Identification of Catalytic Residues of Ca2+-independent 1,2-
-D-Mannosidase from Aspergillus saitoi by Site-directed Mutagenesis*
Yota Tatara,
Byung Rho Lee,
Takashi Yoshida
,
Koji Takahashi
and
Eiji Ichishima ¶
From the
Laboratory of Molecular Enzymology, Graduate School of Engineering, Soka
University, Hachioji, Tokyo, 192-8577, Japan,
Department of Biotechnology, Faculty of
Agriculture and Life Science, Hirosaki University, Hirosaki, 036-8561, Japan,
and
Department of Applied Biological Science,
Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509,
Japan
Received for publication, March 14, 2003
 |
ABSTRACT
|
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The roles of six conserved active carboxylic acids in the catalytic
mechanism of Aspergillus saitoi 1,2-
-D-mannosidase
were studied by site-directed mutagenesis and kinetic analyses. We estimate
that Glu-124 is a catalytic residue based on the drastic decrease of
kcat values of the E124Q and E124D mutant enzyme. Glu-124
may work as an acid catalyst, since the pH dependence of its mutants affected
the basic limb. D269N and E411Q were catalytically inactive, while D269E and
E411D showed considerable activity. This indicated that the negative charges
at these points are essential for the enzymatic activity and that none of
these residues can be a base catalyst in the normal sense.
Km values of E273D, E414D, and E474D mutants were
greatly increased to 1731-fold wild type enzyme, and the
kcat values were decreased, suggesting that each of them
is a binding site of the substrate. Ca2+, essential for the
mammalian and yeast enzymes, is not required for the enzymatic activity of
A. saitoi 1,2-
-D-mannosidase. EDTA inhibits the
Ca2+-free 1,2-
-D-mannosidase as a competitive
inhibitor, not as a chelator. We deduce that the Glu-124 residue of A.
saitoi 1,2-
-D-mannosidase is directly involved in the
catalytic mechanism as an acid catalyst, whereas no usual catalytic base is
directly involved. Ca2+ is not essential for the activity. The
catalytic mechanism of 1,2-
-D-mannosidase may deviate from
that typical glycosyl hydrolase.
 |
INTRODUCTION
|
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In Enzyme Nomenclature Recommendations
(1), three types of
-mannosidases, namely
-D-mannoside mannohydrolase (EC
3.2.1.24
[EC]
), 1,2-
-mannosyl-oligosaccharide
-D-mannohydrolase (EC 3.2.1.113
[EC]
), and 1,3-(1,
6)-mannosyl-oligosaccharide
-D-mannohydrolase (EC
3.2.1.114
[EC]
), are given as systematic names
(2,
3). Aspergillus saitoi
1,2-
-D-mannosidase specifically cleaves the
-1,2-mannosidic linkage in the high mannose type oligosaccharides of
glycoproteins.
Glycosyl hydrolases are classified as retaining and inverting
(46).
The inverting reaction occurs via a single displacement mechanism with
inversion of anomeric configuration. The reaction usually involves two
carboxylic acids. These residues are located
9 Å apart on average.
One acts as a general acid catalyst, donating a proton to the glycosidic
oxygen of the scissile bond. The other acts as a base catalyst, activating
water for nucleophilic attack at the anomeric carbon. The retaining reaction
proceeds via a double displacement mechanism with retention of anomeric
configuration. The nucleophilic carboxylic acid residue attacks the glycosyl
oxygen. A covalent glycosyl enzyme intermediate is formed and hydrolyzed with
general acid-base catalytic assistance. In the retaining enzymes, the two
carboxylic acid residues are
5.5 Å apart.
Recently the three-dimensional structures of
1,2-
-D-mannosidase from Penicillium citrinum were
determined by Lobsanov et al.
(7). The three-dimensional
structures complexed with an inhibitor kifunensin and 1-deoxymannojirimycin
demonstrated that both inhibitors bind to the protein at the bottom of the
cavity in an unusual 1C4 conformation (4C1 is
energetically more stable than 1C4). The inhibitor binding did not undergo
major conformational changes. Three carboxylic acids (Glu-122, Asp-267, and
Glu-409) were potentially involved in the catalytic mechanism. These
correspond to Glu-124, Asp-269, and Glu-411 in the A. saitoi enzyme.
