Journal of Structural Biology xxx (2016) xxxxxx

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Journal of Structural Biology

journal homepage: www.elsevier.com/locate/yjsbi

Crystal structure of the enzyme-product complex reveals sugar ring

distortion during catalysis by family 63 inverting a-glycosidase
Takatsugu Miyazaki a,b, Atsushi Nishikawa b, Takashi Tonozuka b,
a
b

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 Japan
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

a r t i c l e

i n f o

Article history:
Received 4 August 2016
Received in revised form 23 September
2016
Accepted 24 September 2016
Available online xxxx
Keywords:
Glycoside hydrolase family 63
Inverting mechanism
Skew-boat conformation
Glucosylgalactose
Cremer-Pople
Conformational itinerary

a b s t r a c t
Glycoside hydrolases are divided into two groups, known as inverting and retaining enzymes, based on
their hydrolytic mechanisms. Glycoside hydrolase family 63 (GH63) is composed of inverting
a-glycosidases, which act mainly on a-glucosides. We previously found that Escherichia coli GH63
enzyme, YgjK, can hydrolyze 2-O-a-D-glucosyl-D-galactose. Two constructed glycosynthase mutants,
D324N and E727A, which catalyze the transfer of a b-glucosyl fluoride donor to galactose, lactose, and
melibiose. Here, we determined the crystal structures of D324N and E727A soaked with a mixture of
glucose and lactose at 1.8- and 2.1- resolutions, respectively. Because glucose and lactose molecules
are found at the active sites in both structures, it is possible that these structures mimic the enzymeproduct complex of YgjK. A glucose molecule found at subsite 1 in both structures adopts an unusual
1
S3 skew-boat conformation. Comparison between these structures and the previously determined
enzyme-substrate complex structure reveals that the glucose pyranose ring might be distorted immediately after nucleophilic attack by a water molecule. These structures represent the first enzyme-product
complex for the GH63 family, as well as the structurally-related glycosidases, and it may provide insight
into the catalytic mechanism of these enzymes.
2016 Elsevier Inc. All rights reserved.

1. Introduction
Glycoside hydrolases (GHs) are enzymes that catalyze the
hydrolysis of the glycosidic linkages found in various carbohydrates, and these enzymes play a role in both their synthesis and
degradation. To date, GHs have been classified into 129 families
(GH1GH135, among which six families have been deleted) by
their primary sequence identity, and these data are summarized
in the Carbohydrate-Active Enzymes (CAZy) database (http://
www.cazy.org) (Lombard et al., 2014).
The catalytic mechanisms of GHs are generally divided into two
types, the inverting and retaining mechanisms, based on the
anomeric configurations of their products (Koshland, 1953). Both
mechanisms typically use a pair of carboxylate residues, that is,
either aspartate or glutamate residues, for catalysis, and involve
oxocarbenium ion-like transition states (reviewed in Rye and

Abbreviations: EcYgjK, Escherichia coli YgjK; GH63, glycoside hydrolase family

63; Glc1,2Gal, 2-O-a-D-glucopyranosyl-D-galactose; b-GlcF, b-glucopyranosyl
fluoride; PDB, Protein Data Bank; RMSD, root mean squared deviation.
Corresponding author.
E-mail address: tonozuka@cc.tuat.ac.jp (T. Tonozuka).

Withers (2000)). The inverting reaction via a single-displacement

mechanism, and the catalytic acid donates a proton to the departing aglycon, and the catalytic base activates a water molecule that
attacks the anomeric carbon (Fig. 1A). The retaining reaction occurs
via a double-displacement mechanism. In the first glycosylation
step, the catalytic nucleophile attacks the anomeric carbon of the
substrate and forms a covalent glycosyl-enzyme intermediate.
The general acid/base acts as an acid and protonates the glycosidic
oxygen in this step. In the second deglycosylation step, the general acid/base acts as a base and activates the incoming water.
(Fig. 1B). Many retaining GHs also catalyze transglycosylation in
the presence of an acceptor molecule and are used in the production of various glycosides; whereas, inverting GHs do not show this
activity. However, several inverting GHs have been converted into
glycosynthases, which are hydrolytically inactive GH mutants that
catalyze transfer of a glycosyl donor to any acceptor molecule as
effectively as retaining GHs (Fig. 1C) (Honda and Kitaoka, 2006;
Wada et al., 2008; Ohnuma et al., 2012; Miyazaki et al., 2013;
Honda et al., 2016). Because GHs have many physiological roles
and can be efficient tools for synthesis of oligosaccharides and
glycoconjugates, as well as for biomass utilization, the full understanding of their catalytic mechanism is important.

