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A Family 8 Glycoside Hydrolase from Bacillus halodurans C-125
(BH2105) Is a Reducing End Xylose-releasing Exo-oligoxylanase*
Received for publication, August 26, 2004, and in revised form, October 8, 2004
Published, JBC Papers in Press, October 18, 2004, DOI 10.1074/jbc.M409832200
Yuji Honda and Motomitsu Kitaoka‡
From the National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan
The gene encoding family 8 glycoside hydrolases from
Bacillus halodurans C-125 (BH2105), an alkalophilic bac-
terium with a known genomic sequence, was expressed in
Escherichia coli. The protein was expressed with the in-
tact N-terminal sequence, suggesting that it did not pos-
sess a signal peptide and that it was an intracellular en-
zyme. The recombinant enzyme showed no hydrolytic
activity on xylan, whereas it had been annotated as xyla-
nase Y. It hydrolyzed xylooligosaccharide whose degree
of polymerization is greater than or equal to 3 in an exo-
splitting manner with anomeric inversion, releasing the
xylose unit at the reducing end. Judging from its sub-
strate specificity and reaction mechanism, we named the
enzyme reducing end xylose-releasing exo-oligoxylanase
(Rex). Rex was found to utilize only the

-anomer of the
substrate to form

-xylose and
␣
-xylooligosaccharide. The
optimum pH of the enzymatic reaction (6.2–7.3) was found
in the neutral range, a range beneficial for intracellular
enzymes. The genomic sequence suggests that B. halo-
durans secretes two endoxylanases and possesses two
␣
-arabinofuranosidases, one
␣
-glucuronidase, and three

-xylosidases intracellularly in addition to Rex. The ex-
tracellular enzymes supposedly hydrolyze xylan into ar-
abino/glucurono-xylooligosaccharides that are then
transported into the cells. Rex may play a role as a key
enzyme in intracellular xylan metabolism in B. halo-
durans by cleaving xylooligosaccharides that were pro-
duced by the action of other intracellular enzymes from
the arabino/glucurono-xylooligosaccharides.
Endo-

-1,4-xylanases (EC 3.2.1.8) are glycoside hydrolases
(GHs)
1
that catalyze the degradation of xylan, a main compo-
nent of hemicelluloses. The enzyme has been classified mainly
into GH families 10 and 11 based on the amino acid sequences
(1) (Carbohydrate-active Enzymes (CAZy) data base, afmb.
cnrs-mrs.fr/CAZY/). GH10 is known to have a (
␣
/

)
8
barrel
structure to be classified as clan GH-A (2–11), while GH11 has
a jelly roll structure to be classified as clan GH-C (12–17).
Although GH10 and GH11 xylanases have different three-
dimensional structures, both of these enzymes can produce
xylooligosaccharides from xylan in an endo-splitting manner
with anomeric retention.
Recently a GH8 endo-

-1,4-xylanase was found in a culture
supernatant of Pseudoalteromonas haloplanktis and character-
ized (18). Although the GH8 xylanase also hydrolyzes the

-1,4
glycosidic bond of xylan in an endo-splitting manner, its reac-
tion proceeds with anomeric inversion. The GH8 family con-
tains various endoglycoside hydrolases, such as chitosanase
(EC 3.2.1.132), endoglucanase (EC 3.2.1.4), and licheninase
(EC 3.2.1.73) as well as endo-

-1,4-xylanase. These enzymes
also hydrolyze corresponding polysaccharides with anomeric
inversion. The three-dimensional structures of two GH8 en-
zymes, the endo-

-1,4-xylanase from P. haloplanktis and an
endoglucanase from Clostridium thermocellum, were found to
have an (
␣
/
␣
)
6
barrel structure (clan GH-M) (6, 19).
The GH8 endo-

-1,4-xylanase from P. haloplanktis has the
highest amino acid identity (32.6%) with the protein encoded
by the BH2105 gene (GenBank
TM
accession number
BAB05824) of Bacillus halodurans C-125 (18), which is anno-
tated as “xylanase Y.” B. halodurans C-125 is a xylanase-
producing alkalophilic bacterium (20) whose genomic sequence
is available (21). According to the annotation of the genomic
sequence, the alkalophilic microbe supposedly possesses three
endo-

