Báo cáo khoa học: Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18 pdf

Emergence of a subfamily of xylanase inhibitors within
glycoside hydrolase family 18
Anne Durand
1,
*, Richard Hughes
1,
*, Alain Roussel
2
, Ruth Flatman
1,
*, Bernard Henrissat
2
and Nathalie Juge
1,3
1 Institute of Food Research (IFR), Norwich, UK
2 Architecture et Fonction des Macromole
´
cules Biologiques, UMR6098, CNRS et Universite
´
s d’Aix-Marseille I et II, Marseille, France
3 Institut Me
´
diterrane
´
en de Recherche en Nutrition, UMR INRA 1111, Faculte
´
des Sciences et Techniques de St Je
´
ro
ˆ
me, Marseille, France

Recently two classes of plant proteins, designated as
XIP (xylanase inhibitor protein) [1] and TAXI ( Triti-
cum aestivum xylanase inhibitor) [2] have been shown
to inhibit xylanases. XIP-I from wheat (Triticum aesti-
vum) represents the prototype of a novel class of
(b ⁄ a)
8
inhibitors that inhibits reversibly xylanases
belonging to glycoside hydrolase families (GHs) 10 and
11 (CAZY database />[3]. The structural features essential for xylanase inhibi-
tion were recently largely unravelled by the resolution
of the crystal structures of XIP-I in complex with a
GH10 xylanase from Aspergillus nidulans and a GH11
xylanase from Penicillium funiculosum [4]. The inhibi-
tion mechanism is novel since XIP-I possesses two inde-
pendent enzyme-binding sites, allowing binding to two
glycoside hydrolases with different folds [4].
XIP-I belongs to a large protein family (GH18) that
contains mostly chitinases and proteins of unknown
function. The crystal structure of XIP-I confirmed the
structural resemblance to GH18 chitinases [5]. In XIP-
I, however, clear structural differences in the region
corresponding to the active site of chitinases account
for its lack of enzymatic activity towards chitin [5–7].
XIP-type proteins were also isolated from rye,
durum wheat, barley and maize [8], but sequence infor-
mation is limited and the only clones available are
those encoding XIP-I (GenPept, AN: CAD19479) and
XIP-II (GenPept, AN: CAC87260), the other putative
XIP-type inhibitor from wheat (Triticum turgidum ssp.

Durum).
The widespread representation of XIP-type inhibi-
tors in cereals questions further the place ⁄ evolution of
Keywords
chitinase; evolution; family 18 glycoside
hydrolase; proteinaceous xylanase inhibitors;
rice
*Present address
John Innes Centre, Norwich Research Park,
Colney, Norwich NR4 7UH, UK
(Received 16 December 2004, revised 3
February 2005, accepted 9 February 2005)
doi:10.1111/j.1742-4658.2005.04606.x
The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhib-
its xylanases belonging to glycoside hydrolase families 10 (GH10) and 11
(GH11). Sequence and structural similarities indicate that XIP-I is related
to chitinases of family GH18, despite its lack of enzymatic activity. Here
we report the identification and biochemical characterization of a XIP-type
inhibitor from rice. Despite its initial classification as a chitinase, the rice
inhibitor does not exhibit chitinolytic activity but shows specificities
towards fungal GH11 xylanases similar to that of its wheat counterpart.
This, together, with an analysis of approximately 150 plant members of
glycosidase family GH18 provides compelling evidence that xylanase inhibi-
tors are largely represented in this family, and that this novel function has
recently emerged based on a common scaffold. The plurifunctionality of
GH18 members has major implications for genomic annotations and pre-
dicted gene function. This study provides new information which will lead
to a better understanding of the biological significance of a number of
GH18 ‘inactivated’ chitinases.
Abbreviations

