THE JOURNAL OF BIOLOGICAL CHEMISTRY
95979605, 2004
2004 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mechanisms by Which Family 10 Glycoside Hydrolases Bind
Decorated Substrates*
Received for publication, November 10, 2003, and in revised form, December 9, 2003
Published, JBC Papers in Press, December 10, 2003, DOI 10.1074/jbc.M312278200
Gavin Pell, Edward J. Taylor, Tracey M. Gloster, Johan P. Turkenburg,
Carlos M. G. A. Fontes, Luis M. A. Ferreira, Tibor Nagy, Samantha J. Clark,
Gideon J. Davies, and Harry J. Gilbert
From the School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Agriculture Building, Newcastle
upon Tyne NE1 7RU, United Kingdom, Structural Biology Laboratory, Department of Chemistry, University of York,
Heslington, York YO10 5YW, United Kingdom, and CIISA-Faculdade de Medicina Veterinaria, Universidade Tecnica de
Lisboa, Rua Prof. Cid dos Santos, 1300-477 Lisboa, Portugal
Endo--1,4-xylanases (xylanases), which cleave -1,4
glycosidic bonds in the xylan backbone, are important
components of the repertoire of enzymes that catalyze
plant cell wall degradation. The mechanism by which
these enzymes are able to hydrolyze a range of deco-
rated xylans remains unclear. Here we reveal the three-
dimensional structure, determined by x-ray crystallog-
raphy, and the catalytic properties of the Cellvibrio
mixtus enzyme Xyn10B (CmXyn10B), the most active
GH10 xylanase described to date. The crystal structure
of the enzyme in complex with xylopentaose reveals that
at the 1 subsite the xylose moiety is sandwiched be-
tween hydrophobic residues, which is likely to mediate
tighter binding than in other GH10 xylanases. The crys-
tal structure of the xylanase in complex with a range of
decorated xylooligosaccharides reveals how this en-
zyme is able to hydrolyze substituted xylan. Solvent ex-
posure of the O-2 groups of xylose at the 4, 3, 1, and
3 subsites may allow accommodation of the -1,2-
linked 4-O-methyl-D-glucuronic acid side chain in glucu-
ronoxylan at these locations. Furthermore, the uronic
acid makes hydrogen bonds and hydrophobic interac-
tions with the enzyme at the 1 subsite, indicating that
the sugar decorations in glucuronoxylan are targeted to
this proximal aglycone binding site. Accommodation of
3-linked L-arabinofuranoside decorations is observed in
the 2 subsite and could, most likely, be tolerated when
bound to xylosides in 3 and 4. A notable feature of the
binding mode of decorated substrates is the way in
which the subsite specificities are tailored both to pre-
vent the formation of dead-end reaction products and
to facilitate synergy with the xylan degradation-acces-
sory enzymes such as -glucuronidase. The data de-
scribed in this report and in the accompanying paper
(Fujimoto, Z., Kaneko, S., Kuno, A., Kobayashi, H.,
Kusakabe, I., and Mizuno, H. (2004) J. Biol. Chem. 279,
9606 9614) indicate that the complementarity in the
binding of decorated substrates between the glycone
and aglycone regions appears to be a conserved feature
of GH10 xylanases.
* 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.
The atomic coordinates and structure factors (codes 1uqy, 1ur1, 1ur2,
1uqz) have been deposited in the Protein Data Bank, Research Collabo-
ratory for Structural Bioinformatics, Rutgers University, New Bruns-
wick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.: 44-191-
2226962; Fax: 44-191-2228684; E-mail: H.J.Gilbert@Newcastle.ac.uk.
This paper is available on line at http://www.jbc.org
Vol. 279, No. 10, Issue of March 5, pp.
Printed in U.S.A.
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The microbial hydrolysis of the plant cell wall into its con-
stituent sugars is one of the major mechanisms by which or-
ganic carbon is utilized in the biosphere. The enzymes that
catalyze this process are thus of considerable biological and
industrial importance. Endo--1,4-xylanases (xylanases),
which are one of the key components of the repertoire of en-
zymes that catalyze plant cell wall degradation, hydrolyze the
-1,4 glycosidic bonds linking the xyloside units that comprise
the backbone of the polysaccharide xylan (1). Furthermore, the
O-2 and/or O-3 of xylosides within the xylan backbone may be
decorated with acetyl and arabinofuranosyl units, whereas
4-O-methyl-D-glucuronic acid (4-O-MeGlcA)1 is exclusively
-1,2-linked (1). The extent and nature of such decoration
varies between plant species. For complete degradation the
arabinofuranosyl and acetyl side chains are removed from the
polysaccharide by arabinofuranosidases and acetyl xylan ester-
ases, respectively (1), whereas the -glucuronidases, which
release 4-O-MeGlcA, act only on xylooligosaccharides in which
the xylose at the nonreducing end is decorated with the uronic
acid (2, 3).
Xylanases, which are generally located in glycoside hydro-
lase families (GH) 10 and 11 (CAZy website2 and Ref. 4),
hydrolyze glycosidic bonds by acid base-assisted catalysis via a
double displacement mechanism leading to retention of ano-
meric configuration at the site of cleavage (5). The crystal
structures of xylanases show that GH10 enzymes fold into a
(/)8-barrel (6, 7), whereas family 11 enzymes are -jelly roll
proteins (8). Consistent with their endo mode of action, the
substrate binding cleft of xylanases extends along the length of
the proteins and can accommodate from four to seven xylose
residues (9, 10). Each region that accommodates xylose moi-
eties are known as subsites, which are given a negative or
positive number dependent on whether they bind the glycone or
aglycone region of the substrate, respectively, with glycosidic
bond cleavage occurring between the 1 and 1 subsites (11).
The structures of xylanases in complex with oligosaccharides
and inhibitors have revealed detailed information on the inter-
actions of these enzymes with their substrates in the proximal,
2 and 1 glycone subsites (12, 13). The data show that at the
1 subsite the O-3 of xylose makes a hydrogen bond with a
highly conserved lysine and histidine, whereas O-2 interacts
1
The abbreviations used are: 4-O-MeGlcA, 4-O-methyl-D-glucuronic
acid; GH, glycoside hydrolase family; AX2, arabinoxylobiose; AX3, ar-
abinoxylotriose; OX, oat spelt xylan; RAX, rye arabinoxylan; MX3, al-
dotetraouronic acid; Xyn10, xylanase from GH10; HPLC, high pressure
liquid chromatography; PEG, polyethylene glycol.
2
CAZy website: afmb.cnrs-mrs.fr/CAZY/.
9597
9598 GH10 Substrate Complexes
with an asparagine and a glutamate (which acts as the cata-
lytic nucleophile) that are invariant in GH10 xylanases. At the
2 subsite the xylose interacts with residues that are also
highly conserved in family 10 xylanases with the O-2 of the
sugar hydrogen bonding to a glutamate and tryptophan, the
O-3 to an asparagine, and the endocyclic oxygen to a lysine.
There is a paucity of information, however, on the mechanism
by which the aglycone region of the substrate binding cleft of
these enzymes interacts with the xylan backbone, although the
three-dimensional structure of Cellvibrio japonicus xylanase
10A (CjXyn10A) in complex with xylopentaose bound to sub-
sites 1 to 4 has been described (7, 15). These studies showed
that a highly conserved aromatic residue stacks against the
xylose at the 1 subsite, and although hydrophobic stacking
interactions at the 3 and 4 subsites were the primary mech-
anism of protein-substrate recognition, these amino acids are
not invariant in GH10 glycoside hydrolases, suggesting that
xylan binding in the distal aglycone region of this enzyme
family is variable. One of the fundamental differences between
the two major families of xylanases is that the GH11 enzymes
hydrolyze unsubstituted regions of xylan, whereas the corre-
sponding family 10 glycoside hydrolases are able to attack
decorated forms of the polysaccharide (16). The mechanism by
which the side chains of decorated xylans are accommodated in
the active site of GH10 xylanases is largely unknown. Schmidt