-1,4-xylanases (GH8, GH10, and GH11). Enzymatic
characterizations of two secreted xylanases, XynA (BH2120,
GH10) and XynN (probably BH0899, GH11), indicate that
XynA exhibits a broad optimal pH range for activity (pH 4 –10),
whereas XynN (pH 4 – 6) is active only at neutral acidity (20,
22). No other endoxylanases have been reported in B. halo-
durans C125. Thus, we expressed the BH2105 protein in Esch-
erichia coli and characterized the properties of the enzyme.
In this study, we found that the BH2105 protein was not an
endo-

-1,4-xylanase but has a novel activity of hydrolyzing
xylooligosaccharides, releasing xylose from their reducing
ends. This result prompted us to characterize the unique en-
zymatic properties (substrate specificity and reaction mecha-
nism) in detail. Here we describe the characterization of the
newly found enzyme named reducing end xylose-releasing exo-
oligoxylanase (Rex).
EXPERIMENTAL PROCEDURES
Materials—The B. halodurans C-125 (9153) strain was obtained
from the Japan Collection of Microorganisms (Wako, Japan). Restric-
tion endonucleases were obtained from New England Biolabs (Beverly,
MA), and the DNA polymerase from Thermococcus kodakaraensis
KOD1 was obtained from Toyobo (Osaka, Japan). Birch wood xylan was
prepared by lyophilizing the water-soluble fraction of birch wood xylan
(Fluka, Buchs, Switzerland) as described previously (23). Chitosan
(Sigma), curdlan (Wako Pure Chemicals, Osaka, Japan), lichenan
(Sigma), and carboxymethylcellulose (Nacalai Tesque, Kyoto, Japan)
were used as purchased. Cellotriose (G3), cellopentaose, laminaripen-
taose, chitopentaose, and chitosanpentaose were purchased from Seika-
gaku Kogyo (Tokyo, Japan). Xylooligosaccharides (Xnwhere n⫽degree
of polymerization) were purchased from Megazyme (Wicklow, Ireland).
p-Nitrophenyl-

-D-xylopyranoside (X-pNP) was purchased from Sigma.
* This work was supported in part by a grant from the Bio-oriented
Technology Research Advancement Institution. 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”inac-
cordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 81-29-838-
8071; Fax: 81-29-838-7321; E-mail: mkitaoka@nfri.affrc.go.jp.
1
The abbreviations used are: GH, glycoside hydrolase; Rex, reducing
end xylose-releasing exo-oligoxylanase; G, glucose; X, xylose; de, deoxy;
Xn, xylooligosaccharide whose degree of polymerization is n; TAPS,
3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic
acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HPLC, high
pressure liquid chromatography; pNP, p-nitrophenyl.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 53, Issue of December 31, pp. 55097–55103, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 55097
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p-Nitrophenyl-

-D-xylobioside was prepared as described previously
(24).

-(134) hetero-D-glucose and D-xylose-based disaccharides and
trisaccharides (G-X, X-G, G-X-X, X-X-G, G-X-G, X-G-G, G-G-X, and
X-G-X (abbreviations indicate the monosaccharide unit from the non-
reducing end: G, glucose; X, xylose)) were prepared as described previ-
ously (25). 1,5-Anhydroxylotriitol (1-deoxy-xylotriose, X3-de) was syn-
thesized by reducing acetobromoxylotriose, prepared by acetylation and
bromination of X3 with pyridine-acetic anhydride and TiBr
4
, respec-
tively, followed by reduction with lithium aluminum hydride as de-
scribed for 1,5-anhydro-D-glucitol (26, 27). Other reagents were of ana-
lytical grade and were obtained commercially.
DNA Manipulation—Recombinant DNA techniques and agarose gel
electrophoresis were performed as described by Sambrook et al. (28).
Plasmid DNA was prepared using a QIAprep Spin Plasmid kit (Qiagen,
Hilden, Germany). Digestion by restriction enzymes was carried out in
the appropriate buffer at concentrations of 1–10 units/
g of DNA for
0.5–16 h at 37 °C. Completion of the reaction was confirmed by agarose
gel electrophoresis. A QIAEX Agarose Gel Extraction kit (Qiagen) was
used for the extraction and purification of DNA from agarose gels.
Nucleotide Sequence Analysis—The nucleotide sequence was deter-
mined by the dideoxynucleotide chain termination method using an
automated DNA sequencer (Model 310A, Applied Biosystems, Foster
City, CA) with a dRhodamine Terminator kit (PerkinElmer Life Sci-
ences). At least three independent clones of each PCR product were
sequenced. Sequence data were analyzed using GENETYX MAC soft-
ware Version 11.0 (GENETYX Software Development Co., Ltd.,
Tokyo, Japan).
Expression of BH2105 in E. coli—The gene encoding BH2105 was
amplified from the genomic DNA of B. halodurans C-125 by the poly-
merase chain reaction using the forward primer 5⬘-CCT TCC ATG
GAG AAA ACG ACA GAA GGT GCA TTT-3⬘(containing an NcoI site,
denoted by bold type) and the reverse primer 5⬘-GAA CTC GAG GTG
TTC CTC TCT TGG CCC TCA G-3⬘(containing an XhoI site, denoted by
bold type). Because of the addition of the NcoI site, the second amino
acid residue coded was changed into Glu from Lys. The amplified
fragment was digested by the corresponding restriction enzymes. The
digested fragment was ligated into pET28b (Novagen, Madison, WI) at
the corresponding sites, generating the plasmid pET28b-BH2105 en-
coding the BH2105 protein with the His
6
sequence added to its C-
terminal end. Next pET28b-BH2105 was electroporated into E. coli
BL21(GOLD)(DE3) cells, and positive colonies were selected. Resulting
transformants were incubated in Luria broth (100 ml) containing 0.05
mg/ml kanamycin at 37 °C until the optical density, at 600 nm, reached
a level of 0.6. Isopropyl-

