α-Glucosidase mutant catalyzes "α-glycosynthase"-type reaction

Article abstract of DOI:10.1271/bbb.66.928

Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe α-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an α-glucosidic linkage from β-glucosyl fluoride and

Full text of DOI:10.1271/bbb.66.928

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α-Glucosidase Mutant Catalyzes “α-Glycosynthase”-
type Reaction
Masayuki OKUYAMAa, Haruhide MORIa, Kotomi WATANABEa, Atsuo KIMURAa & Seiya CHIBAa
a Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido
UniversitySapporo 060-8589, Japan
Published online: 22 May 2014.
To cite this article: Masayuki OKUYAMA, Haruhide MORI, Kotomi WATANABE, Atsuo KIMURA & Seiya CHIBA (2002) α-
Glucosidase Mutant Catalyzes “α-Glycosynthase”-type Reaction, Bioscience, Biotechnology, and Biochemistry, 66:4,
928-933, DOI: 10.1271/bbb.66.928
To link to this article: http://dx.doi.org/10.1271/bbb.66.928
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Biosci. Biotechnol. Biochem., 66 (4), 928–933, 2002
Preliminary Communication
a
-Glucosidase Mutant Catalyzes ``a-Glycosynthase''-type Reaction
Masayuki OKUYAMA, Haruhide MORI, Kotomi WATANABE
Atsuo KIMURA,† and Seiya CHIBA
,
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
Received November 26, 2001; Accepted December 20, 2001
Replacement of the catalytic nucleophile Asp481 by
the ``
type of
an endo-acting
b
-glycosynthase''-type reaction.4) The second
-glycosynthase has been recently reported on
-glucanase from Bacillus lichenifor-
The mutant of catalytic nucleophile
(Glu134Ala, -glucansynthase)
laminaribiosyl saccharide derivatives from
glycine in Schizosaccharomyces pombe
eliminated the hydrolytic activity. The mutant enzyme
(D481G) was found to catalyze the formation of an
glucosidic linkage from -glucosyl ‰uoride and 4-
nitrophenyl (PNP) -glucoside to produce two kinds of
PNP -diglucosides, -isomaltoside and -maltoside.
The two products were not hydrolyzed by D481G, giv-
ing 41 and 29 yields of PNP -isomaltoside and
maltoside, respectively. PNP monoglycosides, such as
-xyloside, -mannoside, or -glucoside, acted as the
substrate, but PNP -galactoside and maltose could
not. No detectable product was observed in the combi-
nation of -glucosyl ‰uoride and PNP -glucoside. This
study is the ˆrst report on an `` -glycosynthase''-type
reaction to form an -glycosidic linkage.
a-glucosidase
b
b
5)
a-
mis
.
a
b
b
produced
b
a
-
-
a
a
a
a
laminaribiosyl ‰uoride. Until now glycosynthase
research has been limited for
enzymes, and there has been no report of any ``
glycosynthase''-type mutant enzyme from an
glycosylase so far.
b-glycosylase-type
%
a
a-
a-
a-
a
a
b
a
a
-Glucosidase (EC 3.2.1.20,
a
-
D
-glucoside gluco-
hydrolase) is a typical
a-glycosylase which catalyzes
a
a
exo-type hydrolysis, transglucosylation, condensa-
a
tion, and -glucal hydration to form a-anomer con-
D
ˆguration products.6) We reported that three acidic
a
residues (Asp481, Glu484, Asp647) in Schizosac-
Key words:
a
-glucosidase;
catalytic
nucleophile
charomyces pombe a-glucosidase (SPG) were essen-
tial for the hydrolytic reaction,7) in which Asp481 was
found to be a catalytic nucleophile.7,8) To learn the
functions of acidic residues, a series of analyses of
mutant enzymes have been done in our laboratory.