et al. (17) solved the structure of a non-natural decorated
xylooligosaccharide comprising 1,2-(4-deoxy--L-threo-hex-4-
enopyranosyluronic acid)--1,4-D-xylotriose. Although the back-
bone xylose residues were evident at subsites 1 to 3, the side
chain was not observed, presumably because it was highly
disordered.
To probe the structural basis for the capacity of GH10 xyla-
nases to hydrolyze decorated substrates, we have analyzed the
biochemical properties of a family 10 enzyme from Cellvibrio
mixtus (CmXyn10B) and determined its crystal structure in
complex with unsubstituted and decorated substrates at reso-
lutions from 1.7 to 1.4 . CmXyn10B comprises an N-terminal
signal peptide and a 360-residue GH10 catalytic module; the
enzyme does not contain noncatalytic accessory modules typi-
cal of plant cell wall-degrading enzymes (18) and is shown to be
the most active GH10 xylanase described to date. Its capacity
to bind decorated substrates is conferred partly by the exposure
of O-2 and O-3 groups at selected glycone and aglycone subsites
and partly through productive interactions with the 4-O-
MeGlcA side chains. The data provided here and in the accom-
panying paper by Fujimoto et al. (19) focus on a GH10 xylanase
that contains an N-terminal lectin domain (19), reveal comple-
mentarity in the binding of decorated substrates between the
glycone and aglycone regions of the active site, and demon-
strate how these binding modes mediate synergy with the
xylan accessory enzyme, -glucuronidase.
EXPERIMENTAL PROCEDURES
Bacterial Strains, Culture Conditions, and PlasmidsThe Esche-
richia coli strain TUNER:pLysS (Novagen) was used in this study. The
bacterium was cultured in Luria broth (LB) at 37 C with aeration
unless otherwise stated. The plasmid pCF1 is a recombinant of pET21b,
which encodes the mature form of CmXyn10B corresponding to residues
11379. The xylanase gene xyn10B (GenBankTM accession number
AF049493) was inserted into the pET vector at the NdeI and XhoI
restriction sites such that the translation stop codon was removed and
the gene was in-frame with the His6 tag encoding sequence supplied by
the vector (18). Thus recombinant CmXyn10B contains a C-terminal
His6 tag.
Generation of CmXyn10B MutantsDerivatives of CmXyn10B were
generated using the QuikChange site-directed mutagenesis kit (Strat-
agene, La Jolla, CA) according to the manufacturers instructions, using
pCF1 as template DNA and the appropriate primers. The complete
sequences of the DNA encoding the xylanase mutants were determined
using an ABI 377 DNA Sequenator employing T7 forward and reverse
primers and the custom sequencing primers 5-AACGGCGCGCGATG-
TACAAGTCG-3 and 5-CAGCTGGTGAAAGAAGTAGCGC-3 to con-
firm that only the desired mutations had been introduced. During the
course of this study errors in the original manual sequencing of xyn10B,
which resulted in 12 amino acid changes in the encoded enzyme, were
detected and a corrected version of this gene has now been submitted
to GenBankTM.
Expression and Purification of CmXyn10BE. coli strain TUNER:
pLysS harboring pCF1 was cultured in LB supplemented with 50 g
ml1 ampicillin (1000 ml in 2-liter conical baffle flasks) at 37 C and 180
rpm to mid-exponential phase (A600 nm 0.6). The culture was cooled and
incubated at 30 C before expression of CmXyn10B was induced by the
addition of isopropyl -thiogalactopyranoside to a final concentration of
0.2 mM. The culture was incubated for a further 5 h. The cells were then
harvested by centrifugation at 4500 g for 10 min at 4 C and resus-
pended in 1/40th volume 20 mM Tris/HCl buffer, pH 8.0, containing 300
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mM NaCl. Cells were lysed by sonication and centrifuged (25,000 g)
for 15 min at 4 C to produce cell-free extract. CmXyn10B was purified
from cell-free extract by immobilized metal affinity chromatography as
described previously (20) using TalonTM resin (Clontech, Palo Alto, CA).
The protein eluted from the matrix was dialyzed against 3 500
volumes of 10 mM Tris/HCl buffer, pH 8.0 (Buffer A) and was then
applied to a 3-ml DEAE Tris-Acryl column (15 50 mm). CmXyn10B
was eluted with a linear 0 500 mM NaCl gradient in Buffer A. The
recovered protein was concentrated to 1 ml using a Vivaspin 10-kDa
molecular mass cut-off centrifugal concentrator (VIVASCIENCE, Han-
nover, Germany) and then applied to a High LoadTM SuperdexTM 75
column (16 600 mm; Amersham Biosciences) and eluted in Buffer A
containing 150 mM NaCl. The columns were run at 1 ml min1 using the
Bio-Rad Biologic system. The purity of the protein was evaluated by
SDS-PAGE. Protein concentration was determined from the calculated
molar extinction coefficient of the enzyme at 280 nm (67,850 M1 cm1).
Preparation of OligosaccharidesUnsubstituted xylooligosacchar-
ides were purchased from Megazyme International (Bray, County
Wicklow, Ireland) and arabinoxylotriose (AX3) in which arabino-
furanose is -1,3-linked to the internal xylose moiety in xylotriose was
a kind gift from Satoshi Kaneko. Aldotetraouronic acid (MX3) in which
4-O-MeGlcA acid is -1,2-linked to the nonreducing sugar of xylotriose
was prepared as follows. Glucuronoxylan (1 g; Megazyme Int.) was
digested to completion with 1 mg CmXyn10B in a 5-ml reaction in 50
mM sodium phosphate buffer, pH 7.0, at 37 C for 18 h. MX3 was
purified by Dowex X-100 (Sigma) chromatography in which the bound
decorated oligosaccharide was eluted with a 0 500 mM sodium chloride
gradient in 50 mM sodium phosphate buffer, pH 7.0. The MX3 was then
further purified by size exclusion chromatography using a Bio-gel P2
column (2.5 100 cm) in which the oligosaccharide was eluted in
distilled water at a flow rate of 0.2 ml min1. The identity of the product
as MX3 was verified by digestion with the -glucuronidase GlcA67A
from C. japonicus (21) followed by Dionex-HPLC analysis, which re-
vealed xylotriose and 4-O-MeGlcA as the reaction products. Arabinoxy-
lobiose (AX2), in which the arabinofuranose is -1,3-linked to the non-
reducing xylose unit in xylobiose, was prepared as follows. Wheat
arabinoxylan (1 g; Megazyme Int.) was digested to completion with 1
mg of CmXyn10B in a 5-ml reaction in 50 mM sodium phosphate buffer,
pH 7.0, at 37 C for 18 h. AX2 was purified by size exclusion chroma-
tography as described above. The identity of the product as AX2 was
verified by digestion with the arabinofuranosidase Abf51A from
C. japonicus (22) and Dionex-HPLC analysis, which revealed xylobiose
and arabinose as the reaction products.
Enzyme AssaysThe activity of the GH10 xylanases against aryl
-glycosides was determined as described previously (23). Xylanase
activity was performed essentially as described by Charnock et al. (23)
except that the release of reducing sugar was determined using the
Somogyi-Nelson reagent (24). Each assay was performed in triplicate.
To evaluate the activity of the two xylanases against xylooligosacchar-
ides, progress curves were carried out as described previously, and the
data were used to determine kcat/Km following the equation of Matsui et
al. (25). The bond cleavage frequency and kcat/Km data obtained from
these experiments were used to calculate the G of xylose binding at
each of the subsites following the method of Suganuma et al. (26).
Crystallization and Data CollectionPure proteins, as judged by
SDS-PAGE, were washed into water by repeated dilution (40 volumes of
water) and concentrated in a Vivaspin 10-kDa concentrator. The E262S
nucleophile mutant of CmXyn10B was screened by the hanging drop
vapor diffusion method using the PEG/Ion and Crystal screens (Hamp-
ton Research, Aliso Viejo, CA). Crystallization conditions were opti-
mized independently for each xylooligosaccharide complex. Crystals
 GH10 Substrate Complexes 9599
TABLE I
Refinement and structure quality statistics for the Cellvibrio mixtus Xyn10B mutant (E262S) complex structures
r.m.s., root mean square; avg., average; PDB, protein data bank.
Xylopentaose AX2 AX3 MX3