-D-thiogalactopyranoside was then added to
give a final concentration of 1 mM, and the cultures were incubated for
24 h at 25 °C. The BH2105 protein expressed was extracted from the
wet cells (1 g) in 5 ml of 50 mMsodium phosphate buffer (pH 8.0) using
a sonicator (Model 250D sonifier, Branson, Danbury, CT).
Purification of Recombinant BH2105—The cell-free extract was
loaded onto a nickel-nitrilotriacetic acid-agarose (Qiagen) column (1 ⫻
3 cm), and the enzyme was eluted with a stepwise gradient of imidazole
(1, 10 mM;2,20mM; 3, 250 mM)in50mMsodium phosphate buffer (pH
8.0) containing 0.3 MNaCl. The fraction containing the BH2105 protein
was desalted using a PD-10 column (Amersham Biosciences). Next the
protein solution was loaded onto a Q-Sepharose column (2.5 ⫻4 cm),
and the enzyme was eluted with a stepwise gradient of NaCl (1, 0.05 M;
2, 0.2 M;3,0.3M)in50mMsodium phosphate buffer (pH 7.2). The
appropriate fractions were collected, and purity was checked by SDS-
PAGE (29). A 10-kDa protein ladder (Invitrogen) was used as a stand-
ard molecular marker for SDS-PAGE.
Protein concentrations were determined from the absorbance at 280
nm based on the theoretical molar absorption coefficients (106,210
M
⫺1
cm
⫺1
) determined from the amino acid composition of BH2105 (30).
The N-terminal amino acid sequence of the purified recombinant
BH2105 protein was determined using a G1000A protein sequencer
(Hewlett Packard, Palo Alto, CA).
Enzyme Assay—The enzyme activity was routinely determined by
measuring the increase in xylose during the hydrolysis of X3. The
enzymatic reaction was carried out in 50 mMsodium phosphate buffer
(pH 7.1) containing various concentrations of X3 at 40 °C. Periodically
a portion of the reaction mixture was boiled for 5 min to inactivate the
enzyme, and the concentration of xylooligosaccharides (X1-X6) was
quantified by high performance ion exchange chromatography on a
CARBOPAC PA1 column (4 ⫻250 mm, Dionex, Sunnyvale, CA)
equipped with a pulsed amperometric detector (DX-3, Dionex). Chro-
matography was performed with a linear gradient of 0 –0.2 Msodium
acetate in 0.1 MNaOH for 20 min at a flow rate of 1 ml/min.
Effect of pH and Temperature on Enzymatic Activity—Enzymatic
activity was measured under standard conditions of X3 (0.5 mM) hy-
drolysis, while pH of the reaction mixture was changed with each 50 mM
buffer. The pH stability was determined by incubating the enzyme at
30 °C for 30 min at each pH followed by measuring activity under
standard conditions. The buffer systems used were sodium acetate (pH
3.5–5.5) and sodium phosphate (pH 6.0 –8.0), TAPS (pH 8.0–9.0), and
CAPS (pH 9.7–11.0). The final pH values of the reaction solution were
determined after addition of the enzyme and the substrates. The opti-
mum temperature of activity was measured for 10 min under standard
conditions except for temperature. The thermostability was determined
by incubating the enzyme at each temperature for 30 min in 50 mM
sodium phosphate buffer (pH 7.1) followed by measuring the activity
under the standard conditions at 40 °C.
Analysis of the Products—The reaction products from various sub-
strates were separated by TLC on a silica gel 60 F
254
plate (5.0 ⫻7.5 cm,
Merck) with a solvent system of acetonitrile:water (4:1, v/v). Sugars
were detected by baking after dipping the plate in 5% sulfuric acid in
methanol. When necessary, the amounts of the products were quanti-
fied by using high performance ion exchange chromatography as de-
scribed above. The amounts of reducing sugar liberated in the hydrol-
ysis of polysaccharides by the enzyme were determined using the
␥
3,5-dinitrosalicylic acid method (31) or the copper-bicinchoninic acid
method (32).
Analysis of the Anomeric Form of the Products—The anomeric form of
the hydrolytic product from X3 and X4 (50 mM) was determined by
using an isocratic HPLC method (33) described below. The enzymatic
reaction was carried out in 25 mMsodium phosphate buffer (pH 7.1) at
25 °C with an enzyme concentration of 5.5
M. After incubation for 1
and 25 min, an aliquot (10
l) of the reaction solution was immediately
loaded onto a TSK-GEL Amide-80 column (4.6 ⫻250 mm, Tosoh,
Japan), and eluted with acetonitrile:water (7:3, v/v) at a flow rate of 1.5
ml/min at 25 °C, separating the xylooligosaccharides anomers. The
initial substrate and products were detected using a refractive index
monitor (RI Model 504, GL Science, Tokyo, Japan). The retention times
of
␣
- and