http://dx.doi.org/10.1016/j.jsb.2016.09.015
1047-8477/ 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015

T. Miyazaki et al. / Journal of Structural Biology xxx (2016) xxxxxx

Fig. 1. Catalytic mechanism for glycoside hydrolases. (A) Inverting a-glycoside hydrolase, (B) retaining a-glycoside hydrolase, and (C) glycosynthase derived from
a-glycosidase.

Glycoside hydrolase family 63 (GH63) contains three types

of inverting a-glycosidases: (i) processing a-glucosidase I (EC
3.2.1.106), found in various eukaryotes (Kalz-Fller et al., 1995;
Miyazaki et al., 2011; Barker and Rose, 2013), (ii) the a-glucosyl/
mannosyl-D-glycerate hydrolase, found in bacteria (Alarico et al.,
2013), as well as in the plant, Selaginella moellendorffii (Nobre
et al., 2013), and (iii) a glycosidase (YgjK) specific for 2-O-a-Dglucopyranosyl-D-galactose (Glc1,2Gal) from Escherichia coli
(Miyazaki et al., 2013). The crystal structures of E. coli YgjK
(EcYgjK) (Kurakata et al., 2008), Saccharomyces cerevisiae processing a-glucosidase I (Barker and Rose, 2013), and a-mannosyl-Dglycerate hydrolase from Thermus thermophilus HB8 (Miyazaki
et al., 2015) have been determined. Generally, it has been shown
that GH63 enzymes have an (a/a)6-barrel catalytic domain, shared
with proteins belonging to the GH-G (GH37 and GH63) and GH-L
(GH15, GH65, and GH125) clans (Lombard et al., 2014). GH-G
and GH-L enzymes are inverting a-glycosidases with three conserved carboxylic acid residues in their active site: an aspartate

residue as a substrate holder at subsite 1 [the subsite numbering

is based on the literature (Davies et al., 1997)], an aspartate or
glutamate residue as a general acid catalyst, and a glutamate
residue as a general base catalyst (except for GH65 glycoside
phosphorylases).
We previously constructed two glycosynthase mutants, D324N
(subsite 1 mutant) and E727A (catalytic base mutant), of EcYgjK,
which efficiently transfer a b-D-glucopyranosyl fluoride donor
to several sugar acceptors, particularly D-galactose, lactose
(Galb1-4Glc), and melibiose (Gala1-6Glc) (Miyazaki et al., 2013).
The residue corresponding to Asp324 is conserved among GH-G
and GH-L enzymes, and indirectly interacts with a water molecule
that attacks the substrate in the subsequent step (The detailed
description of the role of Asp324 is given in Discussion).
The product of the glycosynthase reaction with D-galactose as
an acceptor was identified as Glc1,2Gal, which is a candidate for
the natural substrate for EcYgjK hydrolysis. The structure of
E727A complexed with Glc1,2Gal (E727A-Glc1,2Gal) further

Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015

T. Miyazaki et al. / Journal of Structural Biology xxx (2016) xxxxxx

revealed that the catalytic domain largely adopts two conformations, the open and closed forms, the latter of which was induced
by Glc1,2Gal binding at the active site. In the present study, we
determined the crystal structures of D324N in complex with
glucose and lactose (D324N-Glc-Lac) and E727A in complex with
glucose and lactose (E727A-Glc-Lac) to further investigate the
substrate specificity and the catalytic mechanism of EcYgjK. These
structures show, for the first time, the enzyme-product complex
and the distortion of glucose at subsite 1 among the GH-G and
GH-L enzymes.
2. Materials and methods
2.1. Preparation and crystallization of EcYgjK mutants
Two glycosynthase mutants of EcYgjK, D324N and E727A, were
expressed and purified as described previously (Miyazaki et al.,
2013). Briefly, the enzymes were expressed in Escherichia coli
BL21(DE3) and purified by hydrophobic interaction chromatography (HiPrep 16/10 Phenyl FF High-Sub column; GE Healthcare,
Little Chalfont, Buckinghamshire, UK), anion-exchange chromatography (HiLoad 16/10 Q-Sepharose HP column; GE Healthcare), and
gel-filtration chromatography (HiPrep 26/60 Sephacryl S-200 HR
column; GE Healthcare). Protein purity was analyzed by
SDS-PAGE, and concentration was determined by measuring absorbance at 280 nm based on the theoretical absorption coefficient
(1 mg mL 1 = 2.13) calculated by ExPASy ProtParam (http://
web.expasy.org/protparam/) (Gasteiger et al., 2005). The purified
enzymes were then crystallized using the hanging-drop vapor
diffusion method, as described previously (Miyazaki et al., 2013).
Table 1
Data collection and refinement statistics.

Data collection
Beamline
Wavelength ()
Space group
Cell dimensions
a ()
b ()
c ()
b ()
Resolution range ()
Measured reflections
Unique reflections
Completeness (%)
I/r(I)
Rmerge
Refinement statistics
Rwork
Rfree
RMSD
Bond lengths ()
Bond angles ()
Number of atoms
Protein
Ligand
Metal ion
Water
Average B (2)
Protein
Ligand
Metal ion
Water
Ramachandran plot
Favored (%)
Outliers (%)
PDB codes

D324N-Glc-Lac

E727A-Glc-Lac

PF AR-NW12A
1.0000
P21

PF AR-NW12A
1.0000
P21

57.4
136.9
81.5
100.7
501.80 (1.861.80)
372,040
110,133
96.8 (90.8)
32.3 (7.2)
0.050 (0.197)

56.7
137.1
81.6
100.0
502.20 (2.282.20)
213,809
60,457
97.3 (91.1)
21.9 (6.0)
0.084 (0.297)

0.149
0.187

0.154
0.208

0.009
1.183

0.009
1.304

12,158
70
5
1286

12,176
70
4
688

15.2
17.8
20.0
26.4

32.6
34.9
35.2
33.0

96.8
0
5CA3

95.8
0
5GW7

The values for the highest resolution shells are given in parentheses.

2.2. Data collection and structure determination

Crystals were soaked for a few seconds in the reservoir solution,
supplemented with 0.8 M glucose and 0.4 M lactose. The crystals
were then cryo-protected with the reservoir solution containing
1.6 M glucose and 0.8 M lactose and then flash-frozen with nitrogen gas at 100 K. The diffraction data were collected at 100 K using
a PF-AR NW12A beamline (Photon Factory, Tsukuba, Japan). All
data were processed and scaled using HKL2000 (Otwinowski and
Minor, 1997). The structure of the complex was solved using the
molecular replacement method with the MOLREP program
(Vagin and Teplyakov, 1997) in the CCP4 suite (Collaborative
Computational Project, 1994). A model of EcYgjK complexed with
mannose (PDB 3W7T) was used as a search model. REFMAC5
(Murshudov et al., 1997) was utilized for refinement, and manual
adjustment and rebuilding of the models were performed with
the COOT program (Emsley et al., 2010). Solvent molecules were
introduced using the ARP/wARP program (Perrakis et al., 1999),
and structure validation was performed with MolProbity (Chen
et al., 2010). Figures were prepared using PyMOL (http://www.
pymol.org/). Data collection and refinement statistics are listed in
Table 1. In Ramachandran plots, 96.496.8% of the residues were
found to be in the favored regions, and no residues were identified
as outliers. The coordinates and structure factors of D324N-Glc-Lac
and E727A-Glc-Lac have been deposited in the Protein Data Bank
(PDB) under the accession codes 5CA3 and 5GW7, respectively.