Recently, the glycosynthase-type reaction was ob-
served in an Asp481Gly mutant of SPG (D481G).
mutant; glycosynthase; oligosaccharide
production
Glycosynthase is an active site nucleophile mutant
enzyme prepared from glycosidase, which is capable
of synthesizing oligosaccharide derivatives without
hydrolysis of the product.1–5) The original glycosyn-
thase has been reported by Withers and co-workers
who constructed the catalytic nucleophile mutant of
This paper describes for the ˆrst time the ``
glycosynthase'' reaction.
a-
All chemicals were purchased from Nacalai Tesque
Chemical (Kyoto, Japan) unless otherwise stated;
and -glucosyl ‰uoride ( -GF and -GF, respec-
Glu358Ala from Agrobacterium sp.
b
-glucosidase
a-
and found a remarkable reaction in which the mutant
enzyme missing hydrolytic activity catalyzed
b
a
b
tively), from Sigma (St. Louis, MO, USA);
restriction endonucleases and Klenow fragment of
Escherichia coli DNA polymerase I, from GIBCO
(Rockville, MD, USA). Mutant SPG of Asp481Ala
(D481A) was prepared and puriˆed as in our previous
paper.7) Quantitative analysis and puriˆcation of the
product diglycosides were done by high performance
liquid chromatography (HPLC) under the conditions
the production of
derivative(s) from two substrates,
galactosyl) ‰uoride and 4-nitrophenyl (PNP) glyco-
side.1,2) We tentatively call the mutant enzyme ``
b
-glycosyl oligosaccharide
a
-glucosyl (or
a-
b
-
glycosynthase''. A Ser mutant, Glu358Ser, increased
the glycosynthase activity and changed the speciˆcity
to PNP glycoside.3) The nucleophile-mutated
b-
×
as follows: column, 4.6 250 mm (5 mm ODS) YMC
glucosidase from Sulfolobus solfataricus also showed
†
To whom correspondence should be addressed. Tel Fax: +81-11-706-2808; E-mail: kimura
＠
abs.agr.hokudai.ac.jp
-glucosidase; D481G and D481A, Asp481Gly and Asp481Ala mutants of SPG,
respectively; ESI-MS, electron spray ionization mass spectrometry; -GF, -glucosyl ‰uoride; -GF, -glucosyl ‰uoride; HPLC, high per-
formance liquid chromatography; TLC, thin-layer chromatography; PNP, 4-nitrophenyl; PNP-Glc, 4-nitrophenyl -glucoside; pPICZA,
Pichia pastoris expression vector; pPICZA SPG and pPICZA D481G, expression vectors for wild-type SPG and D481G in P. pastoris
W
Abbreviations: SPG, Schizosaccharomyces pombe
a
a
a
b
b
a
,
W
W
respectively

a
-Glycosynthase
929
Fig. 1. Courses of Enzymatic Synthesis of PNP
and pH Dependence of -Glycosynthase Reaction by D481G (B).
In panel A, the amounts of PNP -maltoside (OS-A, ) and
-GF, 2 m PNP-Glc, and D481G (230
is the concentration of product formed by wild-type SPG under the same reaction conditions except using
a
-Maltoside and
a
-Isomaltoside Catalyzed by D481G and Product by Wild-type SPG (A),
a
a

a
-isomaltoside (OS-B,
m

) in the 30 C-incubated reaction mixture
9
(0.25 ml) consisting of 20 m
M
b
M
g) in 40 m sodium acetate buŠer (pH 4.5) were measured by
M
$
HPLC. The symbol of
wild-type SPG instead of D481G.
In panel B, the reaction mixture (0.25 ml) containing McIlvaine buŠer (pH 2.5–7.5, prepared by 0.2
M Na2HPO4 and 0.1 M citric
acid), 15 m
9
M b-GF, 3 mM PNP-Glc, and 230 mg D481G was incubated at 30 C.