The four other highly conserved carboxylic residues were also shown (the
equivalent A. saitoi numbering is in parentheses): Glu-271 (Glu-273),
Glu-412 (Glu-414), Glu-472 (Glu-474), and Glu-502 (Glu-504). These four
residues seemed to be too distant from the substrate to be directly involved
in catalytic action. They either are buried at the bottom of the active site
or interact with atoms on the inhibitor far from the anomeric C-1. A molecule
of Ca2+ is located at the bottom of the active site cavity. Glu-271
(Glu-273), Glu-409 (Glu-411), Glu-412 (Glu-414), and Glu-472 (Glu-474) are
hydrogen-bonded to the water molecules that coordinate the Ca2+.
The distance of the Ca2+ and an inhibitor/substrate suggests that
they can directly interact with each other. Moreover, one of the candidates
for catalytic residues, Glu-409 (Glu-411), coordinates the Ca2+ via
a water molecule. The Ca2+ is essential for the enzyme activity of
the mammalian and yeast 1,2-
-mannosidases
(814).
These suggest that Ca2+ may be directly involved in the catalytic
reaction of 1,2-
-D-mannosidases. The amino acid sequence of
A. saitoi 1,2-
-D-mannosidase
(15) is 70% identical with
that of P. citrinum enzyme
(16), and their substrate
specificity to a high mannose type oligosaccharide is the same
(2,
3,
17). Structures of the active
center are believed to be conserved among A. saitoi and P.
citrinum enzymes because of their high homology. Although we previously
demonstrated the probable roles of these acidic residues
(18), their catalytic residues
have not yet been determined experimentally.
In this study, we performed site-directed mutagenesis to determine the
functional role of catalytic residues in the
1,2-
-D-mannosidase from A. saitoi. Ca2+,
potentially involved in the catalysis, was also analyzed with atomic
absorption spectrophotometry. To probe the roles of the three active site
carboxylic acids, Glu-124, Asp-269, and Glu-411, in greater detail, we
generated the alanine mutants of these residues and assayed these enzymes.
Rescue of activity by external anion was expected to provide valuable insight
into the identity of the general base catalyst as well as confirmation of the
identity of the general acid catalyst
(1924).
 |
EXPERIMENTAL PROCEDURES
|
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The nucleotide sequence of the 1,2-
-D-mannosidase gene
(D49827
[GenBank]
), msdS, was described earlier
(15). Aspergillus
oryzae niaD300 and a high level expression vector plasmid pNAN8142 were
used for transformation experiments
(25).
Man-
1,2-Man-OMe1
was purchased from Sigma.
Site-directed MutagenesisSite-directed mutagenesis of the
1,2-
-D-mannosidase gene (msdS) was performed by the
method of Kunkel et al.
(26). The following
oligonucleotides were used as mutagenic primers: E124A,
5'-AGCCTCTTCGcGACCACCATC-3' (msdS position,
361381); E411A, 5'-TGCGCCCGGcAGTGATTGAgAGCTTCTACT-3'
(12221252). The E124Q, E124D, D269N, D269E, E273Q, E273D, E411Q, E411D,
E414Q, E414D, E474Q, E474D, and E504Q mutants were constructed by inserting
the NotI fragment of the mutated genes in pGAM1
(18) into a NotI
restriction site in pNAN8142. The plasmid pNAN-AM1 containing wild type or
mutated msdS was transfected into the A. oryzae niaD300
strain. The transformation of A. oryzae and the expression of the
mutated 1,2-
-D-mannosidase genes were carried out according
to the procedure described previously
(25). Purification of
recombinant enzymes expressed in A. oryzae cells was also described
earlier (25). A pH gradient
was substituted for the NaCl gradient in the cation-exchange chromatography.
The recombinant enzymes were separated by SDS-PAGE gel as described by Laemmli
(27). The proteins were
detected by immunoblotting using anti-A. saitoi
1,2-
-D-mannosidase antibody (raised in rabbit) and
peroxidase-conjugated anti-rabbit IgG secondary antibody.
