Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8

Article abstract of DOI:10.1271/bbb.66.2578

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1- phosphate. The K_m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k_cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D- glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k_cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k_cat of 8.0 s^-1 had the highest (1.1 × 10⁴ M^-1 s^-1) k _cat/K_m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.

Full text of DOI:10.1271/bbb.66.2578

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Characterization of a Cellobiose Phosphorylase from
a Hyperthermophilic Eubacterium, Thermotoga
maritima MSB8
a
a
a
a
Eranna RAJASHEKHARA , Motomitsu KITAOKA , Yeon-Kye KIM & Kiyoshi HAYASHI
a
Enzyme Laboratory, National Food Research Institute 2-1-12 Kannondai, Tsukuba,
Ibaraki 305-8642, Japan
Published online: 22 May 2014.
To cite this article: Eranna RAJASHEKHARA, Motomitsu KITAOKA, Yeon-Kye KIM & Kiyoshi HAYASHI (2002) Characterization
of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium, Thermotoga maritima MSB8, Bioscience,
Biotechnology, and Biochemistry, 66:12, 2578-2586, DOI: 10.1271/bbb.66.2578
To link to this article: http://dx.doi.org/10.1271/bbb.66.2578
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Biosci. Biotechnol. Biochem., 66 (12), 2578–2586, 2002
Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic
Eubacterium, Thermotoga maritima MSB8
Eranna RAJASHEKHARA, Motomitsu KITAOKA,† Yeon-Kye KIM, and Kiyoshi HAYASHI
Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Received May 27, 2002; Accepted July 25, 2002
The cepA putative gene encoding a cellobiose phos-
phorylase of Thermotoga maritima MSB8 was cloned,
expressed in Escherichia coli BL21-codonplus-RIL and
characterized in detail. The maximal enzyme activity
ing b-1,4 glycosidic bonds, but has a relaxed speciˆci-
ty with respect to the reducing sugars that function as
glucosyl acceptors in the synthetic reaction. The ab-
solute recognition of the b-anomeric hydroxyl group
was observed at pH 6.2 and 80
9C. The energy of activa-
of the acceptor molecule was conˆrmed for the en-
tion was 74 kJ mol. The enzyme was stable for 30 min
zyme from Cellvibrio gilvus by using pseudo-glu-
W
9
)
at 70
9
C in the pH range of 6–8. The enzyme phos-
coses. However, b-glucosides are not accepted as
phorolyzed cellobiose in an random-ordered bi bi
mechanism with the random binding of cellobiose and
the acceptor by any reported cellobiose phosphory-
lases. Syntheses of various disaccharides and bran-
ched trisaccharides using the synthetic reaction of
phosphate followed by the ordered release of
and -glucose-1-phosphate. The for cellobiose and
phosphate were 0.29 and 0.15 m respectively, and the
cat was 5.4 s . In the synthetic reaction, -glucose,
mannose, 2-deoxy- -glucose, -glucosamine, -xylose,
and 6-deoxy- -glucose were found to act as glucosyl ac-
ceptors. Methyl- -glucoside also acted as a substrate
for the enzyme and is reported here for the ˆrst time as a