Packed Column AQ303 (YMC; Kyoto, Japan) incu-
A ``megaprimer'' PCR approach9) was used to in-
troduce Gly at the position of Asp481 in SPG. The
9
bated at 50 C; mobile phase, 9:91 CH3CN H2O at
W
the ‰ow rate of 1 ml min; detection, absorbance at
mutagenic antisense primer was 5?-CGCAGAAC-
W
313 nm. The amount of each diglycoside was calcu-
lated from the peak area in the HPLC chromato-
gram. The purity of isolated products was examined
by thin-layer chromatography (TLC; Merck Silica gel
GAGCTCGGTTCGTTCATACCAGTCCAAAT-3?,
in which the underline is the mutated anticodon. The
puriˆed megaprimer PCR product was ligated into
the pPICZA SPG, designated pPICZA D481G. The
W
W
60 plate; development of 1-butanol 2-propanol
cloned product was sequenced to verify the introduc-
tion of the desired mutation. Transformation of P.
pastoris and expression of D481G were done basical-
ly in the same way as for the wild-type SPG.
W
W
H2O, 10:5:4). H- and 13C-NMR spectra of digluco-
sides were recorded on a Bruker AMX500 at 500 and
125 MHz, respectively, using sodium 3-(trimethyl-
silyl)propionate-2,2,3,3-d4 as an external standard.
Molecular masses of diglycosides were conˆrmed by
electron spray ionization mass spectrometry (ESI-
MS, JEOL JMS-SX102A).
1
Recombinant SPG showing a single band in SDS-
PAGE was puriˆed from culture medium of P.
pastoris GS115 harboring the pPICZA SPG or
W
pPICZA D481G by DEAE-Sepharose CL-6B and
W
The original wild-type enzyme was expressed inside
and in the surface of the Saccharomyces cerevisiae
Sepharose 6B at pH 4.5 (Amersham Pharmacia;
Uppsala, Sweden). Enzyme activity, protein concen-
tration and CD spectra of recombinant SPG were
measured as in our previous paper.7) The properties
of the wild-type SPG analyzed were identical to those
cells that harbored the vector of pYES SPG carrying
W
SPG cDNA.7) We found the Pichia pastoris expres-
sion system to secrete the wild-type SPG into the cul-
ture medium, giving the simple enzyme isolation
procedure. For construction of an expression plas-
mid for the new host cell, the SPG cDNA in the
of native enzyme except for sugar contents, 74
z for
the wild-type one and 78
z
for the native one.7) There
was no diŠerence in CD spectra of native and recom-
binant SPG.
pYES SPG was cleaved by HindIII, blunt-ended
W
with the Klenow fragment of E. coli DNA poly-
merase I, and then digested with XhoI. The resulting
3.0-kb DNA fragment containing SPG cDNA was in-
serted under the AOX1 promoter in the P. pastoris
expression vector (pPICZA, Invitrogen; Carlsbad,
CA, USA) that had been treated with XhoI after
being digested with EcoRI and blunt-ended. The
The recombinant enzymes (D481G and D481A)
mutated in the Asp481, which is a nucleophile in the
catalytic reaction of the S. pombe
lost the hydrolytic activity on any carbohydrates,
such as PNP -glucoside (PNP-Glc) and maltose.
However, the D481G was found to synthesize the
products from a reaction mixture consisting of -GF
and PNP-Glc. A reaction mixture of 0.25 ml contain-
ing 20 m -GF, 2 m PNP-Glc, and D481G
(230 g) in 40 m sodium acetate buŠer (pH 4.5) was
incubated at 30 C. Aliquots of reaction mixture were
a
-glucosidase,7)
a
b
resulting plasmid was named pPICZA SPG. Trans-
W
formation of P. pastoris GS155 and expression of
recombinant protein were done according to the
manufacturer's protocol (Invitrogen).
M
b
M
m
M
9

930
M. OKUYAMA et al.
Table 1. PNP Diglycoside Formation by D481G from b-GF and
taken at the indicated reaction times (Fig. 1(A)), and
were analyzed by TLC and reverse-phased HPLC.