Resolution of data (outer shell), 201.72 (1.781.72) 351.43 (1.481.43) 301.6 (1.661.6) 351.55 (1.611.55)
R-mergea (outer shell) 0.038 (0.174) 0.055 (0.334) 0.076 (0.432) 0.057 (0.358)
Mean I/I (outer shell) 46.0 (10.35) 26.02 (1.94) 14.6 (1.93) 30.22 (3.39)
Completeness (outer shell), % 99.6 (98.7) 83.3 (20.5) 98.3 (90.3) 95.4 (71.0)
Multiplicity (outer shell) 6.02 (5.49) 5.62 (1.38) 3.25 (2.23) 5.53 (2.25)
No. unique reflections 34539 49417 42050 45059
No. rejections 21320 603 133 333
No. protein atoms 2819 2858 2869 2841
No. ligand atoms 67 27 56 69
No. solvent waters 561 569 591 583
Rcryst 0.140 0.151 0.152 0.158
Rfree 0.186 0.186 0.189 0.199

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r.m.s. deviation 12 bond () 0.014 0.014 0.010 0.011
r.m.s. deviation 13 angle () 1.539 1.609 1.319 1.333
r.m.s. deviation chiral volume (3) 0.102 0.098 0.083 0.097
Avg. main chain B (2) 12.97 10.68 12.29 9.27
Avg. side chain B (2) 15.37 13.22 13.92 11.31
Avg. substrate B (2) 20.96 16.65 18.437 17.49
Avg. solvent B (2) 28.39 25.05 29.91 24.03
PDB code 1uqy 1ur1 1ur2 1uqz
a
R-merge hkli/Ihkli Ihkl/hkli/Ihkli.

