residues that interact directly with substrate, but are mainly due to the shape of the active cleft composed of the four loops
Roles of Trp356 and Tyr374 Drastic conformational changes of two residues, Trp356 and Tyr374, were observed upon binding with 42-P2 (Fig 3C) In neopullulanase [30] and maltogenic amylase [33], the residues corresponding to Trp356 and Tyr374 are already stacked in the unliganded state The 2Fo–Fcelectron density maps of Trp356 and Tyr374 in unliganded and
Table 2 Hydrogen bond contacts between TVA II and 42-P2.
a Gln112 and Arg44 are located at domain N of MOL-2.
Fig 4 Schematic drawing of the interactions
of 42-P2 bound to the active site Hydrogen
bonds of less than 3.5 A˚ are shown as dashed
lines Water molecu les are shown as spheres.
The residues with asterisks are located in
domain N of the MOL-2 molecule Three
catalytic residues, except for Asn325, which is
aspartic acid in native TVA II, are surrounded
by an elliptical box.
FEBS 2004 Structure of T vulgaris a-amylase II complex (Eur J Biochem 271) 2535
Trang 7Fig 5 Four loops composing the nonreducing region of the active cleft Stereo-views of the four loops that form the active cleft of TVA II (in magenta), which are superimposed on CGTase (PDB code, 1CDG) and TAA (PDB code, 7TTA), drawn with coils in orange and green, respectively Loops I, II, III and IV are located at the nonreducing region of the active cleft (A) The four loops and the residues engaged in substrate binding are shown in a coil and stick model (B) The comparison of the shape of the active cleft based on the molecular surface model of TVA II CGTase and TAA are superimposed and drawn in coils (C) The position of Glc )3 of a-1,4-glucan is predicted using the structure of TAA complexed with acarbose The nonreducing region of 42-P2 (black stick) and acarbose (antique white sticks) are only shown as sites )1, )2 and )3 for 42-P2 and )1¢, )2¢ and )3¢ for acarbose.
Trang 8complexed TVA II were clearly seen (Fig 6) When no
substrates are taken into the active site, Tyr374 is fixed by
Glu98 of MOL-2 with a weak hydrogen bond at a distance
of 3.4 A˚, and the space around Trp356 is observed as a wide
cavity This environment generates the flexibility of Trp356,
allowing suitable interaction with Glc +2 Tyr374 is
continuously rotated to cause the stacking with Trp356,
and thus Tyr374 seems to play an important role as lining
for Trp356
To investigate the role of Tyr374, we constructed Y374A
mutated TVA II, with replacement of tyrosine by alanine,
by site-directed mutagenesis, and carried out kinetic analysis
for starch and pullulan The Kmvalue of Y374A for starch
was almost identical to that of the wild-type enzyme and
that of Y374A for pullulan showed nearly a threefold
decrease compared to that of the wild type Because the
cavity around Trp356 of the Y374A mutant was very wide
in both the unliganded and liganded states, it is likely that in
the mutant, Trp356 was enabled to rotate its side chain to be
appropriate for the position of Glc +2 On the other hand,
the kcatvalue of the Y374A mutant protein was decreased to
less than 10% of the wild-type value (Table 3) This
observation suggests that Tyr374 also participates in the
catalytic activity, in addition to assisting in substrate
binding through the lining of Trp356 Upon hydrolysis, a
water molecule, located near the glucosidic linkages between
Glc)1 and +1, is incorporated into the carbonium cation
intermediate Tyr374 in the substrate binding state catches a water molecule at a distance of 2.6 A˚, and this water is also captured by two catalytic residues, Glu354 and Asp421, at the same distance of 2.7 A˚ Kuriki et al [34] suggested that Tyr377, Met375 and Ser42 of neopullulanase (correspond-ing to Tyr374, Met372 and Ser419 of TVA II) are located
on the entrance path of the attacking water molecule, and these residues are involved in hydrolysis and transglycosy-lation, as shown by using site-directed mutagenesis and computer modeling The replacement of tyrosine by alanine increases the hydrophobicity around the entrance path of the water molecule and makes it impossible to fix the water molecule near the glucosidic linkage to be cleaved Thus, we suggest that Tyr374 is involved in supplying the water that is necessary for substrate hydrolysis
Acknowledgements
This study was supported in part by Grants-in-Aid for Scientific Research (14580621) from the Ministry of Education, Culture, Sports, Science and Technology of Japan The data collection was carried out under the approval of the Photon Factory Advisory Committee, the National Laboratory for High Energy Physics, Tsukuba (2001G341).
We thank Dr Igarashi and Dr Suzuki for help in data collection at the Photon Factory, BL18B We also thank the X-ray crystallography laboratory, Tokyo University of Agriculture and Technology, Fuchu, Tokyo for data collection using an R-AXISIIc.
References
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Fig 6 Rotational change of Trp356 and Tyr374 between unliganded TVA II and complex with 42-P2 (A) Unliganded TVA II (B) Structure of complex with 4 2 -P2 The 2F o –F c electron density maps contoured 1 r are shown for both models Gln112 with the asterisk belongs to the MOL-2 molecule.
Table 3 Kinetic parameters for starch and pullulan.
k cat (s)1) K m (%) k cat /K m (s)1Æ%)1) Starch Wild-type 120 ± 2.9 0.23 ± 0.010 520 ± 26
Y374A 9.4 ± 0.35 0.19 ± 0.020 40 ± 5.5
Pullulan Wild-type 140 ± 6.1 1.4 ± 0.12 100 ± 10
Y374A 4.8 ± 0.082 0.57 ± 0.084 8.4 ± 1.2
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