Two new spots were observed on the TLC plate, and
each spot size was increased with reaction time. As
illustrated in Fig. 1(A), the quantitative HPLC analy-
sis showed that the amounts of two chromogenic
compounds (OS-A and OS-B) increased and reached
plateaus at 100 min. The spontaneous degradation of
PNP Glycoside
Substrate
Yield of PNP diglycosidea
(PNP monoglycoside)
(z)
PNP
a
-glucoside
41 (PNP
29 (PNP
a
a
-isomaltoside),
-maltoside)
82
13
10
0
PNP
PNP
PNP
PNP
a
b
a
a
-xyloside
-glucoside
-mannoside
-galactoside
b
-GF was also observed. In the initial stage of reac-
tion, however, the quantity of ‰uoride ion released
was higher than that in the no-enzyme reaction sys-
tem (control). The combination of b-GF and maltose
did not produce any oligosaccharide.
OS-A and OS-B were puriˆed by HPLC in order to
analyze the structures. The isolated OS-A and -B
were homogeneous on TLC. The Rf and retention
time of OS-A were identical to those of authentic
a
Reaction mixture (0.25 ml) containing 20 m
monoglycoside, and D481G (230 g) was incubated at pH 5.7 and 309C
of b-GF, 2 m of PNP
M M
m
for 80 min. Yield was calculated from the conversion of each PNP
monoglycoside into PNP diglycoside. Molecular mass of each product
was analyzed by ESI-MS.
Under the conditions shown in Fig. 1(A), about
z
of the PNP-Glc was converted to PNP oligosac-
PNP
0.1
maltose from OS-A and isomaltose from OS-B, im-
plying the formation of an -anomeric conˆguration
in the reaction. NMR and MS analyses of OS-A and
a
-maltoside. The partial acid hydrolysis using
50
z
tri‰uoroacetic acid at 100 C for 3 h released
9
charide derivatives. We examined the eŠects of pH
on the glycosynthase reaction to obtain a high yield
a
of oligosaccharide synthesis. The optimal pH for
glycosynthase activity of D481G was around pH 5.7
(Fig. 1(B)), at which the 41 and 29 yields of PNP
isomaltoside and of PNP -maltoside, respectively,
were obtained (Table 1). Table 1 also shows the sub-
strate speciˆcities for PNP monoglycosides. PNP
xyloside was found to be more e‹cient than PNP-
Glc. PNP -mannoside and -glucoside were poor
substrates, and PNP -galactoside was not.
Wild-type enzyme, which was expressed in P.
pastoris, catalyzed -1,4- and -1,6-glucosyl transfer-
ring reactions to produce PNP -maltoside and PNP
-isomaltoside from 20 m -GF and 2 m PNP-
Glc. Native SPG, which was isolated from S. pombe
catalyzed the -1,6-transglucosylation to produce
a-
OS-B were done. PNP
a
-maltoside (OS-A); ESI-MS:
z
a-
m z [M+Na]+ 486, NMR dH and dC (D2O): being
a
W
completely identical to authentic PNP
a
-maltoside.
PNP
a
-isomaltoside (OS-B); ESI-MS: m z
a-
W
[M+Na]+ 486, NMR dH (D2O): 3.39 (1H, dd,
J
＝
9.5, 9.5 Hz, 4
-H), 3.58 (1H, dd,
dd, 9.6, 9.5 Hz, 4-H), 3.65 (1H, m, 5
(2H, m, 6a-H), 3.72 (2H, dd,
?
-H), 3.47 (1H, dd,
J
＝
9.8, 6.7 Hz,
a
b
＝
2?