substrate for cellobiose phosphorylases. -Xylose had
cat followed by 6-deoxy- -glucose
-glucose (16 s ). The natural
D
-glucose
1
0–14)
a-
D
K
m
cellobiose phosphorylase have been reported.
M
Direct synthesis of cellobiose from sucrose by the
combined action of three enzymes including cel-
－
1
k
D
D
-
1
5)
D
D
D
lobiose phosphorylase was also reported, and hence
indicated the possible commercial importance of the
enzyme. The enzyme was used in the selective colori-
metric measurment of cellobiose in the presence of
D
b
-
D
1
6)
D
glucose and cellooligosaccharides, a useful tool in
investigation of biomass degradation.
－
1
the highest (40 s
(
)
k
D
－
1
－
1
17)
17 s ) and 2-deoxy-
D
The enzyme from Cell. gilvus and Cellulomonas
－
1
8)
substrate,
highest (1.1
glucosyl acceptors.
phosphorylase, acted as a competitive inhibitor of the
D
-glucose with the
k
cat of 8.0 s
had the
uda catalyzed the phosphorolytic reaction in an or-
4
－
1
－
1
×
10
M
s
)
k
K
compared with other
-Glucose, a substrate of cellobiose
dered bi bi mechanism as follows: the enzyme at ˆrst
bound cellobiose and then phosphate followed by the
sequential release of glucose and G-1-P. Recently, we
found that the enzyme from Clostridium thermocel-
lum YM-4 also followed an ordered bi bi mechanism,
cat
W
m
D
other substrate,
centrations.
a-D-glucose-1-phosphate, at higher con-
1
8)
but the order of the substrate binding was reverse.
Key words: hyperthermostable enzyme; cellobiose
phosphorylase; Thermotoga maritima
The type strain of Thermotoga maritima, strain
;
MSB8, is a hyperthermophilic fermentative bacteri-
phosphorolytic reaction; synthetic reac-
tion
um that grows optimally at 80 9C and its whole
genome was sequenced for functional genomic stu-
1
9)
dies. Both T. maritima and T. neapolitana are able
to use various carbohydrates for growth, and their
cellulolytic enzymes are reported to be stable at ex-
treme temperatures. Due to the extreme thermal
resistance, there is an increasing interest for applica-
tion of hyperthermostable enzymes in biotechnologi-
Cellobiose, a common intermediate in enzymatic
cleavage of cellulose, can be degraded by hydrolysis
or inorganic-phosphate-dependent phosphorolysis.
Cellobiose phosphorylase (EC 2.4.1.20) catalyzes the
reversible phosphorolysis of cellobiose into
a
-
D
-glu-
2
0–25)
cose 1-phosphate (G-1-P) and -glucose with the in-
D
cal processes.
version of the anomeric conˆguration, but does not
phosphorolyze cellotriose or higher cello-oligosac-
charides. The presence of this enzyme was ˆrst
Recently, a cellobiose phosphorylase from a hyper-
thermophilic bacterium, T. neapolitana, was charac-
terized, but not in detail. Because of the commer-
cial importance of the enzyme and for fundamental
understanding, a detailed functional analyses, phos-
phorolytic- and synthetic reaction of the recombinant
2
6)
1
)
demonstrated in Clostridium thermocellum and
2
–8)
subsequently studied in several organisms. The en-
zyme is absolutely speciˆc for cleaving and synthesiz-
†
To whom correspondence should be addressed. Fax: +81-298-38-7321; E-mail: mkitaoka
＠
nfri.aŠrc.go.jp