-xylose were confirmed by loading a solution of
␣
-xylopy-
ranose immediately after preparation, while the retention times of
anomers of xylobiose were confirmed by loading the products of 1 and 25
min hydrolyzes of phenyl-

-xylobioside by Cex (34), a family 10 xyla-
nase that forms

-xylobiose as its initial product. In addition, the
retention times of xylooligosaccharides were evaluated by the propor-
tion of the equilibrated anomers (
␣
:

⫽⬃4:6) using an equilibrated
solution of xylooligosaccharides. For all xylooligosaccharides, the
␣
-ano-
mers had shorter retention times than the corresponding

-anomers.
Kinetic Analysis—To determine the apparent kinetic parameters,
X3–X6 were subjected to hydrolysis in 50 mMsodium phosphate buffer
(pH 7.1) at 40 °C. The initial rates were measured as the increase in
xylose by using high performance ion exchange chromatography as
described above. The kinetic parameters were calculated by regressing
the experimental data (substrate concentration range; 0.2 ⫻K
m
⫺3⫻
K
m
) with the Michaelis-Menten equation by the curve fit method using
Kaleidagraph
TM
Version 3.51 (Synergy Software).
Site-directed Mutagenesis—Site-directed mutagenesis for E70A,
D128A, D263A, and D128A/D263A was performed using the PCR over-
lap extension method (35). The following mutagenetic oligonucleotide
primers were used (the mismatched bases are underlined): 5⬘-CTC GAT
GTG CGG ACT GCA GGA ATG TCC TAC-3⬘(E70A), 5⬘-GCC CCA GCT
CCG GCC GGA GAG GAA TAT TTT-3⬘(D128A), and 5⬘-CAC TTT TTT
AGC GCT TCT TAT CGT GTG GCT-3⬘(D263A). The mutated enzymes
were prepared and purified as described above.
RESULTS
Characterization of Recombinant BH2105—The recombi-
nant BH2105 protein was expressed in E.coli BL21(GOLD)
and purified to yield a 45-kDa protein on SDS-PAGE. The
enzyme was purified 2.7-fold from the cell-free extract. The
N-terminal sequence of the purified protein was Met-Glu-Lys-
Thr-Thr-Glu-Gly-Ala-Phe-Tyr, corresponding to the deduced
amino acid sequence from the starting codon. This sequence
information suggested that the enzyme has no signal peptide.
This enzyme did not hydrolyze birch wood xylan even at high
concentrations (10 mg/ml) as shown by measuring the increase in
reducing value, clearly indicating that it is not an endo-