J
9.3, 9.4 Hz, 3
?-H), 3.59 (1H,
a
J
＝
?-H), 3.66
＝
J
12.2, 8.7 Hz,
a
a
6
?
a-H), 3.79 (1H, m, 2-H), 3.80 (1H, m, 6
?b-H), 3.88
a
(1H, m, 5-H), 3.89 (1H, dd,
J
＝
11.8, 5.3 Hz, 6b-H),
a
M
a
M
＝
＝
3.93 (1H, dd,
3.5 Hz, 1 -H), 5.82 (1H, d,
9.2 Hz, PNP-3,5-H), 8.26 (2H, d,
PNP-2,4-H); NMR C (D2O): 61.2 (6 -C), 66.2 (6-C),
70.1 (4-C), 70.2 (4 -C), 71.6 (2-C), 72.1 (2 -C), 72.3
(5-C), 72.5 (5 -C), 73.8 (3 -C), 73.9 (3-C), 97.3 (1-C),
98.5 (1 -C), 117.7, 117.7, 126.9, 126.9, 143.3, 162.1
J
9.5, 9.5 Hz, 3-H), 4.86 (1H, d,
J
,
?
J
＝
3.7 Hz, 1-H), 7.3 (2H,
a
＝
＝
9.2 Hz,
d,
J
J
mainly isomaltose from maltose of high concentra-
d
?
tion,10) in which there was a possibility of maltose
?
?
formation (a-1,4-transglucosylation), but it was
?
?
di‹cult to ˆnd the newly synthesized maltose in this
reaction. Therefore, the regiospeciˆcity in D481G
reaction to produce PNP a-isomaltoside and a-mal-
?
(PNP-C). ESI-MS data indicated that two com-
pounds were PNP diglucosides. The NMR data of
OS-A agreed completely with those of authentic PNP
toside was maintained even after replacement of
Asp481 by Gly, implying that the subsites 2 and 3 in
active center of D481G were unchangeable in recog-
nition of glucosyl and PNP groups in PNP-Glc, re-
spectively (Scheme 1(A)). At the middle and ˆnal
reaction stages of native and wild-type enzymes, the
transglucosylation products formed were subse-
quently hydrolyzed and decreased, as the isomaltose
and two PNP diglucosides were favorable substrates
for both enzymes. The best advantage of the D481G
reaction, therefore, is the accumulation of product
without further degradation, providing an e‹cient
approach to synthesize oligosaccharide.
a
-maltoside. OS-B was determined to be a PNP
isomaltoside, in which two-dimensional NMR and
HMBC analysis also supported the occurrence of
a-
a
-
1,6-linkage. A possible reaction is shown in Scheme
1(B). D481G of the catalytic nucleophile mutant
could not hydrolyze two products (PNP
a-maltoside
and -isomaltoside), meaning that the product only
a
accumulated in the reaction system. Under the same
reaction conditions, D481A gave no detectable
product. The mutant enzyme in which Asp481 was
replaced by an amino acid of small size catalyzed the
formation of PNP
a
-diglucosides. The wild-type
-GF did not
D481G synthesized no product from the mixture of
a-GF and PNP-Glc. The synthetic reaction only
SPG showing no hydrolytic activity on
b
synthesize any product (Fig. 1(A)) and the enzyme
hydrolyzed the PNP-Glc only.
occurred by using
b
-GF as a ‰uoride substrate to
-anomeric con-
produce PNP diglucosides having
a

a
-Glycosynthase
931
Scheme 1. Subsite Structure of D481G Recognizing Substrates (A), D481G-Catalyzed
a
-Glycosynthase-type Reaction with
b
-GF and PNP-
a-Maltoside
Glc (B), Hydrolytic (I) and Reverse (II) Reactions of Glucoamylase (C), and Glucoamylase-catalyzed Formation of Methyl
and -Isomaltoside from -GF and Methyl
-Glucoside (D).11)
Glc-PNP, PNP -glucoside; Me, methyl; wedge between subsite 1 and 2 (panel A), catalytic position.