Cellobiose Phosphorylase from Thermotoga maritima
2579
cellobiose phosphorylase from another hyperther-
mophilc bacterium, T. maritima, was studied and
reported in this paper.
shaker in Luria-Bertani (LB) broth (100 ml) with
30 g ml of kanamycin to produce seed culture. This
100-ml seed culture was used as the inoculum for
00 ml of LB broth supplemented with 30 g ml of
kanamycin. When the OD600 of the broth reached ap-
proximately 0.6, the target gene was induced by the
addition of isopropyl thio-galactopyranoside (IPTG)
m
W
9
m
W
Materials and Methods
Genomic DNA, plasmids and bacterial strains.
The genomic DNA of the type strain T. maritima
MSB8 was kindly provided by Prof. Dr. K. O. Stetter
to a ˆnal concentration of 1 m
cells was continued for another 5 h at 30
cells were harvested by centrifugation at 4,000 g for
20 min at 4 C.
M
. Cultivation of the
9C . Then the
(
Lehrstuhl Fuer Mikrobiologie, University of Regen-
sburg, Germany). A plasmid pCR 2.1-TOPO vector
9
(
TOPO TA cloning kit, Invitrogen, USA) was used
to insert the PCR-ampliˆed product of the cepA
Puriˆcation of recombinant enzyme. Approxi-
mately 2.7 g (wet weight) of cells were suspended in
gene described later. TOP 10 electrocompetant
„
Escherichia coli cells F mer
A
D
(
mrr-hsdRMS-
rec 1araD139
ara-leu)7697galUgalKrpsL(str )-endA1nupG ] were
20 m
M
sodium phosphate buŠer, pH 8.0, containing
mcrBC)
q
80lacZ
D
M15
D
lacX74deo
R
A
D
300 m
M
NaCl and 10 m
M
imidazole (buŠer A) and
R
(
the cells were lysed by soniˆcation. The soniˆed sam-
ple was centrifuged at 10,000 g for 30 min to remove
the cell debris. The supernatant containing crude en-
zyme (71 ml) was mixed with 3 mL of fresh NiNTA
resin (Qiagen, Germany) and kept on ice under gentle
shaking for 15–20 min. The enzyme-bound resin was
then packed into a column and eluted the enzyme
used as the host for the transformed by pCR2.1 plas-
mids harboring the cellobiose phosphorylase gene
(
cepA) of T. maritima. The plasmid vector pET
8a(+) from Novogen, USA, was used for the sub-
cloning of the gene orienting the (His) epitope tag at
2
6
the gene's N-terminal end. The E. coli strain
BL21-Codonplus-RIL competent cells [BF„ompT
with a linear gradient of 10–200 m
buŠer A. The active fractions were pooled and dia-
lyzed overnight at 4 C against 5 m 2-[ -mor-
M
of imidazole in
r
hsdS(rB„mB„)dcm+Tet gal endA Hte [argU ileY
r
leuW Cam ] were used for the heterologous expres-
9
M
N
sion of the cepA gene. All the recombinant DNA
pholino]ethane sulfonic acid (MES) buŠer, pH 6.2.
The dialyzed enzyme sample was further puriˆed by
an anion exchange column, Source 30 Q (Pharmacia,
Sweden), of column volume 4 ml. The enzyme pro-
techniques followed were as described by Sambrook
2
7)
et al
.
Cloning, Sequencing of cepA, and Expression.
The ORF TM1848 (AE001822) which codes for the
putative cellobiose phosphorylase of T. maritima
MSB8 was ampliˆed from the genomic DNA by a 25
tein was eluted with a linear gradient of 0–100 m
NaCl in 20 m MES buŠer, pH 6.2.
M
of
M
Protein analyses. Protein concentrations were
measured using Coomassie Plus Protein assay rea-
gent kit (Pierce, Rockford, USA) with bovine serum
cycle PCR (98
ing KOD-DASH DNA polymerase (Toyobo, Osaka,
Japan), a forward primer, 5 CAT ATG GTG CGA
TTC GGT TAT TTT GAT GAC GTC, and a reverse
primer, 5 GAG CTC TCA GCC CAT CAC AAC
TTC AAC CCT GTG with the incorporation of NdeI
N-terminal end) and SacI (C-terminal end) restric-
9 9 9
C, 30 s—55 C, 30 s—72 C, 150 s) us-
?
albumin as a standard. Sodium dodecyl sulfte poly-
W
acrylamide gel electrophoresis (SDS-PAGE) of the
enzyme samples was done and the protein bands were
made visible by staining the gel with Coomassie brilli-
?
2
8)
(
ant blue. A 10-kDa ladder (Life technologies, USA)
was used as a standard molecular mass protein mar-
ker. The puriˆed enzyme (approximately 0.2 mg)
from the Mono Q fraction was put on a gel ˆltration
tion sites (underlined) respectively. The amplicon was
cloned in pCR 2.1-TOPO and transferred to elec-
trocompetent E. coli TOP 10 cells. The nucleotide se-
quence of the cloned gene was conˆrmed using a Big
dye terminator cycle sequencing kit (Perkin-Elmer,
Applied Biosystems, CA) and GENETIX program
column of Superose 6 HR10 30 previously equili-
W
brated with 50 m
M
MES buŠer, pH 6.2, containing
0.1 NaCl, at a ‰ow rate of 0.1 ml per min to meas-
M
(
Software Development Co., Tokyo, Japan). The
ure its native molecular mass with MW-Marker
HPLC (Oriental Yeast Co., Osaka, Japan).
target gene, cepA from the pCR 2.1-TOPO-TmcepA
was digested with NdeI and SacI restriction enzymes
and ligated to pET 28a(+) that had been previously
digested with the above restriction enzymes.
Ligation-High T4 DNA ligase (Toyobo) was used for
gene-vector ligation during subcloning. The pET
Enzyme assays. The reaction of cellobiose
phosphorylase was done at 60
9C in 25 m
M
MES
buŠer, pH 6.2. In the phosphorolytic reaction the
amount of -glucose 1-phosphate (G-1-P) or -glu-
a
-
D
D
2
8a(+) harboring cepA with (His)
6
residues at its N-
cose formed was measured. G-1-P was estimated by
the phosphoglucomutase-glucose-6-phosphate de-
terminal end was transferred to electrocompetent E.
coli BL21-Codonplus-RIL cells. The transformed
2
9)
hydrogenase system.
D
-Glucose was measured by
9
cells were cultured overnight at 37 C over a rotary
the glucose oxidase-peroxidase method using a Glu