E:I
50
, molar ratio enzyme–inhibitor that gives 50% of inhibition; GH, glycoside hydrolase; pRIXI, putative rice xylanase inhibitor; RIXI, rice
xylanase inhibitor; rXIP-I, recombinant XIP-I produced in Pichia pastoris; SPR, surface plasmon resonance; XIP-I, xylanase inhibitor protein I;
XYNC, Penicillium funiculosum xylanase C.
FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1745
this new class of protein within GH18. The existence
of several classes of GH18 chitinases in plants was pre-
viously suggested [9]. However the general impression
was that gene duplications, gene losses and perhaps
also translocations resulted in rather unreliable rela-
tionships for deriving evolutionary conclusions [10]. In
contrast to the abundant genetic information produced
from recent sequencing programmes of plant organ-
isms (rice and Arabidopsis), relatively little is known
about the enzymatic and structural properties of
GH18 plant chitinases. An emergent proportion
of sequences appear to encode plant inactivated chitin-
ases, such as narbonin and concanavalin B, the recep-
tor-like kinase Chrk1, and XIP [11]. Based on the
recent structural data obtained on XIP-I, can we ana-
lyze family GH18 and find other proteins with the
same function as XIP? This has implications for an
improved annotation of plant genes or ESTs and is
particularly important as there is no apparent relation-
ship between the old function (chitinase) and the newly
evolved one (xylanase inhibitor), despite sequence and
structural similarity.
No XIP-type protein was so far identified in rice.
Among the GH18 sequences isolated from the rice

genome [at least 23 – data from the Carbohydrate-
active enzymes database, />CAZY/ accessed 11 January 2005)], only two cDNA
sequences were shown to encode recombinant proteins
having chitinase activity [12] while others were classified
as putative rice class III chitinase(s) based on sequence
homology only [13]. In particular the (GenPept data-
bank; AN: BAA23810.1) clone shares higher similarity
with XIP-I than with ‘active’ chitinases and was thus
selected as a putative rice xylanase inhibitor (pRIXI).
In this work, we report for the first time the func-
tional identification of a rice ortholog of the wheat
XIP, originally classified as a rice class III chitinase
and analyze the features that allow discriminating the
subfamily of xylanase inhibitors within the large GH18
family.
Results
Production and structural characterization
of pRIXI
The pRIXI clone (GenPept databank; AN:
BAA23810.1) is expected to encode a protein of 304
residues with a predicted relative molecular mass of
33 946.8 and a theoretical pI of 9.33 [13]. In order to
address the functionality of this potential inhibitor, its
cDNA sequence and that of XIP-I were expressed in
conditions similar to those used for the production of
active basic chitinase in Pichia pastoris GS115 strain
[12], e.g. under the control of the alcohol oxidase pro-
moter and with an His-tag tail at the C-terminus. Both
recombinant XIP-I (rXIP-I) and pRIXI were produced
in P. pastoris with a high secretion yield of approxi-

mately 250 mgÆL
)1
. The recombinant proteins were
purified to apparent homogeneity from the culture
supernatant as a C-terminal tag fusion protein using
one step affinity chromatography. The purified pro-
teins migrated on SDS ⁄ PAGE as a 33 and 37 kDa
single bands for pRIXI and rXIP-I, respectively. The
relative molecular mass of pRIXI, obtained by ESI-
MS, was 33 446 Da, thus in total agreement with the
predicted calculated mass including the myc epitope
and His-tag in C-terminal. In contrast, rXIP-I showed
an apparent relative molecular mass of 37 000 Da on
SDS ⁄ PAGE, thus higher than the size of the native
protein from wheat (34 076 Da). Native XIP-I has
been reported to be weakly glycosylated and the two
N-glycosylation sites (Asn89 and Asn265) are occupied
[3,5,6]. These glycosylation sites are not present in the
rice homologue. The relative molecular mass discrep-
ancy between the native and recombinant proteins
may be explained by hyperglycosylation of the rXIP-I
in P. pastoris, as confirmed by mass spectrometry,
where five main peaks were identified (37 529, 37 692,
37 853, 37 873 and 38 015 Da). Isoelectric focusing
revealed that rXIP-I consisted of three molecular iso-
forms of pI 6.8–7.2–8.2 with a main band at pI 7.2.
This value differs from native XIP-I of pI 8.7–8.9 [6],
due to the insertion of the myc epitope and His-tag
in C-terminal. The recombinant pRIXI showed a pI
close to pH 9, thus in agreement with the calculated