Enzyme AssayThe purified wild type and mutant enzymes were
assayed for 1,2-
-D-mannosidase activity with
Man-
1,2-Man-OMe as a substrate. Mannose from Man-
1,2-Man-OMe
released by the enzymic reaction was stained by the Somogyi-Nelson
(28,
29) method. One katal of
1,2-
-D-mannosidase activity was defined as the amount of
enzyme required to liberate 1 mol of mannose from Man-
1,2-Man-OMe per
second at 30 °C and pH 5.0.
The initial rates of 1,2-
-D-mannosidase activity were
determined for a Man-
1,2-Man-OMe substrate at 30 °C and pH 5.0 in
50 mM acetate buffer using 8 substrate-concentrations ranging from
0.5x to 2x Km. Values of the
catalytic coefficient (kcat) and Michaelis constant
(Km) values were determined by fitting initial
rates as a function of substrate concentration to the direct linear plot of
Cornish-Bowden.
Chemical Rescue MethodologyActivities of E124A, D269N, and
E411A 1,2-
-D-mannosidase mutants catalyzing
Man6GlcNAc2-PA to Man5GlcNAc2-PA
in the presence of external nucleophilic anion at pH 5.0 were analyzed by HPLC
analysis. Ten pmol of Man6GlcNAc2-PA (Takara Shuzo Co.,
Kyoto) with or without 3 M sodium azide was added to the enzyme
solution. After 72 h at 30 °C, the reaction mixture was stopped by heating
to 100 °C for 3 min and analyzed on HPLC using TSKgel Amide-80 column (4.6
x 250 mm, Tosoh Corp., Tokyo). The solvent and elution conditions were
described by Kondo et al.
(30).
Ca2+ Binding TreatmentThe purified
recombinant A. saitoi 1,2-
-D-mannosidase was used
as a Ca2+-free wild type enzyme. NaCl was added to the purified
enzyme solution in the final concentration of 1 M. The solution was
dialyzed against 10 mM sodium acetate buffer, pH 5.0, containing 50
µM CaCl2 for 16 h at 4 °C and then dialyzed
against the same buffer without CaCl2. To analyze the content of
Ca2+ binding to the enzyme, the solution was applied to atomic
absorption spectrophotometry (Shimadzu model AA-660 (P/N206 1000-02)
apparatus).
Thermal Stability StudiesThe stability of the
Ca2+-free and Ca2+-bound
1,2-
-D-mannosidase against thermal-induced unfolding was
studied. The unfolding transition curves were obtained by a circular dichroism
measurement done in a Jasco J-720 spectropolarimeter. The protein solution was
dialyzed against 50 mM formate-KOH buffer, pH 4.0, then diluted in
the same buffer to adjust the A280 to 0.2 0.3. Path
length of the optical cuvette was 2 and 10 mm for the measurement of the far
and near UV regions, respectively. The fraction of protein in the unfolding
state (fU) was determined using the equation
fU = (
T
N)/
D
N), where
T is the
ellipticity at temperature T, and
N and
D represent the ellipticity for the native and
denatured protein, respectively.
 |
RESULTS
|
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Specific Activities of Mutant and Wild Type EnzymeSeven
carboxylic acid residues to be studied were constructed in the cloned A.
saitoi 1,2-
-D-mannosidase gene (msdS) by
site-directed mutagenesis. Recombinant enzymes were purified homogeneously on
SDS-PAGE gels (Fig.
1A) and identified as A. saitoi
1,2-
-D-mannosidase by Western blot analysis
(Fig. 1B). Purified
forms of all recombinant enzymes were assayed by the Somogyi-Nelson method
(28,
29). No activity was detected
under standard assay conditions for any of the mutant enzymes except E504Q
mutant, and the specific activity could only be determined after extended
incubation, typically at 60 90 min
(Table I). From
Table I, it appears obvious
that the mutations generated resulted in enzymes with significantly reduced
ability to hydrolyze Man-
1,2-Man-OMe, as their specific activity is at
least 103-fold less than that of the wild type enzyme. The specific
activities of D269N and E411Q mutants in particular were not detected
completely. The results of several experiments suggest that there is no
significant contamination by wild type 1,2-
-D-mannosidase in
the low activity of mutant enzymes (D269N and E411Q).