2
580
E. RAJASHEKHARA et al.
Table 1. Summary of Puriˆcation of T. maritima Cellobiose
Phosphorylase
Total
Total Speciˆc
Activity
recovery
Puriˆcation
step
Puriˆcation
fold
activity protein activity
(
units)
(mg) (U mg)
(z)
W
Crude ex-
tract
NiNTA
92.3
54.4
19.7
208.7
22.6
7.4
0.44
2.44
2.66
1
5.4
6
100
59
21
Source 30 Q
ICII kit (Wako Pure Chemicals, Osaka, Japan). In
the synthetic reaction the product, phosphate was es-
3
0)
timated by the method of Lowry and Lopez. One
unit of activity was deˆned as the amount of enzyme
that produced one
m
mol of phosphate per minute
glucose at 60 C.
Fig. 1. SDS-PAGE Analysis of Recombinant T. maritima Cel-
lobiose Phopshorylase at DiŠerent Puriˆcation Steps.
lane M, molecular mass markers; lane 1, crude extract; lane 2,
NiNTA fraction, lane 3, ``Source 30 Q'' fraction.
from 10 m G-1-P and 10 m
M
M
9
Optimum pH and temperature. To ˆnd the opti-
mum pH, the phosphorolytic reaction was done at
6
0
9
C for 30 min with 10 m
phosphate in 50 m
C such as sodium citrate (pH 2.2 to 4.1), sodium
acetate (pH 3.7 to 5.7), MES (pH 5.1 to 7.2),
-[ -morpholino]propane sulfonic acid (MOPS)
pH 6.4 to 8.8), -[2-hydroxyethyl]piperzine-
2-ethanesulfonic acid] (HEPES) (pH 6.5 to 8.6),
M
cellobiose and 10 m
M
M
of various buŠers prepared at
tivity at 60
cose formed. The inhibition eŠects of the products,
-glucose (0–1 m ) and G-1-P (0–2 m ), against the
9C was estimated by measuring the -glu-
D
25
9
D
M
M
3
(
[
N
substrates, cellobiose and phosphate, were also stud-
ied at several ˆxed concentrations of one substrate
N
N?-
(2.5, 5, 20 m
M
) and varying the concentration of
) by measuring G-1-P
Tris[hydroxymethyl]aminomethane hydrochloride)
Tris HCl) (pH 7.1 to 9.1), 2-[ -cyclohex-
yamino]ethane sulfonic acid (CHES) (pH 8.2 to
0.3), and 3-[cyclohexylamino]-1-propane sulfonic
acid (CAPS) (pH 9.6 to 11.5), and the amount of
other substrate (0.25–2.5 m
M
(
N
and -glucose, respectively, to ˆnd the orders of both
D
W
substrates binding and products release.
1
D
-
Synthetic reaction. Various sugars and their
derivatives mentioned in Table 2 were examined for
the glucosyl acceptor of the cellobiose phosphorylase
glucose formed was measured as mentioned earlier.
To ˆnd the optimum temperature, the phosphoro-
lytic reaction was done at temperatures ranging from
at 10 m
25 m MES (pH 6.2) buŠer. The kinetic parameters
were measured for those glucosyl acceptors that
showed a rate higher than 1 mole min mg.
M
concentration with 10 m
M
9
G-1-P at 60 C in
2
1
5 to 95
0 m cellobiose and 10 m
-glucose formed after the reaction was measured
9
C for 30 min in 25 m
M
MES buŠer with
M
M
M
phosphate. The amount
of
D
m
W
W
and used for evaluating the average activity for
3
0 min.
Kinetic analyses. Kinetic parameters were calculat-
ed by regressing the experimental data to the ap-
propriate rate formula based on the curve ˆt method
by using a computer program, Graˆt-Ver.4 (Erithaus
Software, UK).
pH and thermal stability. The puriˆed enzyme
(
9
13.3 mg ml) was incubated for 30 min at 70 C in
W
various buŠers as mentioned above and the residual
enzyme activity was measured at 60 C for 30 min in
MES buŠer, pH 6.2 with 10 m of cellobiose and
0 m phosphate. To measure the thermal stability,
the enzyme (13.3 g ml) was incubated for 30 min in
5 m MES buŠer (pH 6.2) at diŠerent temperatures
ranging from 30 to 100 C and the residual enzyme
activity was measured at 60 C for 30 min in MES
buŠer, pH 6.2 containing 10 m of cellobiose and
0 m phosphate.
9
M
Results
1
M
m
Puriˆcation and properties of the enzyme
W
2
M
The recombinant cellobiose phosphorylase
was puriˆed 6-fold to a ˆnal speciˆc activity of
9
9
2.66 units mg protein with an activity recovery of
W
M
21z through two puriˆcation steps (Table 1). Ap-
1
M
proximately 7.4 mg of pure enzyme was obtained
from 1000 ml of culture. The puriˆed cellobiose
phosphorylase gave a single protein band on SDS-
Reaction mechanism. In the phosphorolytic direc-
tion, the reaction mechanism was measured using
diŠerent combinations of cellobiose and phosphate
PAGE gel corresponding to a molecular mass (
r
M )
of approximately 85 kDa (Fig. 1), which agreed with
the size calculated (94,322) on the basis of the prima-
concentrations from 0.25 to 2.5 m . The enzyme ac-
M