grew in 2 4 days at 18 C. Drops contained 1 or 2 l of protein (25 mg
ml1), 0.5 or 1 l of xylooligosaccharide (10 mM), and 1 l of mother
liquor. Crystallization conditions for each xylooligosaccharide complex
were as follows: xylopentaose, 0.20 M MgCl2, 0.1 M Tris, pH 8.0, and 30%
PEG 4000; AX2, 0.35 M MgCl2, 0.1 M Tris, pH 6.5, 30% PEG 4000, and
5% PEG 400; AX3, 0.20 M MgCl2, 0.1 M Tris, pH 8.5, 30% PEG 4000, and
5% PEG 400; MX3, 0.10 M MgCl2, 0.1 M Tris, pH 6.5, 30% PEG 4000,
and 5% isopropanol. Crystals in the form of plates were harvested in
rayon fiber loops and cryoprotected in 15% 2-methyl-2,4-pentanediol,
85% mother liquor prior to flash freezing in liquid N2.
All data were collected from single crystals at 120 K, over an oscil-
lation range of 135 with a of 0.5. Data for CmXyn10B E262S in
complex with xylopentaose were collected in the home laboratory using
a MarResearch image plate detector on a Rigaku rotating anode RU-
200 x-ray generator with a Cu target operating at 50 kV and 100 mA
and focusing x-ray optics (Osmics). Data for CmXyn10B E262S in
complex with AX3 were collected at the Daresbury Synchrotron Radia-
tion Source (SRS) on beamline PX-14.2. Data for CmXyn10B E262S in
complex with AX2 and MX3 were collected on the European Synchrotron
Radiation Facility (ESRF) beamline ID-29. SRS and ESRF data were
collected using ADSC Quantum-4 and Quantum-210 charge-coupled
device detectors, respectively.
Structure Solution and RefinementAll data were processed and
scaled with the HKL suite (27). All other computing used the CCP4
suite (28) unless otherwise stated. All crystals belonged to space group
P212121 with approximate unit cell dimensions a 47 , b 68 , and
c 105 .
The structure of CmXyn10B E262S in complex with xylopentaose
was solved by molecular replacement with the program AMoRe and
with data in the resolution range 20 3.0 and an outer radius of
Patterson integration of 25 . The protein atoms from the catalytic core
domain of the C. japonicus Xyn10A (protein data bank 1clx) was used as
the search model. AMoRe generated one solution corresponding to one
molecule in the asymmetric unit. This gives a VM of 2.0 A3 D1 and a
solvent content of 36.6%. Prior to refinement and model building, 5% of
the observations were set aside for cross-validation analysis (29) and
were used to monitor various refinement strategies such as the weight-
ing of geometrical and temperature factor restraints as well as the
insertion of solvent water during maximum likelihood refinement.
REFMAC and ARP/wARP (CCP4 suite) were used to build the correct
sequence into electron density automatically. Manual corrections of the
model using the X-FIT routines of the program QUANTA (Accelrys)
were interspersed with cycles of maximum likelihood refinement using
REFMAC. Solvent molecules were added automatically using ARP/
wARP and inspected manually prior to deposition.
The refined structure of the CmXyn10B E262S complexed with xy-
lopentaose was used as the starting model for the refinement of the
decorated oligosaccharide complexes. The same 5% of observations was
maintained as the Rfree set in each case. Ideal coordinates for stereo-
chemical dictionaries for the substituted xylooligosaccharides were gen-
erated using the energy minimization CHARMm function in QUANTA.
Structures were validated using PROCHECK (30). Coordinates have
been deposited with the Macromolecular Structures Database (Table I).
RESULTS
Biochemical Properties of CmXyn10BCmXyn10B was pu-
rified to electrophoretic homogeneity, and its biochemical prop-
erties were determined (Tables II and III). The enzyme dis-
plays catalytic properties typical of a GH10 xylanase. The
activity of CmXyn10B against xylooligosaccharides increases
with the degree of polymerization (d.p.) of the substrate, al-
though the difference in the hydrolysis rate between xylo-
hexaose and xylopentaose is modest, implying that the sixth
binding site (4 subsite) interacts only weakly with the oligo-
saccharide substrate. The hydrolysis of xylotetraose exclusively
into xylobiose demonstrates that the enzyme contains two sub-
sites on either side of the point of cleavage, but as no xylotriose
was generated, the xylanase does not contain a significant 3
subsite (Fig. 1). The catalytic efficiency of CmXyn10B and its
mode of action against different xylooligosaccharides and aryl-
-xylosides (Table IV) shows that the 2 and 2 subsites have
binding energies of 9.0 and 3.7 kcal mol1, respectively. The
maximum binding energy at the 3 is 1.1 kcal mol1, assuming
that the increased activity of CmXyn10B against xylopentaose,
compared with xylotetraose, is due entirely to binding at this
location.
Against both poorly and highly decorated xylans, CmXyn10B
initially generates a range of oligosaccharides that progres-
sively become smaller in size as the enzyme reactions progress
(data not shown). Against both wheat arabinoxylan and glucu-
ronoxylan, the unsubstituted end products of the reactions
comprise xylose and xylobiose. An oligosaccharide that did not
co-migrate with linear xylooligosaccharides was also observed
in the two reactions, and these products were purified by size
exclusion chromatography and treated with a GH51 arabino-
furanosidase, a GH67 -glucuronidase, and a GH39 -xylosi-
dase. The oligosaccharide produced from glucuronoxylan was
hydrolyzed by the -glucuronidase into xylotriose and 4-O-
MeGlcA but was not cleaved by the other two enzymes. The
arabinofuranosidase cleaved the saccharide generated from ar-
abinoxylan into xylobiose and arabinose with a stoichiometry of
1:1, whereas the GH67 and GH39 enzymes did not attack the
oligosaccharide (data not shown). Thus the major decorated
oligosaccharide released from glucuronoxylan by CmXyn10B
consists of xylotriose with 4-O-MeGlcA linked -1,2 to the xy-
9600 GH10 Substrate Complexes
TABLE II
Biochemical properties of CjXyn10A and CjXyn10C
The substrates used were as follows: X3, xylotriose; X4, xylotetraose; X5, xylopentaose; X6, xylohexaose; PNPX2, 4-nitrophenyl--D-xylobioside;
PNPX, 4-nitrophenyl--D-xylopyranose; PNPG2, 4-nitrophenyl--D-cellobioside; OX, oat spelt xylan; RAX, rye arabinoxylan; WAX, wheat arabi-
noxylan; GX, glucuronoxylan; , not determined; ND, no activity detected.
Enzyme kcat/Kma Specific activityb
X3 X4 X5 X6 PNPX2 PNPX PNPG2 OX RAX WAX GX

min1 M
1
min1
CmXyn10B 6.0 104 3.2 107 2.0 108 2.7 108 8.2 107 2.1 102 2.4 104 11420 5882 7996 17782
F340A 1.3 103 7.1 105 6.2 106 1.5 107 1.7 104
Y200F 2.8 107 2.3 108 2.7 106 1.0 102 1.3 104 8823 2627 3652 6347
E262S ND ND ND ND ND ND ND
CjXyn10A 9.2 103 7.3 105 1.3 107 1.2 108 5.1 106 2.9 101 3.2 103 988 794 833 1226
CjXyn10Cc 7.3 102 8.5 104 2.9 106 1.0 107 2.3 106 3.4 101 1.2 101 1159 1223 1497 1359
CfXyn10Ac 2.5 104 1.8 107 1.1 108 1.9 108 1.5 108 5.2 102 6.2 105 615
a