3. Results
3.1. Structures of glycosynthase mutants in complexes with glucose
and lactose
To further clarify the catalytic mechanism of EcYgjK hydrolysis
and glycosynthase activity, the crystal structures of D324N and
E727A in complex with both glucose and lactose, D324N-Glc-Lac
and E727A-Glc-Lac, respectively, were determined. In each case,
the crystals were soaked in solution containing glucose and
lactose, and they diffracted to 1.80 and 2.10 resolutions, respectively. Both belong to the space group P21 and contain two molecules (named Mol-A and Mol-B) in the asymmetric unit, similar
to the previously determined EcYgjK crystal structures (Kurakata
et al., 2008; Miyazaki et al., 2013). The electron density maps (2|
Fo| |Fc|) for both complexes contoured at 1 r show continuous
density for almost all amino acid residues. The structure of EcYgjK
consists of two domains, the b-sandwich N-domain and the
(a/a)6-barrel A-domain, which contains the active site, and they
are connected by a two-helix linker. In both structures, clear
electron density associated with the ligands was observed in the
Mol-A and Mol-B active sites: a lactose molecule is located at
subsites +1 and +2 in each active site of the all structures, whereas
a glucose molecule is bound to subsite 1 in D324N-Glc-Lac Mol-B
and in E727A-Glc-Lac Mol-A and Mol-B. The electron density for
glucose was not clear in D324N-Glc-Lac Mol-A.
The catalytic A-domain of EcYgjK adopts two conformations, an
open and closed form, and the latter is induced by occupation of
both subsite 1 and +1 with the substrate Glc1,2Gal (Miyazaki
et al., 2013). The A-domain in E727A complexed with Glc1,2Gal
(E727A-Glc1,2Gal, PDB 3W7W) adopts the closed form, whereas
those in the other structures (PDB 3D3I, 3W7S, 3W7T, 3W7U,
and 3W7X), in which the ligands do not occupy both subsites 1
and +1, adopt the open form. The root mean square deviations
(RMSDs, average values of Mol-A and Mol-B for Ca atoms) between
the A-domains of D324N-Glc-Lac and E727A-Glc1,2Gal, and
between those of E727A-Glc-Lac and E727A-Glc1,2Gal, were
0.329 and 0.382 , respectively. In contrast, the RMSDs between

Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015

T. Miyazaki et al. / Journal of Structural Biology xxx (2016) xxxxxx

the A-domains of D324N-Glc-Lac and E727A-Glc-Lac, and those of

the open forms, in which the ligands do not bind both subsites 1
and +1, were 1.071.22 . These results suggest that the Adomains of D324N-Glc-Lac and E727A-Glc-Lac adopt the closed
form.
3.2. Glucose binding at the subsite

The |Fo| |Fc| maps for the ligands in Mol-B of D324N-Glc-Lac,

and in Mol-A and Mol-B of E727A-Glc-Lac were calculated without
the ligands. The |Fo| |Fc| maps indicate that the glucose molecules
at subsite 1 (named Glc 1) adopt the b-anomer (Fig. 2). Glc 1
in Mol-A of D324N-Glc-Lac could not be modeled due to poor electron density (Fig. 2B). The side chain of the mutated residue,
Asp324 ? Asn, in Mol-A of D324N-Glc-Lac has two different

conformations; one is oriented in the same direction as Asn324

in Mol-B, forming hydrogen bonds with O4 and O6 atoms of
Glc
1, and the other is directed away from subsite
1
(Fig. 2A and C). This conformational change was similarly observed
in the structure of D324N-melibiose (PDB 3W7X) and was suggested to affect substrate binding and hydrolytic activity
(Miyazaki et al., 2013).
Interestingly, in both D324N-Glc-Lac and E727A-Glc-Lac, the
sugar rings of Glc 1 in Mol-B (Glc 1B) are distorted, whereas
those in Mol-A (Glc 1A) of E727A-Glc-Lac are in a stable chair
conformation (Fig. 2CH). The puckering of Glc 1 was analyzed
in more detail using the Cremer-Pople system (Cremer and Pople,
1975). We found that the Cremer-Pople puckering parameters
for Glc
1A of E727A-Glc-Lac were u = 185.1, h = 2.2, and
Q = 0.572, indicating that it was in a 4C1 chair conformation. Rather,