Cellobiose Phosphorylase from Thermotoga maritima
2581
Fig. 2. EŠects of pH on the Activity of the T. maritima Cel-
Fig. 4. EŠects of pH on the Stability of the T. maritima Cel-
lobiose Phosphorylase.
lobiose Phosphorylase.
The activity was measured at 60
buŠers of various pH levels. Values are shown as a percentage of
the maximum activity, which is deˆned as 100 . BuŠers used
were sodium citrate ( ), sodium acetate ( ), MES ( ), MOPS
), HEPES ( ), Tris HCl ( ), CHES ( ), and CAPS ( ).
9
C for 30 min in 25 m
M
The stability of the enzyme was examined by treating the en-
zyme at 70
residual enzyme activity was measured at 60
buŠer of pH 6.2. Values are shown as percentages of the original
activity (measured in 25 m MES, pH 6.2, without incubation
at 70 C) and this is deˆned as 100 . BuŠers used were sodium
citrate ( ), sodium acetate ( ), MES ( ), MOPS ( ), HEPES
), Tris HCl ( ), CHES ( ), and CAPS ( ).
9
C for 30 min in buŠers at various pH levels. The
z
9
C in 25 m MES
M



(

#
$
%
&
M
W
9
z




(
#
$
%
&
W
The optimum pH for the maximal enzyme activity
was found at 6.2 and, the enzyme showed more than
8
0
z
of the maximal activity between pH 4.5 to 7.5
(
Fig. 2). The optimum temperature for the maximal
enzyme reaction for 30 min was approximately 80
9C
(
Fig. 3). From the linear part of the Arrhenius plot
between 25 and 80
9
C, the activation energy for the
phosphorolytic reaction was found to be 74 kJ mol.
W
The enzyme retained more than 90
imal activity in the pH range of 5 to 9 after 30 min of
heating at 70 C (Fig. 4). The enzyme retained its
original activity at temperatures lower than 70 C,
but approximately 25 of the activity was lost upon
incubating at 90 C and the activity was completely
lost at 100 C after a 30-min incubation period
Fig. 5). It is notable that the reaction rate at 95
for 30 min (35 of the maximum) was much lower
than the value expected from the stability data (71
z
of its max-
9
9
z
9
9
(
9
C
Fig. 3. EŠects of Temperature on the Activity of the T. maritima
Cellobiose Phosphorylase.
z
The activity was measured at various temperatures for 30 min
z
in 25 m
M MES buŠer, pH 6.2. Values are shown as a percentage
remaining after 30 min). The fact may suggest some
reversible change in the enzyme structure to an inac-
tive form at higher temperatures.
of the maximum activity, which is deˆned as 100
z
.
The course of the thermal inactivation of the en-
1
9)
ry structure of the enzyme. The native molecular
mass was estimated to be 150 kDa by gel ˆltration,
indicating that the recombinant cellobiose phos-
phorylase existed in a dimer form. The enzyme phos-
phorolyzed cellobiose, but did not phosphorolyze
cellotriose or cellotetrose to form G-1-P even with a
long incubation time.
zyme at 60 and 80
any substrate or stabilizer. At 60
found to be stable for a period of 5 h, while at 80
there was a 45 reduction in the enzyme activity af-
9
C was studied in the absence of
9
C the enzyme was
9C
z
ter 60 min with the enzyme displaying a half-life of
70 min.