a
b
a
a
ˆguration, meaning that the anomeric conˆguration
is opposite between the ‰uoride substrate ( ) and the
products ( ). A similar reaction catalyzed by D481G
and isomaltose from
b-glucose by condensation, that
b
is, the reverse reaction (Scheme 1(C)-II), and the
a
formation of methyl
a
-maltoside and methyl
-GF and methyl
a
a
-
-
(Scheme 1(B)) has been reported in the ``inverting en-
zymes'' like glucoamylase, trehalase, glucodex-
isomaltoside (plus HF also) from
b
glucoside (Scheme 1(D)).11) Glucodextranase also
tranase, and
b
-amylase,11–16) which catalyzed two
produced the same methyl
from -GF and methyl
formed -trehalose through two kinds of reaction
mixtures: the ˆrst mixture composed of -glucose
and -glucose (condensation) and the second one
composed of -GF and -glucose at which HF was
also released.12) The last example is a
-amylase to
produce maltotetraose from -maltose by condensa-
tion15,16) and to produce
-maltose from -maltosyl
‰uoride.13) In the latter reaction catalyzed by
-amy-
lase, -maltotetraosyl ‰uoride intermediate was ex-
pected to be synthesized at ˆrst and subsequently to
be split to -maltose and -maltosyl ‰uoride by
a
-diglucosides and HF
inverting-type reactions, such as hydrolysis and
oligosaccharide production. In the hydrolysis
b
a
-glucoside.11) Trehalase
a,a
(Scheme 1(C)-I), the
substrate is inverted to release a
glucose or
-maltose.17–20) In the oligosaccharide
a
-glucosidic linkage in each
b
b
-product, namely
b
-
a
b
b
a
production, Hehre and co-workers have shown that
glucoamylase, glucodextranase, and trehalase of
b
b
inverting enzymes synthesized
products from -glucose or
-GF,11,12) and that
amylase synthesized an -conˆgurational product
from -maltose or
-maltosyl ‰uoride.13) Glucoamy-
lase, for example, catalyzed the formation of maltose
a
-conˆgurational
b
b
b
b
b
-
b
a
b
b
b
b
b
b-

932
M. OKUYAMA et al.
2) Mackenzie, L. F., Wang, Q., Warren, R. A. J., and
Withers, S. G., Glycosynthases: mutant glycosidases
amylase-catalyzed hydrolysis, that is, the whole reac-
tion involved two combined steps of i) -mal-
totetraosyl ‰uoride and HF formation from two
molecules of -maltosyl ‰uoride and ii) hydrolysis of
b
for oligosaccharide synthesis. J. Am. Chem. Soc.
120, 5583–5584 (1998).
,
b
3) Mayer, C., Zechel, D. L., Reid, S. P., Warren, R. A.
J., and Withers, S. G., The E358S mutant of
resultant tetrasaccharide derivative to give
b-mal-
tose.13) The mechanism in the former step is similar to
that of maltotetraose formation in the condensation
Agrobacterium sp.
b-glucosidase is a greatly im-
proved glycosynthase. FEBS Lett., 466, 40–44 (2000).
4) Trincone, A., Perugino, G., Rossi, M., and Moracci,
M., A novel thermophilic glycosynthase that eŠects
of b
-maltose.13,15,16) Also the other three inverting en-
zymes, glucoamylase, trehalase and glucodextranase,
have been found to catalyze the condensation and the
oligosaccharide formation by using a ‰uoride sub-
strate through the same reaction path,11,12) meaning
that the oligosaccharide production from ‰uoride
substrate is the condensation.
branching glycosylation. Bioorg. Med. Chem. Lett.
,
10, 365–368 (2000).
5) Malet, C. and Planas, A., From
b-glucanase to b
-
glucansynthase: glycosyl transfer to
a
-glycosyl ‰uo-
rides catalyzed by a mutant endoglucanase lacking its
catalytic nucleophile. FEBS Lett.
,
440, 208–212
The reaction in D481G to form the inverted
a-
(1998).
anomer-product from -GF and PNP-Glc (Scheme
b
6) Chiba, S., Molecular mechanism in
a-glucosidase and
1(B)) is considered to be the condensation itself in the
inverting enzymes-catalyzed oligosaccharide produc-
tion mention above, implying that D481G changes
from a ``retaining enzyme'' to an ``inverting en-
glucoamylase. Biosci. Biotechnol. Biochem.