-1,4-
xylanase even though it was annotated as xylanase Y. Further-
more the enzyme did not hydrolyze any other polymeric sub-
strates for GH8 enzymes (chitosan, lichenan, curdlan, and
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carboxymethylcellulose) determined by increases in reducing
value. Various pentasaccharides (xylopentaose, cellopentaose,
laminaripentaose, chitopentaose, and chitosanpentaose) were ex-
amined for the substrate, and the enzyme showed hydrolytic
activity only on xylopentaose, producing initially X1 and X4 and
finally X1 and X2. To obtain further information on the hydrol-
ysis of xylooligosaccharides (Xnwhere n⫽2– 6), the products
were analyzed by TLC. In the initial stage, the enzyme released
X1 and X(n⫺1) from Xn, and the final products were X1 and X2
when nⱖ3. It hydrolyzed X2 into X1 at a much slower rate than
that of X3 (less than 0.01% rate). On the other hand, the enzyme
exhibited no activity on X-pNP and X2-pNP even at extended
incubation time, suggesting the possibility of hydrolysis at the
reducing end. Oligosaccharides larger than the initial substrates
in the enzymatic reaction were not detected, indicating that the
enzyme had no transglycosidation activity.
Enzymatic properties as a function of pH and temperature
were determined with the X3 hydrolysis (Fig. 1). The optimum
pH for activity was between 6.2 and 7.3, and the enzyme was
completely stable between pH 5.0 and 9.8 at 30 °C for 30 min.
The enzyme was stable at temperatures up to 40 °C, and the
optimal temperature was 50 °C.
Substrate Specificity: Recognition of Sugar Residue—To de-
termine the position of hydrolysis and further substrate spec-
ificity of the enzyme, various derivatives of X3 were examined.
When

-(134) D-glucose and D-xylose-based trisaccharides (G-
X-X, X-X-G, G-X-G, X-G-G, G-G-X, X-G-X, and G3) were exam-
ined as the substrate, the enzyme hydrolyzed only G-X-X, X-
X-G, and G-X-G at the linkage of the reducing end side, judging
from the products, with a much slower rate than X3 (Table I).
The reducing end specificity is completely different from that of

-xylosidase, which liberates xylose from the non-reducing end.
The substitution of the middle xylose unit into glucose caused
complete loss of activity, indicating that the sugar moiety lo-
cated at the ⫺1 subsite (Fig. 2) must strictly be xylose. Substi-
tution at the reducing end and the non-reducing end caused
drastic decreases in activity, indicating that both the ⫹1 and
⫺2 subsites (Fig. 2) strongly prefer xylose but were not as strict
as the subsite ⫺1. Judging from the specificity, the enzyme is
very specific to homo-xylooligosaccharides. It hydrolyzed X3-de
into X2 and 1,5-anhydroxylitol (X-de) at a rate that was 3.2% of
the hydrolysis of X3 (Table I). This result again confirms that
the enzyme hydrolyzes the reducing end glycosyl linkage. The
result also suggests that the enzyme recognizes one of the
anomeric hydroxyl groups at the reducing end. The small hy-
drolysis rates for the derivatives of xylotriose were due to their
higher K
m
values evidenced by their linear S-vrelations in the
range lower than 2.6 mM, whereas K
m
for X3 was 2.4 mM.
Anomeric Hydroxyl Group Recognition by the Enzyme—The
anomeric composition of the degradation products of X3 and X4
by the BH2105 protein were analyzed by HPLC. Fig. 3 shows
the HPLC profiles of the hydrolytic products from X3 and X4
obtained with the enzymes. The standard equilibrium ratio of
␣
:

anomers for X3 and X4 were ⬃4:6 (Fig. 3).
As shown in Fig. 3A, the enzyme produced

-anomer of X1
and
␣
-anomer of X2 from X3 in the reaction for 1 min. Further-
more the
␣
-anomer of X3 was the predominant anomer remain-
ing in the reaction. Small amounts of
␣
-X1 and

-X2 were also
observed, which may have been due to the mutarotation of the
initial products over the short term. This result strongly sug-
gests that the enzyme hydrolyzed only the

-anomer of X3 at
the linkage of the reducing end side with anomeric inversion to
form
␣
-X2 and

-X1. The preference of the

-anomer explains
the reason why the enzyme hydrolyzed X3-de at a much slower
rate. After 25 min of the reaction, the substrate (X3) disap-
peared, and the anomeric composition of the product (X and X2)
reached equilibrium. This result suggests that the enzyme
hydrolyzed
␣
-X3 after the mutarotation that converted
␣
-X3
into