2
582
E. RAJASHEKHARA et al.
Fig. 5. EŠect of Temperature on the Stability of the T. maritima
Fig. 7. Detailed Reaction Mechanisms in Sequential bi bi
Mechanisms.
Cellobiose Phosphorylase.
The enzyme was incubated in 25 m
MES, pH 6.2 buŠer at diŠerent temperatures for 30 min. Resid-
ual enzyme activities were measured at 60 C for 30 min. The
residual activity are shown as percentages of the maximum ac-
tivity (measured at 60 C for 30 min without a prior incubation
step) taken as 100
M ˆnal concentration of
(
1) The typical sequential bi bi mechanisms appeared in
31)
Cleland's report.
(2) Inhibition pattern and postulated
9
mechanism of T. maritima cellobiose. phosphorylase.
Left column, scheme of each mechanism; center column, name
of each mechanism; right column, table of inhibition pattern of
each product against each substrate. A and B are substrates and
P and Q are products. In the table, c, m, and u mean competi-
tive, mixed-type, and uncompetitive inhibitions, respectively.
Inhibition patterns out of and in the parenthesis mean the inhi-
bition pattern observed with unsaturated and saturated concen-
trations of the other substrate, respectively. The concentrations
9
z
.
of the other substrate used in the table of (2) are 2.5, 5 m
saturated), and 20 m (saturated).
M (un-
M
quential bi bi mechanism were calculated by regress-
ing the experimental data with the following equation
as shown below:
[
＝
k
cat[E][A][B] (
K
iA
mB
K +KmA[B]+KmB[A]
W
+
[A][B])
where [A] is the initial concentration of cellobiose,
[B] is the initial concentration of phosphate, and [E]
is the enzyme concentration.
Fig. 6. Double Reciprocal Plot of the Phosphorolytic Reaction
of Cellobiose.
The enzyme activity was measured as a function of the con-
centration of cellobiose at various concentrations of phosphate
„
1
k
cat
＝
5.4 s
0.15 m
The inhibition patterns of the products, G-1-P and
glucose, against the substrates, -cellobiose and
;
K
mA
＝
0.29 m
M
;
K
iA
＝
1.7 m
M
;
at 60
9
M
C for 30 min. Concentrations of phosphate were

,
K
mB
＝
M
.
0
.25 m
;

, 0.5 m , 1.0 m , 1.5 m , 2.5 mM.
M
;

M
;

M
;
#
D
-
D
phosphate, are summarized in the table of Fig. 7(2).
G-1-P acted as a competitive inhibitor and glucose as
a mixed-type inhibitor against both cellobiose and
phosphate at all of the three concentrations of the
opposite substrates examined. This result did not
match with any of three possible mechanisms in the
Reaction mechanism
Kinetic analyses of the phosphorolytic reaction
were undertaken to discover the reaction mechanism
of the enzyme. When the concentration of cellobiose
was varied, the 1 v„1 [cellobiose] plot obtained
W
W
with selected phosphate concentrations showed the
lines crossing at a point in the upper left-hand quad-
rant (Fig. 6), indicating that the phosphorolytic reac-
tion occurred in a sequential bi bi mechanism. The
kinetic parameters in the rate equation for the se-
31)
sequential bi bi mechanism as shown in Fig. 7(1).
A
possible explanation of the result is the ``random-
ordered bi bi mechanism'' as described in Fig. 7(2),
where the sequence of the substrate binding is ran-

Cellobiose Phosphorylase from Thermotoga maritima
2583
Table 2. Substrate Acceptor Speciˆcity of T. maritima Cellobiose Phosphorylase in the Synthetic Reaction
Substrate
Rate (
m
mol min mg)
Substrate
-Glucono-
Rate ( mmol min mg)
W W
W
W
D
D
-Glucose
-Mannose
8.7
D
d
-lactone
—
1.1
2.9
2.9
—
—
—
—
—
—
Methyl-
Methyl-
b
-
-
D
-glucoside
-glucoside
1.1
—
—
—
—
—
—
—
—
—
—
—
2
-Deoxy-D-glucose
-Glucosamine
-Mannosamine
a
D
D
D
D
D
D
-Cellobiose
-Glucitol
-Mannitol
-Arabinose
-Arabinose
-Ribose
D
N
N
-Acetyl-
-Acetyl-
D
-glucosamine
-mannosamine
D
D
D
D
-Allose
-Galactose
-Fucose
L
D
L
L
L
L
-Sorbose
-Glucose
-Fucose
-Xylose
6
-Deoxy-
D
-glucose
13.8
15.0
—
D
D
D
-Xylose
-Glucuronamide
-Fructose
—
Values were measured using the concentrations of 10 m
, not detected (less than 0.6).
9
M G-1-P and 10 mM acceptor substrate at 60 C.
—
Table 3. Kinetic Parameters for Various Glucosyl Acceptors of
T. maritima Cellobiose Phosphorylase
K
m
m
k
(s
cat
„1
k
(M
cat
K
s
m
W
Substrate
„1 „1
(
M
)
)
)
a
4
D
D
D
-Glucose
0.69
67
5.7
47
4.1
14
135
8.0
4.4
5.2
16
17
40
1.1
6.5
9.1
3.4
4.3
2.8
4.8
×
10
×
10
×
10
×
10
×
10
×
10
×
10
b
1
2
2
3
3
1
-Mannose
-Glucosamine
c
b
c
2
6
-Deoxy-
-Deoxy-
D
-glucose
-glucose
D
D
-Xylosed
e
Methyl-
b
-
D
-glucoside
6.5
Concentrations used: a, 0.5–5 m
; e, 20–200 m
; b, 10–100 m ; c, 1–10 m ; d, 5–50
M M M
m
M
M
.
dom and the product release is ordered (glucose ˆrst
and then G-1-P).
Substrate speciˆcity for the synthetic reaction
Out of 27 compounds tested, seven were found to
act as glucosyl acceptors for the cellobiose phos-
phorylase (Table 2). It is notable that the enzyme
Fig. 8. The v-[
Cellobiose Phosphorylase at Fixed Concentrations of G-1-P.
Concentrations of G-1-P were , 1 m , 2 m , 5 m
The theoretical lines were obtained by regressing the experimen-
D
-glucose] Plot of the Synthetic Reaction of the