pI of 8.7. The predominant N-terminal sequences,
EAEAEFAGGK for rXIP-I and EFGPAMAAGK for
pRIXI indicated that the two proteins were correctly
processed at the Kex2 and Ste13 signal cleavage sites,
respectively. Both recombinant proteins were recog-
nized by antibodies raised against His-tag. However,
although the rXIP-I was recognized by antibodies
raised against native XIP-I, there was no cross-reaction
with pRIXI (data not shown).
Functionality of the recombinant proteins
The recombinant proteins were tested for their chitinase
activity using two different size substrates. Using chitin
azure, a long and insoluble substrate, no chitinase activ-
ity could be detected at pH 5.5 and 8.0 for both pRIXI
and rXIP-I, confirming the lack of chitinase activity pre-
viously reported for native XIP-I at pH 5.5 in the same
conditions [6]. Interestingly, no activity could be detec-
ted using this substrate with the recombinant basic
A novel GH18 xylanase inhibitor identified in rice A. Durand et al.
1746 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS
chitinase (GenPept databank; AN: BAA22266.1) al-
though Streptomyces griseus chitinase, used as control,
was active. The activity of the proteins were then further
tested on a short and soluble substrate, 4-nitrophenyl
b-d-N,N¢,N¢¢-triacetylchitotriose [p-nitrophenol-(Glc-
NAc)
3
]. Using this substrate, the recombinant basic chi-
tinase showed a specific activity of 31.3 and 9.9 UÆmg
)1

at pH 5.5 and 8.0, respectively. However, neither pRIXI
nor rXIP-I showed any evidence for chitinase activity
even in a presence of a molar excess of inhibitors, 3.5 : 1
and 10 : 1 (inhibitor–basic chitinase) for pRIXI and
rXIP-I, respectively.
The specificity of pRIXI towards fungal and bacter-
ial GH10 and GH11 xylanases was compared to that
of rXIP-I (Table 1). The pattern of inhibition of rXIP-
I towards GH11 xylanases was similar to that previ-
ously reported for the native inhibitor (E : I
50
values,
Table 1). All the fungal GH11 xylanases were inhibited
by both pRIXI and rXIP-I up to a molar ratio E : I of
1 : 30 (Table 1), although the E : I
50
of pRIXI were
higher than those of rXIP-I. The lowest molar ratio
(1 : 6.5) was obtained for the Trichoderma longibrachi-
atum (M3) xylanase. Indeed, for the GH11 Aspergil-
lus niger xylanase the value of the E : I
50
is greater
than 1 : 52, in these conditions 34% of inhibition was
observed. As for native XIP-I, no inhibition was
observed for pRIXI and rXIP-I against two bacterial
GH11 xylanases from Bacillus subtilis and rumen
microorganism (M6) (Table 1). In contrast to both
native and recombinant XIP-I, none of the GH10 xy-
lanases from A. niger, A. aculeatus and A. nidulans

(fungal) or from Cellvibrio japonicus (bacterial) were
inhibited by pRIXI (Table 1).
Interaction of the inhibitors with xylanases
The relative affinities and pH dependencies of the inter-
action of XIP-I with xylanases were studied using titra-
tion curves. The GH11 XYNC from P. funiculosum
interacted with both pRIXI and rXIP-I across the
entire range of pH (Fig. 1B). However, although GH11
A. niger (Fig. 1C) and GH10 A. nidulans (Fig. 1D) xy-
lanases interacted with rXIP-I, no complex formation
was observed with pRIXI (Fig. 1C,D), in agreement
with the activity assays data, suggesting that pRIXI is
a weaker inhibitor than XIP-I. The interaction between
rXIP-I and A. niger xylanase occurred across a narrow
range of pH, as previously demonstrated with native
XIP-I [3]. In contrast, no complex was observed with
bacterial GH11 xylanases from rumen microorganism
M6 and B. subtilis (data not shown) in agreement with
the reported specificity of XIP-type inhibitors.
The molecular interaction between xylanases and
pRIXI or rXIP-I was further studied in real time by
using a biosensor based on surface SPR. The inhibitor
proteins were immobilized as a ligand on the dextran
surface of a chip whereas the P. funiculosum XYNC
xylanase was used as an analyte over the surface. The
sensorgrams for the interaction with XYNC are shown
in Fig. 2. The increase of RU from the baseline repre-
sents the binding of the xylanase to the surface-bound
inhibitor. The plateau line represents the steady-
state ⁄ equilibrium phase of the XYNC–inhibitor inter-