-X3.
In the case of X4 hydrolytic reaction after 1 min,

-X1 and
FIG.2.Schematic drawing of BH2105 subsite structure. Num-
bers represent the subsites.
FIG.1. Enzymatic activity and stability at various pH and
temperatures. A, the effect of pH on activity (open circles) and stability
(solid circles). B, the effect of temperature on activity (open circles) and
stability (solid circles).
TABLE I
Activity of BH2105 toward various trisaccharides
关S兴
0
⫽2.6 mM. The products were analyzed by high performance ion
exchange chromatography.
Substrate Product Specific activity
s
⫺1
X-X-X (X3) X-X ⫹X 84.0 (100%)
X-X-X-de (X3-de) X-X ⫹X-de 2.7 (3.2%)
G-X-X G-X ⫹X 0.94 (1.1%)
X-X-G X-X ⫹G 0.42 (0.5%)
G-X-G G-X ⫹G 0.003 (0.004%)
X-G-G —
a
G-G-X —
X-G-X —
G-G-G (G3) —
a
—, not detectable.
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␣
-X3 were produced with a decrease in

-X4 (Fig. 3B). Again
small amounts of the opposite anomers were also observed that
were probably due to mutarotation. It should be noted that X2,
the hydrolytic product of X3, was hardly detected even though
half of X4 had already been hydrolyzed in the reaction, sug-
gesting that the enzyme did not act on the
␣
-anomer. The
␣
-X4
remaining and the
␣
-X3 produced must be converted into their

-anomers before the hydrolysis. After 25 min of reaction, the
products were equilibrated X1 and X2. The hydrolytic mecha-
nism is schematically summarized in Fig. 4.
Kinetic Property—The S-vcurve of X3–X6 hydrolysis by the
enzyme indicates a typical Michaelis-Menten type relationship.
The kinetic parameters are summarized in Table II. The K
m
value increased with the increase in degree of polymerization,
while k
cat
/K
m
decreased with the increase in K
m
. These results
suggest that X3 is the most suitable substrate for the enzyme
and that the role of the enzyme is to hydrolyze smaller xylo-
oligosaccharides. Subsites downstream of subsite ⫺2 (Fig. 2)
may be postulated, but such subsites must have negative bind-
ing energies.
Mutational Analysis—The BH2105 amino acid sequence
analysis indicated a 32.6% identity with P. haloplanktis GH8
xylanase (GenBank
TM
accession number AJ427291). As found
in the sequence alignment between these enzymes, the cata-
lytic residues (Glu-70, Asp-128, and Asp-263; BH2105 number-
ing) proposed for the xylanase were strongly conserved in the
BH2105 amino acid sequence (19). Therefore, we examined the
enzymatic activity of alanine mutants (E70A, D123A, and
D263A) and found that, as expected, the hydrolytic activity of
E70A and D263A was 10
⫺4
orders less than that of wild type
FIG.4. Reaction mechanism of X3
and X4 hydrolysis by the BH2105
protein.
FIG.3. Anomer analysis of the hy-
drolytic products from X3 and X4. A,
X3 degradation. B, X4 degradation. For
experimental methods see the text. RI,
refractive index.
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(Table III). On the other hand, D128A retained slightly higher
activity than that of E70A and D263A, although it was still
approximately three hundredths of wild type (Table III). The
residues were predicted to act as an acid catalyst (Glu-70) and
a base catalyst (Asp-263) and to stabilize the
2,5
Bconformation
of the sugar moiety bound at subsite ⫺1 (Asp-123) (6, 19).
These results suggest that these amino acid residues are con-
served and are essential for the catalytic reaction of the
enzyme.
DISCUSSION
In this study, we determined that the GH8 glycosidase from
B. halodurans C-125 has novel enzymatic properties, specifi-
cally (i) the enzyme hydrolyzes xylooligosaccharides but not
xylan, (ii) the enzyme releases xylose from the reducing end of
a xylooligosaccharide in an exo-splitting manner, and (iii) the
enzyme strictly recognizes the

-anomeric hydroxyl group at
the reducing end of the substrate. Considering the reaction
mechanism and the substrate specificity, we propose that the
name of the enzyme be Rex. Unique enzymatic properties and
the role of Rex are discussed below.
Reaction Mechanism of Rex—Rex is a GH8 enzyme found to
be an exoenzyme recognizing reducing ends. It shows 32%
amino acid sequence identity with endo-

-1,4-xylanase from
P.haloplanktis with at least six subsites at the substrate
binding groove, both sides of which are open (18, 19). Con-
sidering the substrate specificity of Rex, presuming three
subsites (⫺2, ⫺1, and ⫹1) should be enough to explain the
results. Also the substrate binding groove should be close to
the upstream subsite ⫹1 (Fig. 2) to be such an exoenzyme.
The structural analysis of Rex will reveal the real mechanism
of the exohydrolysis.
It is interesting to note that Rex essentially recognizes the