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kcat/Km was determined by measuring progress curves at a substrate concentration below Km.
b
Specific activity is expressed as mol of product produced/mol of enzyme/min.
c
The biochemical properties of CjXyn10C and CfXyn10A have been reported elsewhere by Pell et al.3 and in Ref. 10, respectively.

TABLE III be modeled as xylotriose and xylotetraose, respectively. The

Kinetic constants of CmXyn10B and the mutant F340A against xylans nonreducing sugar of the trisaccharide does not appear to con-
CmXyn10B Substratea Km kcat Km/kcat tact CmXyn10B; its conformation is fixed by the intrachain
1 1 1 hydrogen bonding network of the xylooligosaccharides.
mg/ml min min mg1 ml
At the 2 subsite the O-2 of the xylose moiety hydrogen
Wild type GX 3.79 12,364 3,262
Wild type OX 6.17 19,900 3,225 bonds with the side chains of Glu-67 and Trp-328, and the O-3
F340A GX 13.4 4,235 316 interacts with Asn-68. The endocyclic oxygen of the internal
a
GX, glucuronoxylan; OX, oat spelt xylan. xylose makes hydrogen bonds with Lys-71 and Gln-111. These
interactions are almost invariant in GH10 xylanases, and sub-
stitution of any of these residues with alanine greatly reduces
lose at the nonreducing end (the GH67 enzyme removes side
the activity of these enzymes against xylooligosaccharides (23).
chains only from the nonreducing end of xylooligosaccharides;
Similar to the 2 subsite, the interactions of the 1 subsite
(Ref. 21)), whereas the major substituted product derived from
of CmXyn10B with the reducing sugar of xylotriose are very
wheat arabinoxylan is an arabinofuranose moiety linked -1,3
similar to other GH10 xylanases. The xylose residue is perpen-
to xylobiose (wheat arabinoxylans are decorated with arabi-
dicular to the ring plane of the highly conserved tryptophan
nose primarily at the 3 position; Ref. 1)). Typical of GH10
Trp-336. The sugar O-1 (in conformation) interacts with
enzymes, CmXyn10B hydrolyzes aryl--cellobiosides (10) but
Gln-231 and His-233. O-2 hydrogen bonds with Asn-156 and a
displays no significant activity against cellulosic substrates.
water, which provides a link to the nucleophile mutant (Ser-
Comparison of the Activity of CmXyn10B with Other GH10
262), whereas the O-3 interacts with Lys-71 and His-104. The
XylanasesCmXyn10B, similar to Cellulomonas fimi Xyn10A
activity of CmXyn10B against 4-nitrophenyl--D-cellobioside is
(CfXyn10A; formerly known as Cex), displays considerably
2000 times lower than against the corresponding xylobioside,
higher activity against small oligosaccharides compared with
indicating that the C-5 hydroxymethyl group of glucose cannot
other reported GH10 enzymes (Table II). Against xylohexaose,
easily be accommodated at the 1 and 2 subsites. It is well
however, CmXyn10B exhibits similar activity to other family 10
established that accommodation of glucosides at the 1 subsite
xylanases such as CjXyn10A, which likely reflects stronger bind-
demands conformational change in a flanking tryptophan res-
ing in the distal subsites of the C. japonicus enzyme (Table II). A
idue (12). In CmXyn10B, the conformation of this residue,
particularly interesting feature of CmXyn10B is that against
Trp-336, is restricted by a network of hydrophobic interactions
both poorly substituted and extensively decorated xylans, the
with Phe-340, Leu-337, and Pro-341 (Fig. 3), explaining why
enzyme is considerably more active than all other GH10 enzymes
the enzyme displays poor activity against 4-nitrophenyl--D-
analyzed to date (Table II). These data are rather surprising, as
cellobioside. In CfXyn10A Trp-281, which corresponds to Trp-
the xylanase displays similar activity against xylohexaose to
336 in CmXyn10B, is more flexible, as the indole side chain
CjXyn10A, implying that the C. mixtus Xyn10B enzyme contains
does not interact with other amino acids in the xylanase (32).
features that specifically facilitate xylan hydrolysis.
This conformational freedom of the aromatic residue provides
Crystal Structure of CmXyn10B in Complex with Linear Xy-
an explanation of why the C. fimi enzyme is more active
looligosaccharidesTo investigate the mechanism by which
against aryl--cellobiosides than the C. mixtus glycoside hydro-
CmXyn10B interacts with substrate, the crystal structure of an
lase. The poor activity of C. japonicus Xyn10C3 against 4-nitro-
inactive mutant of the enzyme in which the catalytic nucleo-
phenyl--D-cellobioside compared with CmXyn10B may further
phile had been substituted with serine (E262S) was solved in
reflect structural differences at the 2 subsite. In the C. japoni-
complex with xylopentaose was by molecular replacement us-
cus enzyme, Tyr-340 makes a steric clash with the C-5-CH2OH of
ing CjXyn10A as the search model (Fig. 2a). The model refined
glucose, whereas in the C. mixtus xylanase the equivalent resi-
to a final R-factor of 0.14 and Rfree of 0.19. The enzyme has a
due, Gln-111, does not restrict the binding of glucose at this
classic (/)8-barrel fold typical of Clan GH-A enzymes, which
subsite.
include GH10 xylanases (6, 7). The three-dimensional struc-
The 1 subsite interacts with the nonreducing sugar of
ture of CmXyn10B and the catalytic domain of the xylanase