Kinetic AnalysisKinetic characterization of E124Q, E124D,
D269E, E273D, E411D, E414D, and E474D mutants and wild type
1,2-
-D-mannosidase was performed at pH 5.0 using
Man-
1,2-Man-OMe as a substrate. Results of these kinetic experiments
are shown in Table II. Kinetic
parameters for D269N and E411Q mutants could not be measured, since the
reaction rates were below the detectable limit. The kcat
value for E124Q mutant was 0.0078 s1, almost 0.3%
that of the wild type enzyme. E124D mutant enzyme also decreased the
kcat value (0.9% of wild type enzyme). The
Km values of E124Q and E124D mutant enzymes were
essentially unchanged. Compared with wild type
1,2-
-D-mannosidase, the D269E and E411D mutant enzymes
showed 17- and 28-fold increases in Km values,
respectively, whereas the kcat values were slightly lower
than that of wild type enzyme. The Km values of
E273D, E414D, and E474D mutant enzymes were greatly increased to
2331-fold that of wild type enzyme, and the kcat
values were decreased (115% of wild type enzyme).
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TABLE II Kinetic parameters of wild type and mutant
1,2- -D-mannosidases S.E in kinetic parameters:
Km (±2.6-10.4 %) and kcat
(±1.8-4.8 %).
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pH-Activity Profiles of E124A and E124D Mutant
1,2-
-D-MannosidaseFig.
2 shows the pH dependence of the reaction rate for
Man-
1,2-Man-OMe hydrolysis for wild type
1,2-
-D-mannosidase and E124A and E124D mutant enzymes. The
optimum pH of E124D and E124A mutant enzymes shifted from 5.0 to 4.0 and 4.5,
respectively. The activity of E124A mutant enzyme was found to be lower than
that of wild type enzyme over the whole pH range studied, but the effect was
more pronounced for the basic limb.
Effects of an External Nucleophile on Alanine Mutant
EnzymesMutation (Glu or Asp to Ala) at the general base catalyst
creates a cavity in the active site that can accommodate a small external
nucleophile. Sodium salts of azide at 3 M were used as external
nucleophiles in the enzyme-catalyzed hydrolysis of
Man6GlcNAc2-PA sugar chain. The reaction mixture was
analyzed by HPLC (Fig. 3). The
activity of wild type 1,2-
-D-mannosidase was inhibited by a
high concentration of azide. The E124A, D269N, and E411A mutant enzymes were
inactive in the absence of azide, as a peak corresponding to a
Man5GlcNAc2-PA was not detected. In the presence of
azide, these mutant enzymes could not be reactivated.

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FIG. 3. Effects of an external nucleophile on catalytic residue mutants of
A. saitoi 1,2- -D-mannosidase using
Man6GlcNAc2-PA as a substrate. A mixture (20 µl)
of 10 pmol Man6GlcNAc2-PA, 3 M sodium azide,
and a mutant or the wild type enzyme was incubated at pH 5.0 (50 mM
acetate buffer) and 30 °C for 72 h. A, 0.25 µM wild
type enzyme. B, 9.7 µM E124A mutant enzyme. C,
5.9 µM D269N mutant enzyme. D, 10.5 µM
E411A mutant enzyme.
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Atomic Absorption Spectrophotometric Analysis and Kinetic Analysis of
Wild Type
1,2-
-D-MannosidaseConcentrations of
Ca2+ were determined by atomic absorption spectrophotometric
analysis. The purified recombinant wild type
1,2-
-D-mannosidase almost completely did not contain
Ca2+ (data not shown). Other divalent metal cations including
Mg2+, Mn2+, Co2+, Cu2+, and
Zn2+ were also not detected. After Ca2+ treatment (see
"Experimental Procedures"), Ca2+ content was determined
as 0.9 mol/mol of wild type 1,2-
-mannosidase. Kinetic parameters of
Ca2+-free and Ca2+ binding
1,2-
-D-mannosidase were determined
(Table III); the
kcat and Km values were
little affected by the Ca2+ binding.
EDTA InhibitionEDTA inhibited the Ca2+-free wild
type 1,2-
-D-mannosidase. The inhibition was investigated by
fixed concentrations of EDTA in the assay buffer and by varying the
concentrations of substrate. A Lineweaver-Burk plot showed a pattern
consistent with competitive inhibition
(Fig. 4) and gave a
Ki of 0.91 mM for EDTA.