Fig. 2. Active sites of D324N-Glc-Lac and E727A-Glc-Lac. (A and B) D324N-Glc-Lac Mol-A, (C and D) D324N-Glc-Lac Mol-B, (E and F) E727A-Glc-Lac Mol-A, (G and H)
E727A-Glc-Lac Mol-B. (A, C, E and G) Stereo views of the active sites. Side chains of residues around the active site and the main chains of Asn384 and Gly399, as well as
ligands, are shown in stick models. Hydrogen bonds are indicated by black dashed lines. Amino acid residues, glucose, and lactose are colored in yellow, green, and cyan,
respectively. The catalytic residues Asp501 and Glu727(Ala) are highlighted in red. (B, D, F and H) Stereo views of the |Fo| |Fc| omit maps for the ligands. The difference
Fourier maps are calculated excluding the ligands, and the resulting |Fo| |Fc| omit maps (blue mesh) are contoured at 3.0 r.

Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015

T. Miyazaki et al. / Journal of Structural Biology xxx (2016) xxxxxx

Fig. 2 (continued)

the parameters for Glc 1B of D324N-Glc-Lac were u = 214.2,

h = 82.3, and Q = 0.711, and those for E727A-Glc-Lac were
u = 213.4, h = 81.6, and Q = 0.714, indicating that they adopt 1S3
skew-boat conformations.
Glc 1A forms hydrogen bonds with atoms NE1 of Trp323, OD1
and OD2 of Asp324, O of Gly399, and NE1 of Trp680, as well as
Glc 1B (Fig. 2C, E, and G). Atom O1 of Glc 1A (4C1) does not
interact with any amino acid residues in subsite 1, whereas Glc
1B (1S3) in D324N-Glc-Lac forms a hydrogen bond with OE2 of
the catalytic base, Glu727. In contrast, Glc 1B in E727A-Glc-Lac
does not directly interact with any residues, but indirectly associates with Tyr679 via a water molecule. The conformations of all
residues interacting with Glc 1A or Glc 1B are almost identical
to each other, except for Tyr679, which in Mol-A is pointed away
from subsite 1. We previously reported that there were two
states in the closed form of EcYgjK, semi-closed and fully-closed,
depending on the orientation of Tyr679 (Miyazaki et al., 2013).

Thus, Mol-A adopts the semi-closed state, whereas in both the

structures of D324N-Glc-Lac and E727A-Glc-Lac, as well as in
E727-Glc1,2Gal, Mol-B adopts the fully-closed state.
3.3. Lactose binding at the subsite +1 to +2
In both the structures of D324N-Glc-Lac and E727A-Glc-Lac,
lactose molecules are located in the same position, subsite +1 to
+2 (Fig. 2). The galactose component of lactose at subsite +1 (Gal
+1) is recognized by Trp321, Asp368, Lys391, and the catalytic acid
Asp501 via hydrogen bonds and by Trp496 through hydrophobic
stacking. In contrast, a reducing-end glucose at subsite +2 (Glc
+2) does not directly interact with any amino acid residue, but
O6 atoms of Gal +1 and Glc +2 interact with carbonyl O of
Asn384 via a water molecule. Superimposition of D324N-Glc-Lac
and E727A-Glc-Lac with E727A-Glc1,2Gal indicates that the orientation of Gal +1 is identical to that of the galactose moiety of

Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015

T. Miyazaki et al. / Journal of Structural Biology xxx (2016) xxxxxx

Glc1,2Gal in E727A-Glc1,2Gal, and O2 of Gal +1 is pointed to subsite 1. It has been demonstrated that the EcYgjK glycosynthase
can transfer b-GlcF to lactose as well as to galactose (Miyazaki
et al., 2013), and therefore O2 of Gal +1 is the most likely target
of glucosylation by the EcYgjK glycosynthase.
4. Discussion
The conformational changes that occur during the hydrolytic
reaction have been reported in a variety of GHs and are called
the conformational itinerary (Speciale et al., 2014). It was proposed
that the reactions of inverting and retaining GHs undergo the following two and three steps, respectively: substrate-bound (namely
Michaelis complex), covalent intermediate (if retaining GH), and
product-bound, and there are oxocarbenium ion-like transition
states between each step. Because the conformational itineraries
cannot be directly observed, structures complexed with substrate
analogues and inhibitors, which mimic the sugar conformation in
those steps, are often analyzed using X-ray crystallography.
Michaelis complexes have been also obtained using inactive
mutants in complex with substrates.
In the present study, we found that a glucose at subsite 1 in
Mol-B of both E727A-Glc-Lac and D324N-Glc-Lac was distorted
to a 1S3 skew-boat conformation. GHs acting on b-glycosidic
linkages generally require a large distortion of the sugar at
subsite 1 to change the glycosidic bond (C1O) to a pseudoaxial position (Davies et al., 2012). Additionally, sugar distortion
at subsite
1 was found in a crystal structure of GH14
b-amylase, an inverting a-glycoside hydrolase. In the structure of
soybean b-amylase maltose complex, two maltose molecules are
bound in subsites 2 to 1 and in subsites +1 to +2, and the pyranose ring at subsite 1 forms a boat or half-chair conformation
(Hirata et al., 2004).
The 1S3 skew-boat conformation was found in the covalent
intermediates of retaining GH-D clan (GH27 and GH31) enzymes
(Lovering et al., 2005; Guce et al., 2010; Larsbrink et al., 2012)
and Michaelis complexes of retaining GH5 endoglucanases
(Davies et al., 1998). Superimposition of Glc 1B of D324N-GlcLac with the covalent intermediates of E. coli GH31 a-xylosidase
YicI (PDB 1XSK) (Lovering et al., 2005) and Cellvibrio japonicus
GH31 a-transglucosylase CjAgd31B (PDB 4BA0) (Larsbrink et al.,
2012) shows that the pyranose conformations of the ligands are
almost identical to each other (Fig. 3). In the covalent intermediate
structures of GH31 enzymes, the anomeric carbon forms a covalent
bond with each catalytic nucleophile in a pseudo-axial orientation
(Fig. 3B and C). Conversely, O1 of Glc 1 in D324N-Glc-Lac is in a

Fig. 3. 1S3 skew-boat pyranose conformations. (A) Glc 1 of D324N-Glc-Lac Mol-B.

(B) The covalent intermediate of E. coli GH31 a-xylosidase (PDB 1XSK). (C) The
covalent intermediate of C. japonicus GH31 a-transglucosylase (PDB 4BA0).

Fig. 4. Structural comparison of the active sites in D324N-Glc-Lac, E727A-Glc-Lac,

and E727A-Glc1,2Gal. The side chains of amino acid residues and the ligands are
indicated by thick and thin stick models, respectively. The putative nucleophilic
water identified in the E727A-Glc1,2Gal structure is shown as a sphere. The
conformational difference between the Tyr679 side chain in the semi-closed and
fully-closed states is indicated by a double-headed arrow. Colors: D324N-Glc-Lac
Mol-B, green; E727A-Glc-Lac Mol-A, red; E727A-Glc-Lac Mol-B, yellow; E727AGlc1,2Gal Mol-A, magenta; E727A-Glc1,2Gal Mol-B, cyan.