xylotetraose located in the aglycone region of the substrate
SoXyn10A described in the accompanying paper (19) are very
binding cleft of CmXyn10B. The xylopyranose moiety is sand-
similar. The catalytic acid-base (Glu-157) and nucleophile (Glu-
wiched between two hydrophobic walls, with Phe-336 and Phe-
262) residues are at the ends of -strands 4 and 7, respectively,
a feature that is conserved in all Clan GH-A glycoside hydrolases
(31). The data in Fig. 2b reveal the electron density for molecules 3
G. Pell, L. Szabo, S. J. Charnock, H. Xie, T. M. Gloster, G. J. Davies,
located in both the glycone and aglycone binding sites, that can and H. J. Gilbert, manuscript submitted for publication.
 GH10 Substrate Complexes
FIG. 1. Activity of CmXyn10B against xylooligosaccharides. CmXyn10B was incubated with xylotriose (A), xylotetraose (B), xylopentaose
(C), and xylohexaose (D), and the reaction products generated were determined by HPLC. The reaction products generated are as follows: xylose
(
), xylobiose (), xylotriose (), xylotetraose (), xylopentaose (f), and xylohexaose ().
TABLE IV
Binding energies at the subsites of CmXyn10B
The glycosides in parentheses refer to the substrates used to deter-
mine binding energies at each subsite, and the superscript values
define the kcat/Km values for cleaving the specific glycosidic bond (which
are numbered from the reducing end; data were obtained from Fig. 1
and Table II), which were used to obtain subsite binding energies as
described under Experimental Procedures. PNPX2, 4-nitrophenyl--
D-xylobioside; PNPX, 4-nitrophenyl--D-xylopyranose; X3, xylotriose;
X4, xylotetraose; X5, xylopentaose.
G 2 subsite G 2 subsite G 3 subsite
1
kcal mol
9.0 (PNPX2 and PNPX) 3.7 (X42 and X31) 1.1 (X42 and X53)
340 on one side and Tyr-200 on the other. In contrast, xylose
located at the 1 subsite of the other GH10 xylanases charac-
terized to date stack against a single aromatic residue (either
phenylalanine or tyrosine), which is equivalent to Tyr-200 (6,
7). The hydrophobic interactions between CmXyn10B and both
the and faces of the sugar located at the 1 subsite may
explain why this enzyme displays unusually high activity
against small xylooligosaccharides. The binding mode at 1
represents the major difference in the network of interactions
in the proximal subsites between the C. mixtus enzyme and
other GH10 xylanases. Subsite mapping shows that the en-
zyme does not display unusually high binding energy at the 2
subsite (Table IV), and thus interactions at the 1 and/or 1
subsites are likely to mediate increased catalytic efficiency
against small substrates. As the interactions with substrate at
the 1 subsite of CmXyn10B appear to be identical to other
GH10 xylanases, we propose that the additional hydrophobic
interactions between Phe-336 and Phe-340 and the xylose res-
idue in the 1 subsite are responsible for the increased activity
of the C. mixtus xylanase against small substrates; to investi-
gate this hypothesis we have characterized the mutant F340A.
The mutant F340A does indeed display greatly reduced activity
9601
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against xylotriose, xylotetraose, xylohexaose, glucuronoxylan,
and 4-nitrophenyl--xylobioside (Tables II and III). It is inter-
esting to note that the mutation causes only a small decrease in
activity against 4-nitrophenyl--D-cellobioside, and this may
well reflect increased mobility of Trp-336, which must undergo
a conformational change to accommodate the C-5 hydroxy-
methyl group of glucose at the 1 subsite, as Phe-340 restricts
the mobility of the indole side chain of the tryptophan (dis-
cussed above).
At the 2 and 3 subsites Trp-272 stacks against the
xylosyl moiety. The equivalent residue in CjXyn10A, Tyr-255,
interacts with substrate only at the 3 subsite (10), illustrat-
ing the variation in the structure of the aglycone region of the
substrate binding cleft in GH10 enzymes. At the 3 subsite, in
addition to the stacking interaction with Trp-272, Glu-273
makes a hydrogen bond with the O-2 of the xylose moiety. At
the 4 subsite the O-3 of the xylose residue interacts only with
the side chain of Ser-270. The variation in substrate recogni-
tion in the distal region of the aglycone binding cleft of GH10
xylanases, alluded to above, is further illustrated by the accom-
panying paper (19), which demonstrates a lack of substrate
binding at the 3 subsite of SoXyn10A. The structure of
CmXyn10B in complex with xylopentaose bound to both the
glycone and aglycone region of the substrate binding cleft pro-
vides the first glimpses of the conformation of xylan when
bound to all subsites of a GH10 xylanase. Xylotriose bound at
subsites 3 to 1 is twisted into a 3-fold helical structure (Fig.
2b), consistent with the conformation of xylan as determined by
fiber x-ray crystal analysis (33). At subsites 1 to 3 the
xylotetraose substrate is also in a 3-fold helical conformation.
However, at the 4 subsite (the reducing end) the sugar is at
an angle to the rest of the chain (Fig. 2b). This twist in the
substrate chain reflects the observation that the distal region of
the aglycone binding cleft is considerably more enclosed than
other GH10 enzymes. This is due to a large excursion at the
9602 GH10 Substrate Complexes