action, whereas the decrease in RU from the plateau
represents the dissociation phase. The slow dissociation
phase observed on the SPR sensorgrams for the com-
plex between rXIP-I and XYNC suggests that the
interaction is stronger than the one previously reported
between native XIP-I and the A. niger xylanase [3], in
agreement with the inhibition constants reported for
these enzymes (K
i
¼ 3.4 nm and 317 nm for XYNC
and A. niger xylanases, respectively) [3]. XYNC exhib-
ited a faster dissociation with pRIXI compared to
rXIP-I, in agreement with the lower value obtained
from E : I
50
for pRIXI (1 : 45 for pRIXI against
1 : 2.3 for rXIP-I and 1 : 1.6 for native XIP-I). SPR
analysis thus demonstrated that a faster dissociation
rate probably accounts for the weaker interaction
between pRIXI and XYNC compared to XIP-I.
Taken together, these data demonstrate that pRIXI
is not a chitinase but a novel XIP-type inhibitor in rice
and herein named RIXI for ‘RIce xylanase inhibitor’.
Table 1. Xylanase inhibition specificity of XIP-I and RIXI towards
xylanases.
RIXI XIP-I
Recombinant Recombinant Native
Family 11 xylanases
Fungal
A. niger Yes

a
1:3.6
b
1 : 2.1
XYNC P. funiculosum 1 : 45 1 : 2.3 1 : 1.6
T. longibrachiatum (M3) 1 : 6.5 1 : 2.3 1 : 1.1
Bacterial
B. subtilis No
c
No No
Rumen microorganism (M6) No No No
Family 10 xylanases
Fungal
A. nidulans No Yes 1 : 0.6
d
A. niger No Yes 1 : 0.7
A. aculeatus No No No
Bacterial
P. fluorescens No No No
a
Inhibition observed within the limit defined earlier (> 10% inhibi-
tion at E : I molar ratio up to 1 : 30 maximum [3]).
b
E:I
50
, molar
ratio of enzyme to inhibitor that gives 50% of inhibition.
c
No inhibi-
tion within the detection limit described in

a
.
d
From [3].
A. Durand et al. A novel GH18 xylanase inhibitor identified in rice
FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1747
Discussion
RIXI is a novel xylanase inhibitor from rice
Our data clearly show that the rice putative chitinase
sequence (GenPept databank; AN: BAA23810.1) in
fact encodes a xylanase inhibitor. The previous lack
of detection of xylanase inhibitor in rice extracts can
be explained by the methodology used in the
previous reports [14,15]. Indeed the absence of detec-
tion by Western blotting is due to the lack of cross-
reactivity between purified RIXI and anti-XIP-I Igs
[14]. Furthermore, the weak interaction between
RIXI and GH11 A. niger xylanase explains why
affinity chromatography failed to interact with the
rice inhibitor [15] and why no xylanase inhibitor
activity was detected in rice extracts using the same
enzyme [14]. The observed weaker interaction is not
A
B
C
D
Fig. 1. Interaction of RIXI and rXIP-I with
xylanases. (A) Titration curves showing the
inhibitors. (i) RIXI; (ii) rXIP-I. (B) Titration cur-
ves showing the interaction between GH11

XYNC from P. funiculosum and the two rec-
ombinant inhibitors. (i) XYNC; (ii) a mixture
of XYNC and RIXI; (iii) a mixture of XYNC
and rXIP-I. (C) Titration curves showing the
interaction between GH11 A. niger xylanase
and the two inhibitors. (i) A. niger xylanase;
(ii) a mixture of A. niger xylanase and RIXI;
(iii) a mixture of A. niger xylanase and rXIP-I.
(D) Titration curves showing the interaction
between GH10 A. nidulans xylanase and the
two inhibitors. (i) A. nidulans xylanase; (ii) a
mixture of A. nidulans xylanase and RIXI; (iii)
a mixture of A. nidulans xylanase and rXIP-I.
For each experiment the molar ratio E : I
was identical (1 : 1) and 116 pmol of each
protein were loaded on the gel.
A novel GH18 xylanase inhibitor identified in rice A. Durand et al.
1748 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS
expected to be due to the lack of glycosylation of
RIXI, as glycosylation in XIP-I does not affect inhi-
bition specificity [4,5].
The presence of xylanase inhibitor in rice is not
surprising as hemicellulose in the cell walls of rice
cells is composed mainly of arabinoxylan [16] and the
ability to degrade xylan represents an important
attribute for a rice pathogen to infect plant tissues.
Indeed, secretion of xylanases by rice pathogens was
reported for Magnaporthe grisea, the fungal pathogen
that causes rice blast disease [17], and Xanthomonas
oryzae pv. oryzae, the causal agent of bacterial leaf