Ca2+ Binding Properties of Mutant
1,2-
-D-Mannosidases Ca2+
concentration of the mutant 1,2-
-D-mannosidase was
determined by atomic absorption spectrophotometry after Ca2+
binding treatment as described under "Experimental Procedures." As
shown in Table IV, the
Ca2+-free wild type enzyme bound 0.9 mol of calcium/mol of protein,
1 mol/mol. E124D, E411D, and E504Q mutants also contained 0.8, 1.1, and
0.9 mol of calcium/mol of protein, respectively. E124Q, D269E, D269N, E474Q,
and E474D contained less calcium than 1 mol of calcium/mol of protein. E411Q,
E273Q, E273D, E414Q, and E414D mutants demonstrated an almost complete loss of
binding of Ca2+.
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TABLE IV Ca2+ binding properties of wild type and mutant
1,2- -D-mannosidases after Ca2+
-treatment determined by atomic absorption spectrophotometry
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Effect of Ca2+ Binding on
1,2-
-D-Mannosidase Thermal StabilityThe
spectra of Ca2+-free and Ca2+-bound
1,2-
-D-mannosidase at far and near UV region were consistent
with each other (data not shown). The Ca2+ binding did not undergo
a large conformational change. Thermally induced loss of secondary and
tertiary structure was monitored at 222 and 291 nm, respectively
(Fig. 5).
Tm, the temperature at which half of the
molecules are unfolded, for Ca2+-free
1,2-
-D-mannosidase was 56 and 58 °C at 222 and 291 nm,
respectively. The Ca2+ binding contributed to the thermal stability
increasing Tm to 58 and 62 °C at 222 and 291
nm, respectively.
 |
DISCUSSION
|
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Identification of Catalytic Amino Acid ResiduesDrastic
reductions in the values of the catalytic coefficient
(kcat) were observed in the E124Q and E124D mutant enzymes
(Table II). This may result
from significant impairment of the chemical steps in the catalytic reaction.
The Km value was essentially unchanged from wild
type enzyme. It is, thus, clear that Glu-124 participates in glucosidic bond
hydrolysis. E124D shifted the optimum pH to be more acidic
(Fig. 2). This result is shown
by the lower pKa of Asp than that of Glu and
supports the assumption that Glu-124 is directly involved in the catalytic
mechanism. It could not be determined whether the Glu-124 residue is a
carboxylate group (COO) or carboxyl group (COOH). The pH
dependence of E124A affected the basic limb, which was generally assumed to be
due to the general acid catalyst. An external nucleophile sodium azide did not
reactivate the E124A mutant enzyme (Fig.
3). Thus the Glu-124 may act as an acid catalyst. The mechanism of
the all carbohydrate hydrolases usually involves a pair of carboxylic acid
residues. In P. citrinum 1,2-
-D-mannosidase, two of
the three residues (Glu-124, Asp-269, and Glu-411 in A. saitoi
enzyme) are believed to be potentially catalytic residues
(7). Glu-124 was suggested to
act as an acid catalyst as discussed above. The most likely candidates for the
other catalytic residues are Asp-269 and Glu-411. However, D269E and E411D did
not show a considerable decrease in the kcat values
(Table II). Recently, a base
catalyst of an inverting
-amylase (EC 3.2.1.2
[EC]
) from Bacillus
cereus var. mycoides was determined by the chemical rescue
methodology for a catalytic site mutant (E367A) by azide
(19). In the case of A.
saitoi 1,2-
-D-mannosidase an external nucleophile did
not reactivate the D269N and E411A mutant enzymes
(Fig. 3). Neither Asp-269 nor
Glu-411 residues can be a base catalyst in the normal sense. Although the
D269N and E411Q mutant enzymes were catalytically inactive, the D269E and
E411D mutant enzymes showed considerable activity. The results indicate that
the carboxyl groups at these positions may be required for the
1,2-
-D-mannosidase activity. Structural and modeling studies
on cellobiohydrolase Cel6A (EC 3.2.1.91
[EC]
) from Trichoderma reesei
suggest that the catalytic mechanism may not directly involve a catalytic base
(31). The catalytic mechanism
of 1,2-
-D-mannosidase deviates from the typical glycosyl
hydrolase.