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FIG. 2. Three-dimensional structure of the Xyn10B from C. mixtus. a, the protein schematic is color-ramped from the N terminus (blue)
to the C terminus (red) and is shown in divergent (wall-eyed) stereo. The single Mg2 ion is shown as a shaded sphere and the two xylooligosac-
charide ligands in ball-and-stick representation. This figure was drawn with MOLSCRIPT (34). b, the electron density of xylopentaose in complex
with E262S. The map, which is shown in divergent (wall-eyed) stereo, is a maximum likelihood-weighted 2Fobs Fcalc synthesis contoured at 1
(0.45 electrons 3 and 0.47 electrons 3, respectively), and is drawn with BOBSCRIPT (35).

FIG. 3. The 1 subsite of C. mixtus

Xyn10B. A possible explanation for the
poor tolerance of glucosides in the 1 sub-
site comes from the steric restraints be-
hind Trp-336, which prevent the move-
ment of this residue necessary to
accommodate the C-5CH2OH group of
glucosides (14, 32). This figure, shown in
divergent (wall-eyed) stereo, was drawn
with BOBSCRIPT (35).

end of strand -7, comprising residues 268 304, which brings a
number of residues into contact with the 2 to 4 subsites,
notably Trp-272 which stacks above the xylose 2, Glu-273,
which hydrogen bonds to the sugar at 3 and Ser-27, which
interacts with the 4 saccharide.
Crystal Structure of CmXyn10B in Complex with Decorated
XylooligosaccharidesTo investigate how decorated xylans are
accommodated in GH10 xylanases, the crystal structures of the
inactive nucleophile mutant, E262S, of CmXyn10B in complex
with various decorated xylooligosaccharides were solved. The
oligosaccharides comprised 4-O-MeGlcA -1,2 linked to the
xylose moiety at the nonreducing end of xylotriose (MX3), ar-
abinofuranose -1,3 linked to the central xylose of xylotriose
(AX3), and arabinofuranose -1,3 linked to the nonreducing
xylose in xylobiose (AX2). A schematic of how these substrates
bind to Xyn10B in the crystal complexes is displayed in Fig. 4.
 GH10 Substrate Complexes 9603

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FIG. 4. Schematic representation of the C. mixtus Xyn10B complexes. The schematic provides insight into the binding mode of
xylopentaose, MX3 and AX3 bound in the substrate binding cleft of CmXyn10B, for which electron density is shown in Figs. 2 and 5. Note that
residues that are not apparent in each substrate are disordered in the crystal structure.

CmXyn10B-MX3 ComplexThe structure of CmXyn10B
E262S in complex with MX3 (Fig. 5a) was refined to a final
R-factor of 0.16 and Rfree of 0.20. The 4-O-MeGlcA of the dec-
orated xylooligosaccharide is accommodated in the 1 subsite,
which is entirely consistent with the biochemical properties of
not only CmXyn10B but other GH10 xylanases, including
SoXyn10A described in the accompanying paper (19), as MX3 is
the major substituted product generated from glucuronoxylan
by these enzymes (16). No 4-O-MeGlcA can be modeled in the
3 subsite, as the electron density is diffuse; this is consistent
with the observation that it does not interact with the enzyme
but is simply accommodated (the O-2 of the xylose moiety at
this location is solvent-exposed). It is interesting to note, how-
ever, that although the uronic acid also does not interact with
the 3 subsite of SoXyn10A, clear electron density for 4-O-
MeGlcA is evident at this subsite (19). In CmXyn10B the elec-
tron density of 4-O-MeGlcA at the 1 subsite is clearly defined,
indicating that the side chain has a more restricted conforma-
tion because of direct interactions with the aglycone region of
the active site (Fig. 5a). The hydroxyl group of Tyr-200 and the
OD-2 of Asp-161 make hydrogen bonds with the O-2 of 4-O-
MeGlcA, and the pyranose sugar ring in the 1 subsite makes
hydrophobic interactions with the aromatic ring of Phe-340. In
contrast, studies reported in the accompanying paper show
that when MX3 and MX2 are bound in the aglycone region of
the substrate binding cleft of SoXyn10A, the electron density
for the 4-O-MeGlcA is not apparent, suggesting that the side
chain is disordered (19). The difference between CmXyn10B
and SoXyn10A may reflect generally weaker binding of the
decorated substrate to the distal aglycone binding sites of the
Streptomyces enzyme, as evidenced by the absence of a xylose
moiety at the 3 subsite. The tighter binding of substrate at
the 1 subsite of CmXyn10B through the additional hydropho-
bic interactions mediated by Phe-340, both with the backbone
xylose and the 4-O-MeGlcA side chain, may well contribute to
the clear electron density for the uronic acid.
In GH10 xylanases the equivalent residue to Tyr-200 (stacks
against the xylose sugar at the 1 subsite; see above) is either
tyrosine or phenylalanine. To investigate whether the identity
of the aromatic residue at the 1 subsite influences the cata-
lytic activity of CmXyn10B, the biochemical properties of
Y200F were investigated. Removal of the hydroxyl group does
not influence activity against xylooligosaccharides; however, it
does cause a 3-fold reduction in catalytic efficiency against
glucuronoxylan (Table II). The crystal structure of CjXyn10B
reveals a hydrogen bond between the OH of Tyr-200 and the
OD2 of Asp-161, and the resultant restriction in the mobility of
the respective side chains may facilitate their capacity to in-
teract with the uronic acid side chain. These results indicate
that the tyrosine-phenylalanine polymorphism at the 1 sub-
site of GH10 xylanases may influence the capacity of these
enzymes to hydrolyze xylans decorated with 4-O-MeGlcA.
Inspection of CmXyn10B E262S in complex with substrates
shows that the O-2 hydroxyls of the xylose moieties at the 2,
9604 GH10 Substrate Complexes

FIG. 5. Electron density for MX3 (a),

AX2 (b), and AX3 (c) in complex with
the mutant E262S of C. mixtus
Xyn10B. The maps, shown in divergent
(wall-eyed) stereo, are maximum-likeli-
hood weighted 2Fobs Fcalc synthesis con-
toured at 1 (0.52 electrons A3 and 0.48

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electrons A3, respectively). This figure
was drawn with BOBSCRIPT (35).

FIG. 6. Solvent accessibility of the hydroxyls of xylan. The 3 to 3 subsites of the C. mixtus Xyn10B are revealed by the crystal structure
of the E262S mutant in complex with xylopentaose. In the 3, 1 and 3 subsites (labeled) the O-2 and O-3 hydroxyls point into solvent,
permitting the accommodation of xylan side chains. In the 1 and 2 subsites, however, the hydroxyls are directed into the protein surface, and
binding of side-chain decorations is occluded. At the 2 subsite the O-2, but not the O-3 hydroxyl, is directed into the protein surface; hence
substitution at O-3 only is possible at this subsite. The 4 sugar bends around the protein surface and is occluded on this figure. This figure, shown
in divergent (wall-eyed) stereo, was drawn using Pymol (DeLano Scientific).