blight, a serious disease in rice [17–19]. The recent
demonstration that xylanases secreted by rice patho-
gens are important factors of their virulence agrees
with a potential role of RIXI in plant defence, as
proposed for XIP-I [20]. This hypothesis is reinforced
by the homology of RIXI with chitinases, which are
known to act in response to invading pathogens by
degrading polysaccharides of their cell wall. Class III
chitinases have been classified into pathogenesis-rela-
ted proteins (PR-8) because of their inducible expres-
sion upon infection by pathogens [21,22]. Plant
chitinases exhibit rapid evolution by acting as prime
targets for the coevolution of plant–pathogen interac-
tions. XIP-type proteins could have evolved from
chitinases as part of the plant defence pathway to act
both on the xylanases secreted by pathogens and on
the pathogen itself. In both cases, the function is ori-
entated towards a general role in plant defence and
the production of inhibitors prevents the plant to
undergo unnecessary metabolic costs.
XIP-type inhibitors represent a subfamily of GH18
GH18 includes chitinases from various species, inclu-
ding bacteria, fungi, nematodes, insects plants, and
mammals, but also a growing number of nonchitinase
proteins, the latter making genome and ESTs annota-
tions particularly unreliable (for instance RIXI was
thought to be a basic chitinase). Sequence-based famil-
ies such as those in CAZy, PFAM, etc., group together
proteins that have sometimes different functions. Here
the case is particularly tricky as the novel function has

been acquired relatively recently (in such a case, only
functional and structural characterization can help
building the necessary knowledge to enable prediction
methods). In the present work, novel biochemical and
structural information of XIP-type inhibitors are used
to test whether it is possible to better predict function-
ality within the GH18 family.
Although the overall sequence similarity between
GH18 chitinases is not particularly high (average pair-
wise 21%), their active site regions contain many
residues that are fully or highly conserved. The
most prominent motif dictating chitinase activity is
DxxDxDxE that includes the glutamate acting as the
catalytic acid. The GH18 members devoid of chitinase
or known enzymatic activity, all have nonconservative
substitutions of one of the acidic amino acid residues
in the catalytic region (Fig. 3). The XIP-type inhibitors
all have the third aspartic acid DxxDxDxE mutated
into an aromatic residue (Phe126
XIP-I
) whereas the cat-
alytic glutamate residue is only conserved in XIP-I
(Glu128
XIP-I
). The substitution of the critical Asp aci-
dic amino acid by a bulky residue thus is a major
determinant for the lack of chitinase activity reported
for XIP-I and RIXI. This suggests that another GH18
sequence (GenPept, AN: BAC10141.1) could be an
additional xylanase inhibitor in rice.

The inhibition specificity of RIXI can be explained
on the basis of the recently solved 3-D structure of
XIP-I in complex with a GH10 xylanase from A. nidu-
lans and a GH11 xylanase from P. funiculosum [4].
The inhibition of GH10 xylanase occurs through
extensive interactions between the two proteins. XIP-I
a7 helix (232–245) interacts with the loops forming the
xylanase groove; side chains emerging from the helix
point into the heart of the cleft and occupy the four
central subsites: )1 (Lys234
XIP-I
), +1 (Asn235
XIP-I
),
)2 (His232
XIP-I
), and +2 (Tyr238
XIP-I
), whereas
Lys246
XIP-I
sterically blocks access to subsite )3. Two
additional regions (loop b
6
a
6
from residue 193–205
and a8 helix 268–272) make contact with the enzyme.
These three regions are determinants for the inhibitory
activity. Although a7 and a8 helixes are pretty well