pseudo-axial orientation and forms a hydrogen bond with the

catalytic base, Glu727 (Fig. 3A). Furthermore, superimposition of
D324N-Glc-Lac Mol-B and E727A-Glc1,2 Gal shows that the position of the pseudo-axial O1 of Glc 1 in D324N-Glc-Lac Mol-B is
almost identical to that of the putative nucleophilic water in
E727A-Glc1,2Gal Mol-A (Fig. 4). Based on these observations, the
1
S3 skew-boat sugar conformation seems to be the state arising
immediately after nucleophilic attack by a water molecule
activated by the catalytic base Glu727 (Fig. 5A). Additionally, the
distorted sugar-ring conformation may mimic that of the donor
substrate, b-GlcF, of the Michaelis complex in the EcYgjK glycosynthase reaction (Fig. 5B).
The torsion angle of C2-C1-O5-C5 of Glc 1 (4C1) in Mol-B of
E727A-Glc1,2Gal is 32.0 while that of Glc 1 (1S3) in Mol-B of
E727A-Glc-Lac is 54.0. Because of the double-bond character
between the O5-C1 atoms in the oxocarbenium ion-like transition
state, a flat conformation of C2-C1-O5-C5 would be present during
the 4C1 ? 1S3 conformational change (Uitdehaag et al., 1999). E3,
4
H3, and 4E conformers satisfy the requirement, and considering
a minimal movement pathway from 4C1 to 1S3, the transition state
is likely to adopt a half-chair 4H3 conformation (Fig. 5C). During the
enzymatic reaction of human a-galactosidase, a GH27 retaining
enzyme, a pyranose at subsite 1 has been proposed to have a conformational itinerary 4C1 ? 4H3 ? 1S3. (Guce et al., 2010). Together
with our observation, a common conformational change is likely to
occur during the enzymatic reaction despite the difference
between retaining and inverting glycosidases.
The residue equivalent to Asp324 at subsite 1 in EcYgjK is
highly conserved among GH-G and GH-L enzymes. In complex
structures of GH15 glucoamylases from Aspergillus awamori and
Thermoanaerobacterium thermosaccharolyticum with a pseudotetrasaccharide acarbose, an aspartic acid residue (Asp55 in
A. awamori; Asp344 in T. thermosaccharolyticum) interacts with a
putative nucleophilic water via O6 atom of the valienamine residue
of acarbose (Aleshin et al., 1994, 2003). It is likely that the residue
equivalent to Asp324 in clan GH-G and GH-L enzymes involves in
assisting electron transfer between the catalytic base residue and
nucleophilic water via O6 atom of the substrate (Fig. 5). Similarly,
some other inverting glycosidases also have a carboxylic acid residue near the catalytic base. For instance, clan GH-N (GH28 and
GH49) inverting a-glycosidases have been reported to have two
aspartic acid residues in the active site and there is a controversy
which residue acts as the catalytic base (van Santen et al., 1999;
Larsson et al., 2003). It is likely that either residue functions similar

Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015

T. Miyazaki et al. / Journal of Structural Biology xxx (2016) xxxxxx

Fig. 5. Proposed mechanisms for hydrolysis (A), glycosynthase reaction (B), and conformational itinerary (C) in GH63 a-glycosidase. The hydrogen bonds are shown in dotted
lines (A and B). The proposed conformational trajectory of GH63 enzymes is indicated as a grey double-headed arrow and the pyranose ring configurations are highlighted in
red.

to that of Asp324 in EcYgjK, which assists the catalytic base residue

in activating the water molecule.
To our knowledge, this is the first report showing the distorted
conformation of the pyranose ring in the enzyme-product complex
immediately after catalysis among GH63 and related inverting
a-glycosidases of clan GH-G and GH-L. Further studies will be
necessary, however, to prove the conformational itinerary and
the reaction mechanism for GH63, as well as for the inverting
glycosidases.

Acknowledgements
The authors thanks to Dr. Shinya Fushinobu for providing the
CremerPople parameter calculator. This work was supported, in
part, by a Grant-in-Aid for Scientific Research (T.T., No.
16K07687; T.M., No. 25-7279) and a Research Fellowship (T.M.)
from the Japan Society for the Promotion of Science. This work
has been performed under the approval of the Photon Factory
Program Advisory Committee (Nos. 2014G512 and 2016G013).

Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015

T. Miyazaki et al. / Journal of Structural Biology xxx (2016) xxxxxx

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Please cite this article in press as: Miyazaki, T., et al. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by
family 63 inverting a-glycosidase. J. Struct. Biol. (2016), http://dx.doi.org/10.1016/j.jsb.2016.09.015