-anomeric hydroxyl group at the reducing end of the substrate
(Figs. 3 and 4). To our knowledge, such a strict substrate
recognition of the anomeric hydroxyl group opposite to the
catalytic position has been reported only with maltose phos-
phorylase (36) and cellobiose phosphorylase (37, 38). The re-
ducing end sugar residue has one fewer hydroxyl group than
the non-reducing end sugar residue if the anomeric hydroxyl
group is not taken into account. In the case of Rex as the
reducing end-specific enzyme, the recognition of the anomeric
hydroxyl group is probably necessary to increase the recogni-
tion position for fixing the reducing end xylose unit.
The anomeric specificity of Rex necessitates the mutarota-
tion of the products required for the next step as shown in
Figs. 3 and 4. Under the conditions used in the experiments
shown in Fig. 3, mutarotation would be the rate-limiting step
to complete the cleavage of X4 into X2 ⫹2 X1. A mutarotase
for xylooligosaccharides may accelerate the Rex reaction in
vivo. However, the mutarotation seems fast enough to com-
plete the hydrolysis within 25 min (Fig. 3B), suggesting that
it is probably not the rate-limiting step of the Rex reaction in
vivo because the enzyme concentration in vivo is not expected
to be so high.
The reducing end-specific monomer-forming exoglycosyl-
ase is not so widely known. To our knowledge, this activity
has been reported only with two amylolytic enzymes, one
from E. coli K12 (39) and one from Thermotoga maritima
(40). Although the enzymes released glucose from the reduc-
ing end of the substrate, the enzymes could also release
p-nitrophenol from p-nitrophenyl
␣
-maltooligosaccharides
(such as G2-pNP or G5-pNP) and could hydrolyze cyclodex-
trins. Thus, these enzymes have some endohydrolytic
activities and are not complete reducing end-specific exogly-
cosylases. In contrast, Rex does not hydrolyze p-nitrophenyl
xylobioside at all, indicating that it specifically liberates the
reducing end sugar moiety.
It is necessary to mention the confusing nomenclature of these
reducing end-specific enzymes. The term “
␣
-glucosidase” was
used to describe amylolytic enzymes that release glucose from
maltooligosaccharides (39, 40). However, in our opinion, this
term is incorrect for these enzymes because they hydrolyze the
linkage at the reducing end, an
␣
-glycoside of a maltooligosac-
charide, releasing glucose from the reducing end, but they do not
hydrolyze real
␣
-glucosides, meaning a linkage at the non-reduc-
ing end. We propose that the term monosaccharide glycosidase
(e.g. glucosidase or xylosidase) should be used for enzymes that
liberate the corresponding monosaccharide from the non-reduc-
ing end as clearly stated in Enzyme Nomenclature (www.
chem.qmul.ac.uk/iubmb/enzyme/). Thus, careful naming of the
reducing end-specific exoglycosylase is required. Considering
this, Rex should not be named a

-xylosidase; thus we gave the
name of “reducing end xylose-releasing exo-oligoxylanase.”
Roles of Rex in Xylan Metabolism in B. halodurans C-125—
Our research reveals that Rex has no signal peptide and that
neutral pH is optimal for hydrolytic activity, indicating that the
enzyme is an intracellular enzyme of B. halodurans C-125. To
understand the role of Rex in xylan metabolism of B. halo-
durans C-125, related enzymes are listed in Table IV. The
microbe possesses 11 possible genes encoding xylan-related
enzymes. However, none of the genes of the related enzymes
seemed to form an operon with the gene of Rex. The locations of
the enzymes were judged as to whether they had signal pep-
tides predicted using computational analysis (41) (www.cbs.
dtu.dk/services/SignalP/). As shown in Table IV, signal pep-
tides were predicted with all the acetylxylan esterases and
endoxylanases, suggesting that they were extracellular en-
zymes. All of the other enzymes,
␣
-arabinofuranosidases,
␣
-glucuronidase, Rex, and

-xylosidases, were predicted not to
have signal peptides, suggesting that they were intracellular
enzymes.
Many microorganisms are known to secrete
␣
-arabino-
furanosidase and
␣
-glucuronidase as well as endoxylanase,
which digest xylan into unsubstituted xylooligosaccharides or
xylose (42, 43). However, B. halodurans seems to secrete en-
zymes to digest xylan only up to arabino/glucurono-xylooligo-
saccharides according to the predictions. The proposed path-
way of xylan metabolism in B. halodurans C125 is illustrated
in Fig. 5. Xylans, with
␣
-arabinofuranosyl and/or
␣
-glucuronyl
side chains, are hydrolyzed to form arabino/glucurono-xylo-
oligosaccharides with a larger degree of polymerization in the
main