, 61,
1233–1239 (1997).
7) Okuyama, M., Okuno, A., Shimizu, N., Mori, H.,
Kimura, A., and Chiba, S., Carboxyl group of
residue Asp647 as possible proton donor in catalytic
zyme''. Catalytic nucleophile mutants of some
retaining enzymes, -glucosidase and -glucanase,
have been designated glycosynthases thus far.1–5)
These `` -glycosynthase''-type enzymes catalyze the
b-
b
b
reaction of a-glucosidase from Schizosaccharomyces
pombe. Eur. J. Biochem., 268, 2270–2280 (2001).
8) Kimura, A., Takata, M., Fukushi, Y., Mori, H.,
Matsui, H., and Chiba, S., A catalytic amino acid
and primary structure of active site in Aspergillus
b
production of
and HF from
b
-anomer oligosaccharide derivatives
a
-glycosyl ‰uoride and suitable PNP
niger
a-glucosidase. Biosci. Biotechnol. Biochem.,
(or 4-methylumbelliferyl) glycoside, and the synthetic
reaction is considered to be the transglycosylation. It
seems, however, to be rational that the glycosynthase
61, 1091–1098 (1997).
9) Datta, A. K., E‹cient ampliˆcation using
`megaprimer' by asymmetric polymerase chain reac-
tion. Nucleic Acids Res., 23, 4530–4531 (1995).
reactions involving b-glycosidases and a-glucosidase
are interpreted on the basis of the condensation
rather than the transglycosylation by the catalytic
nucleophile mutant enzyme which are changed from
``retaining'' to ``inverting'' enzymes.
10) Chiba, S. and Shimomura, T., Comparative
biochemical studies on
a
-glucosidases: part III.
-glucosidase from
Transglucosidation action of an
a
Schizosaccharomyces pombe. Agric. Biol. Chem., 30,
536–540 (1966).
In this study, we found that the D481G also
11) Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai,
T., and Hehre, E. J., Scope and mechanism of carbo-
hydrase action. Stereocomplementary hydrolytic and
glucosyl-transferring actions of glucoamylase and
D
glucodextranase with a- and b- -glucosyl ‰uoride. J.
Biol. Chem., 256, 6017–6026 (1981).
12) Hehre, E. J., Sawai, T., Brewer, C. F., Nakano, M.,
and Kanda, T., Trehalase: stereocomplementary
showed an ``
GF and PNP-Glc to form
charide derivatives. The D481G enzyme is the ˆrst ex-
ample of an `` -glycosynthase''. It is of interest that
the anomeric structures of the ‰uoride substrate and
product are completely opposite in -glycosynthase
and
-glycosynthase.1–5) Therefore, it is preferable to
divide the names of glycosynthase into `` -glycosyn-
thase'' and `` -glycosynthase''.
a-glycosynthase''-type reaction with b-
a
-conˆgurational oligosac-
a
a
b
a
hydrolytic and glucosyl transfer reactions with
-glucosyl ‰uoride. Biochemistry, 21, 3090–3097
(1982).
a- and
b
b-
D
13) Hehre, E. J., Brewer, C. F., and Genghof, D. S.,
Scope and mechanism of carbohydrase action.
Acknowledgment
Hydrolytic and nonhydrolytic actions of
b-amylase
We are grateful to Dr. E. Fukushi and Mr. K.
Watanabe in our Graduate School for measuring the
NMR and MS data.
on a- and b-maltosyl ‰uoride. J. Biol. Chem., 254,
5942–5950 (1979).
14) Hehre, E. J., Okada, G., and Genghof, D. S., Con-
ˆgurational speciˆcity: unappreciated key to under-
standing enzymic reversions and de novo glycosidic
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