M
;

M
;

M
.
used
product from G-1-P and
identiˆed as -methyl-cellobioside by NMR analysis
data not shown). Detailed kinetic studies were done
by using these substrates, and their kinetic
b
-methyl-
D
-glucoside as the acceptor. The
1
2)
tal data to the formula of competitive substrate inhibition.
b
-methyl- -glucoside was
D
b
(
which one substrate, namely
inhibited the other substrate, G-1-P. Therefore, the
possibility of inhibition by -glucose was studied at
concentrations of -glucose ranging from 1 m to
100 m with several ˆxed concentrations of G-1-P.
As shown in Fig. 8, the [ -glucose]-v curves gave
maxima around a -glucose concentration of 10 m
D
-glucose, competitively
12)
parameters are presented in Table 3.
D
-Xylose had
D
„
1
the highes„t
k
cat (40 s ) followed by 6-deoxy-
D
-glu-
D
M
1
„1
cose (17 s ) and 2-deoxy-
natural substrate,
but it had the highest
pared with other glucosyl acceptors.
D
-glucose (16 s ). The
M
„
1
D
-glucose gave the4kcat of 8.0 s
,
D
„1
„1
k
cat
K
W
m
(1.1 10 ) com-
×
M
s
D
M
,
demonstrating that a similar inhibition also occurred
in the synthetic reaction by the cellobiose phosphory-
lase from T. maritima. The theoretical lines obtained
by regressing the experimental data with the formula
Inhibition pattern of
A substrate inhibition by
synthetic reaction was observed previously for the
D
-glucose
D
-glucose in the reverse
1
2)
explaining the competitive substrate inhibition, as
shown below, well explain the result (Fig. 8).
1
2)
cellobiose phopshorylases from Cellv. gilvus and
1
8,32)
Clost. thermocellum
.
These inhibitions were
v
＝
k
cat[E][Q][P]
s
K
iQ
K
mP+(
K
mQ
+
K
iQ
K
mP
KI1)[P]
W
W
explained as ``competitive substrate inhibition'', in