-1,4-xylosyl chain, due to intact side chains, compared
TABLE II
Kinetic parameters of xylooligosaccharides hydrolysis
catalyzed by BH2105
Substrate k
cat
K
m
k
cat
/K
m
s
⫺1
mMs
⫺1
mM
⫺1
X3 163 ⫾5 2.4 ⫾0.2 66.9 ⫾3.5
X4 162 ⫾6 5.0 ⫾0.4 32.3 ⫾1.4
X5 73 ⫾2 4.4 ⫾0.3 16.7 ⫾0.5
X6 175 ⫾19 18.5 ⫾1.3 18.5 ⫾1.3
TABLE III
Specific activity of BH2105 mutants
Substrate is X3 (2.6 mM).
Enzyme Specific activity Relative activity (mutant/wild type)
s
⫺1
Wild type 84.0 1.0
E70A 0.0088 1.1 ⫻10
⫺4
D128A 0.29 3.5 ⫻10
⫺3
D263A 0.019 2.3 ⫻10
⫺4
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with the xylan metabolism systems of other microorganisms.
The resulting oligosaccharides are then incorporated into the
cell by an unidentified transporter system. Inside the cells, the
side chains are removed by
␣
-arabinofuranosidases and
␣
-glu-
curonidases to give unsubstituted xylooligosaccharides fol-
lowed by the hydrolysis of Rex and

-xylosidases from the
reducing end and the non-reducing end, respectively. Although
it has not been shown experimentally, Rex probably does not
directly hydrolyze xylooligosaccharides having side chains as
suggested by the finding that the GH8 endoxylanase from
P. haloplanktis, which is expected to have a similar substrate
binding groove due to its high sequence similarity to Rex, does
not produce short xylooligosaccharides having side chains from
arabino/glucurono-xylans (18). It is likely that a larger degree
of xylooligosaccharide polymerization, caused by the extracel-
lular hydrolysis of xylan such that the decoration remains
intact, requires the action of intracellular Rex to utilize the
xylooligosaccharides more efficiently.
From another point of view, such intracellular enzymes may
play a role in B. halodurans C125 strategies to acquire alkalo-
philic features. Although this microbe lives under alkaline con-
ditions, the inside of the cell retains a neutral environment (44).
To minimize changes that promote enzymatic function in alka-
line conditions, B. halodurans C125 may have adopted a strategy
that sequesters such enzymes in the intracellular compartment,
making it unnecessary to develop alkalophilic enzymes.
Acknowledgment—We are grateful to Setsuko Koyama for technical
assistance during the course of this study.
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TABLE IV
A list of enzymes involved in xylan degradation in B. halodurans C-125
Enzyme Locus Family
a
Signal peptide Group
b
Acetylxylan esterase BH3326 CE7 Yes 1
Acetylxylan esterase BH3902 CE4 Yes 1
Endo-

-1,4-xylanase BH0899 GH11 Yes 1
Endo-

-1,4-xylanase BH2120 GH10 Yes 1
␣
-L-Arabinofuranosidase BH1861 GH51 No 2
␣
-L-Arabinofuranosidase BH1874 GH51 No 2
␣
-Glucuronidase BH1061 GH67 No 2
Rex BH2105 GH8 No 3
Xylosidase BH1068 GH39 No 4
Xylan

-1,4-xylosidase BH1867 GH43 No 4
Xylan

-1,4-xylosidase BH3683 GH43 No 4
a
CE, carbohydrate esterase. For further details see CAZy web site (afmb.cnrs-mrs.fr/CAZY/).
b
The group numbers correspond to the enzymes in Fig. 5.
FIG.5.Schematic proposed pathway for metabolism of xylan
in B. halodurans C-125. The numbers in circles indicate the group of
enzymes. The enzymes in each group are given in Table IV.
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Yuji Honda and Motomitsu Kitaoka
Reducing End Xylose-releasing Exo-oligoxylanase
C-125 (BH2105) Is aBacillus haloduransA Family 8 Glycoside Hydrolase from
doi: 10.1074/jbc.M409832200 originally published online October 18, 2004
2004, 279:55097-55103.J. Biol. Chem.
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