Role of Glu-273, Glu-414, and Glu-474 ResiduesE273D, E414D,
and E474D mutant enzymes decreased kcat values and
increased Km values
(Table II). The reduction of
the kcat value is assumed to be due to the destabilization
of the sugar ring. It has become increasingly clear that ring distortion at
the 1 site is crucial in the catalytic mechanism of cellobiohydrolase
(32,
33). The catalytic reaction of
-amylase from Pseudomonas stutzeri involves three acidic
residues; two residues act as a proton donor and a proton acceptor. The other
residue works to tightly bind the substrate, giving a twisted and deformed
conformation of the glucose ring at position 1
(34). Glu-273, Glu-414, and
Glu-474 may bind the substrate to change the conformation of the mannose ring
into more reactive one. The three-dimensional structure of P.
citrinum 1,2-
-D-mannosidase indicates that Glu-271,
Glu-412, and Glu-472 residues (Glu-273, Glu-414, and Glu-474 in A.
saitoi enzyme) in the active site are located at the bottom of the cavity
and are not directly involved in the catalytic mechanism. It is demonstrated
that these residues are hydrogen-bonded to the Ca2+ via water
molecules, and the Ca2+ is involved in the stability of a
substrate. But the recombinant wild type A. saitoi
1,2-
-D-mannosidase contained no Ca2+ and other
divalent metal cations. Despite the Ca2+ having no effect on the
1,2-
-mannosidase activity (Table
III), its binding sites were conserved in A. saitoi
1,2-
-D-mannosidase. This also indicates that the drastic
decrease of the activities of E273D, E414D, and E474D mutants
(Table IV) did not parallel the
loss of the Ca2+ binding ability. These residues are involved in
the substrate binding sites.
Ca2+ BindingIt was reported earlier
that the recombinant A. saitoi 1,2-
-D-mannosidase
has a molecule of Ca2+
(25). In this study, the
recombinant wild type A. saitoi 1,2-
-D-mannosidase
contained no Ca2+. The purified wild type enzyme did not bind
Ca2+ with an addition of CaCl2 (data not shown). We
found that the Ca2+-free enzyme bound 1 mol of Ca2+ per
1 mol of protein after the addition of a high concentration of NaCl. It was
also demonstrated that the Ca2+ binding site is conserved
(Table IV). 1,2-
-Mannosidase generally requires a divalent metal cation for the
activity (13,
14,
3537).
In the case of A. saitoi 1,2-
-D-mannosidase, the
Ca2+ had no effect on either the kcat or the
Km value
(Table III). Thus, it is clear
that Ca2+ is not essential for the activity of A. saitoi
1,2-
-D-mannosidase, although the Ca2+ binding
site is conserved. EDTA was shown to behave as a competitive inhibitor of
A. saitoi 1,2-
-D-mannosidase
(Fig. 4); this indicates that
the enzyme contains no divalent metal cation. 1-Deoxymannojirimycin inhibits
1,2-
-mannosidase as a substrate analog. However, EDTA is not a
substrate analog. Tris is a competitive inhibitor for 1,2-
-mannosidase
from rabbit liver (14). Tris
and other buffers containing primary hydroxyl groups substantially decreased
its activity (38,
39). Carboxyl groups of EDTA
may have an affinity for the active site of A. saitoi
1,2-
-D-mannosidase. A molecule of Ca2+
contributed to thermal stability (Fig.
5). The role of Ca2+ for A. saitoi
1,2-
-D-mannosidase is to protect the enzyme against thermal
denaturation, and it was observed to have the same role in Saccharomyces
cerevisiae-processing 1,2-
-mannosidase
(12). Although Ca2+
is essential for S. cerevisiae enzyme activity
(12,
37), A. saitoi
1,2-
-D-mannosidase did not require Ca2+ for the
activity (Table III). This is
the first report demonstrating that 1,2-
-D-mannosidase
requires no divalent metal cation for its activity.
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶
To whom correspondence should be addressed. Fax: 81-426-91-9312; E-mail:
ichisima{at}t.soka.ac.jp.
1 The abbreviations used are: Man, mannose; Man-
1,2-Man-OMe, methyl
2-O-
-D-mannopyranosyl
-D-mannopyranoside or
-D-Man-[1
2]-
-D-Man-1
OMe; GlcNAc, N-acetylglucosamine;
OMe, methyl ester; PA, pyridylaminated. 
 |
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