1, and 2 subsites point directly into the enzyme surface
(Fig. 6), demonstrating that the enzyme most likely cannot
accommodate O-2-substituted sugars in these subsites. In con-
trast, the O-2 hydroxyls of the xylosides at the 4 and 3
subsites are pointing into solvent. Thus one would predict that
these hydroxyl groups, in addition to those in 3 and 1, may
accommodate an O-2 substituent, which is entirely consistent
with the data presented in the accompanying paper (19).
CmXyn10B-Arabinoxylooligosaccharide ComplexesThe
model for CmXyn10B E262S in complex with AX2 was refined
to a final R-factor of 0.15 and Rfree of 0.19. The structure
reveals xylose moieties bound in the 1 and 2 subsites, with
arabinofuranose -1,3 linked to the nonreducing xylose (Fig.
5b). The electron density shows that the arabinofuranose moi-
ety is statically disordered in two conformations. In one of these
conformations only, O-2 hydrogen bonds with Glu-67 (a highly
conserved residue in GH10 xylanases); this is the only interac-
tion the arabinofuranose makes with the protein. In the second
conformation (which corresponds to the orientation seen in the
AX3 complex below), the arabinofuranose makes no direct in-
teractions with the protein. In the aglycone region there is very
weak density for a xyloside in 2 and perhaps an arabino-
furanoside at 1, but the density is too weak to allow modeling
with any confidence. These data are consistent with the accom-
panying paper, in which the electron density for the arabino-
furanosyl side chain was not observed in the SoXyn10A-AX2
complex (19).
The model for CmXyn10B E262S in complex with AX3 was
refined to a final R-factor of 0.15 and Rfree of 0.19. The struc-
ture shows xylotriose bound in the 3 to 1 and 1 to 3
subsites, with arabinofuranose bound only in the 2 subsite
(Fig. 5c), similar to the SoXyn10A-AX3 complex described in
the accompanying paper (19). The arabinofuranoside moiety is
in one discrete ordered conformation and does not interact
directly with the protein but hydrogen bonds both to solvent
and through a 2.7- hydrogen bond from O-3 to the endocyclic
O-5 of the 3 subsite xyloside. Xylotriose bound at subsites 1
to 3 subsites may be a trace contaminant; AX3 would not be
able to bind at these subsites, as steric clashes would prevent
decoration of the O-3 of xylose at the 2 subsite (Fig. 5c). It
 GH10 Substrate Complexes
should be noted, however, that as SoXyn10A was used to gen-
erate AX3, a decorated xylose does appear to bind at the 2
subsite of this enzyme. Indeed, in the crystal structure reported
in the accompanying paper (19) the xylose at 2 in the
SoXyn10A/AX3 complex is displaced out of the active site,
which would enable the arabinose side chain to be accommo-
dated, and it is likely that such flexible binding of xylose at this
subsite may be facilitated by the absence of a 3 subsite. In
CmXyn10B the xylose is located in a deeper position, within the
2 subsite, and is likely to display reduced flexibility as adja-
cent sugars interact at the 1 and 3 subsites. Thus the
Cellvibrio enzyme is less likely to accommodate a decorated
xylose at 2 and will therefore generate AX3 less frequently
than SoXyn10A, if at all.
ConclusionsCmXyn10B is the most active GH10 xylanase
reported to date. The increased catalytic activity displayed by
this enzyme is unlikely to reflect interactions in the glycone
region of the substrate binding cleft, as the 1 and 2 subsites
of CmXyn10B are extremely similar to other GH10 enzymes.
Although additional hydrophobic interactions at the 1 and 2
subsites may mediate enhanced activity against small oligosac-
charides, the reasons for the enhanced activity against xylans
are unclear. This report, in conjunction with the accompanying
paper (19), reveals the binding mode of decorated xylooligosac-
charides to GH10 xylanases. The three-dimensional structure
reveals how the uronic acid side chains linked to the O-2 of
xylose moieties can be accommodated at the 1 subsite. Sol-
vent accessibility of O-2 in 3 and 3/4 suggests that these
subsites could also accommodate O-2 decorations. It is signifi-
cant that this pattern of xylan decoration tolerated by
CmXyn10B facilitates synergy with the accessory enzyme
-glucuronidase, illustrating how the substrate specificities of
these enzymes have co-evolved. Thus, by ensuring that 4-O-
MeGlcA can be accommodated at 1 but not 2, CmXyn10B
cleaves glucuronoxylan to release oligosaccharides in which the
sugar at the nonreducing end is decorated with 4-O-MeGlcA.
This is entirely consistent with the mode of action of -glucu-
ronidases, which only remove the uronic acid when it is linked
to the nonreducing end of xylooligosaccharides (2, 21). If the
side chain was accommodated at 2, then a -xylosidase would
be required to remove xylose moieties from the nonreducing
end before the -glucuronidase could hydrolyze the -1,2-
linked 4-O-MeGlcA. A GH39 -xylosidase was recently shown
not to be capable of removing the nonreducing xylose when
linked to a decorated xylose moiety (data not shown); thus it is
possible that decorated xylooligosaccharides in which the
uronic acid side chain is linked to sugar n 1 (when n is the
sugar at the nonreducing end) may be a dead end reaction
product that cannot be further attacked by either the xylanases
(4-O-MeGlcA cannot be accommodated at 1 or 2) or the
accessory enzymes. The accommodation of a xylose at the 4
and 1 subsites that is decorated with the uronic acid will
generate a reaction product that can be further hydrolyzed, as
the 4-O-MeGlcA side chains at n and n 3 of the xylooligosac-
charide can now be located at subsites 3 and 1. A similar
accommodation of decorated substrates by the Streptomyces
xylanase is described in the accompanying paper (19). It is
clear that the enzymatic consortia that exist to degrade this
9605
complex and recalcitrant substrate have co-evolved to facilitate
a remarkable synergy. The subtle accommodation of decora-
tions, honed by the topology of the substrate binding cleft,
ensures complementary between glycone and aglycone regions
such that the reaction products derived from glucuronoxylan
can be further attacked by the xylanase and are the optimal
substrates for the accessory enzymes.
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 The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated
Substrates
Gavin Pell, Edward J. Taylor, Tracey M. Gloster, Johan P. Turkenburg, Carlos M. G. A.
Fontes, Luis M. A. Ferreira, Tibor Nagy, Samantha J. Clark, Gideon J. Davies and Harry
J. Gilbert
J. Biol. Chem. 2004, 279:9597-9605.
doi: 10.1074/jbc.M312278200 originally published online December 10, 2003

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