2
584
E. RAJASHEKHARA et al.
the glucosyl acceptors for the enzyme. The results in-
+KmP[Q]+[Q][P]
dicated that the hydroxyl groups at positions 3 and 4
were essential for the recognition of acceptors. The
kinetic parameters (Table 3) showed that the natural
2
+
+
(
(
K
iQ
K
mP
K
I1
K
I2
+
K
mQ
KI1)[P]
W
W
K
mQ
K
I1
K
I2 )[P]3t
W
substrate of the enzyme,
have the lowest (0.69 m
glucose (4.1 m ). The substitution or deletion of
hydroxyl group at C1, C2 or C6 conˆgurations of
glucose resulted in signiˆcant increases in , in-
dicating that these hydroxyl groups were also recog-
nized by the enzyme. -Xylose acted as a good
substrate with the maximum
compared to -glucose (8.0 s ). The cellobiose
phosphorylases from diŠerent organisms that we
studied strictly recognized the C1 conˆguration of
D
-glucose, was found to
where [Q] is the initial concentration of G-1-P, [P] is
the initial concentration of
enzyme concentration.
K
M
m
M
) followed by 6-deoxy-
D
-
D
-glucose, and [E] is the
D
-
„
1
k
cat
＝
13 s
;
K
mQ
＝
0.29 m
M
;
K
iQ
＝
1.7 m
M
;
K
m
K
mP
＝
0.15 m
M
;
K
I1
＝
7.3 m
M
;
K
I2
＝
190 m
M
.
D
„
1
k
cat value of 40 s as
„1
Discussion
D
The genome sequence results indicated the
b
-
presence of a putative cellobiose phosphorylase in T.
8
,9,12)
1
9)
D
-glucopyranose
zymes did not recognize
However, the cellobiose phosphorylase of T. mariti-
ma was found to accept methyl- -glucoside as a
substrate in the synthetic reaction to produce methyl-
-cellobioside. This unique speciˆcity has not been
reported for any other cellobiose phosphorylases.
The substitution of a hydroxyl group at C1 of -glu-
cose with a methoxy group resulted in a 196-fold in-
and it was believed that the en-
maritima
.
According to the primary structure, cel-
b
-glucosides as the acceptor.
lobiose phosphorylase, cellodextrin phosphorylase,
and chitibiose phosphorylase are grouped in the
3
3)
b
-
D
family 36 of glycosyl transferases. (http: afmb-
WW
¿
mrs.fr pedro CAZY gtf.html) The percent amino
W
W
W
(-
D
acid similarities among cellobiose phosphorylases
found increasing from mesophilic (61 in Cellv.
gilvus, AB010707) to thermophilic (73 Clost.
thermocellum, AB013109, and Clost. stercorarium
U56424), and hyperthermophilic (92 T.
z
z
D
,
crease in the
cose (0.69 m
k
K
M
m
values compared with that of
D
-glu-
z
,
) without signiˆcant diŠerence in the
neapolitana AF039487 and Z99777) organisms. With
the cellodextrin phosphorylase of Clost. stercorarium
U60580) the amino acid sequence similarity was
found to be approximately 40
The phosphorolytic reaction by the cellobiose
phosphorylase of T. maritima was found to proceed
in a sequential mechanism. However, the product in-
hibition pattern did not match with the possible three
cat values obtained.
Competitive inhibition by the natural substrate,
-glucose (Fig. 8), was again observed for the
(
D
z
.
cellobiose phosphorylase from T. maritima. The
phenomenon is supposed to be common in cellobiose
phosphorylases.
In summary, the characterization of the cellobiose
phosphorylase from T. maritima revealed a high
degree of thermal stability and a distinct substrate
speciˆcity for reactions in the synthetic direction.
The mechanism of the reaction was slightly diŠerent
from other cellobiose phosphorylases reported. In
the future, this hyperthermophilic cellobiose phos-
phorylase may ˆnd application in the syntheses of
various oligosaccharides.
3
1)
sequential mechanisms presented by Cleland. Judg-
ing from the inhibition pattern, a possible explana-
tion of the reaction is a random-ordered bi bi
mechanism. In this mechanism, the binding sequence
of cellobiose and phosphate is random, and the
release sequence of the glucose and G-1-P is ordered.
1
7)
The reaction mechanisms of Cellv. gilvus
,
Cellu.
8
)
18)
Uda
,
and Clost. thermocellum cellobiose phos-
phorylases were reported to be ordered bi bi mechan-
isms. The ˆrst two enzymes bind cellobiose ˆrst and
then phosphate followed by the sequential release of
glucose and G-1-P. However, the Clost. thermocel-
lum YM-4 enzyme binds phosphate before cellobiose
and releases glucose and G-1-P with the same se-
Acknowledgments
We thank Prof. K.O.Stetter of the Univ.of Regen-
sberg, Germany for providing the genomic DNA of
T. maritima. We also thank Dr. P. Selvakumar and
Dr. Kshamata Goyal for their suggestions during this
work. The STA fellowship of Japan awarded to the
senior author is gratefully acknowledged. This work
was supported in part by a grant from the Program
for Promotion of Basic Research Activities for In-
novative Biosciences in Japan.
1
8)
quence of the other two enzymes. The random-
ordered mechanism of the T. maritima cellobiose
phosphorylase seems intermediate between the above
two types of ordered bi bi mechanisms because the
diŠerence in the mechanism is only in the sequence of
the substrate binding.
In the synthetic reaction, out of 27 sugars or their
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