Fig. 2. SPR sensorgrams showing the interaction between XYNC ⁄
RIXI (A) and XYNC ⁄ rXIP-I (B). In both panels, XYNC (14 l
M)was
injected at a flow rate of 50 lLÆmin
)1
. The signal is indicated in
resonance units (RU) and time 0 corresponds to the injection of
XYNC.
A. Durand et al. A novel GH18 xylanase inhibitor identified in rice
FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1749
conserved, the lack of inhibition activity of RIXI
against GH10 xylanases can be explained by differ-
ences in the loop b
6
a
6
, a region at the interface
between XIP-I and A. nidulans xylanase (Fig. 4A).
This loop contains two strictly conserved residues,
Cys195 (involved in a disulfide bond with Cys164) and
Trp205 separated by a variable number of amino acids
(see alignment, Fig. 3). In both RIXI and hevamine,
an active GH18 chitinase from Hevea brasiliensis, the
loop contains 13 amino acids, as compared to nine in
XIP-I (see alignment, Fig. 3). When the b
6
a
6
loop of
hevamine for which the three-dimensional structure is

known (pdb code 2HVM), is superimposed on the cor-
responding loop of XIP-I, the extra residues introduce
steric clashes in the interaction with GH10 xylanases,
thus preventing binding with the xylanase (Fig. 4A). A
similar situation is thus expected to occur for RIXI
but also for XIP-II, the other wheat XIP-type inhib-
itor, which contains also four additional amino acids
in b
6
a
6
loop as compared to XIP-I. In contrast, the
BAC10141.1 sequence shows a shorter loop, suggesting
a possible inhibition of GH10, although a clash result-
ing of an insertion before the Cys195 residue cannot
be ruled out.
The XIP-I strategy for inhibition of GH11 xylana-
ses consists of an inhibitory head (the P-shaped La
4
b
5
loop, amino acids 148–153) blocking the entrance to
the active site. The main inhibition determinant
involves a functional arginine side-chain (Arg149
XIP-I
)
projecting into the glycone subsites of the enzyme’s
active site. The top of the inhibitor loop is slightly
twisted, allowing the GGP(150–152) segment to
extend closely parallel to the )3 subsite plane whereas

the main-chain segment 149–150 occupies subsite )2.
This loop is three residues shorter in hevamine, pre-
venting an interaction with xylanase (Fig. 4B) but of
similar length in RIXI, allowing interaction of the
rice inhibitor with GH11 xylanases. However the
important determinant RGG(149–151) in XIP-I is
replaced by MYR(149–151) in RIXI, which might
explain the observed weaker interaction (Fig. 3). The
same characteristic feature is also observed in XIP-II,
predicting interaction with GH11 xylanases, although
weaker. However, in the BAC10141.1 sequence, the
loop is shorter, which might prevent binding to
GH11 xylanases, as also observed with hevamine.
The additional interacting regions of XIP-I; the C-ter-
minal extremity helix a2 and the loops a
5
b
6
and a
6
b
7
,
corresponding to residues 64–70, 179–185 and 213–217
[4] are well conserved among the sequences (Fig. 3).
The structural data thus agree with the inhibition
specificity pattern observed for RIXI and predict
BAC10141.1 to be another xylanase inhibitor from rice
with no chitinase activity and a specificity pattern
opposite to that of RIXI, inhibiting GH10 but not

GH11 xylanases.
Fig. 3. Amino acid sequence alignment of selected GH18 plant members: XIP-I (CAD19479.1); BAC (BAC10141.1); RIXI (BAA23810.1); XIP-II
(CAC87260.1); basic chitinase (BAA22266.1); hevamine (CAA07608.1). Residue numbering is given for the XIP-I sequence [5]. Grey shading
shows residues conserved in all sequences. Boxes show consensus residues involved in chitinase activity and residues involved in complex-
ing with a GH10 or GH11 xylanase [4].
A novel GH18 xylanase inhibitor identified in rice A. Durand et al.
1750 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS
XIP-type inhibitors have evolved from
a common scaffold
A phylogenetic tree is presented for the known plant
members of the GH18 family (Fig. 5). The sequences
were retrieved from the CAZy database [23]. Clearly,
four major subfamilies can be distinguished: (a)
hevamine-type chitinases; (b) putative chitinases; (c)
narbonins; and (d) putative chitinases. Out of the four
major subfamilies that appear from the phylogenetic
analysis, only the one that contains hevamine actually
contains enzymes of demonstrated activity. XIP-type
inhibitors emerged from the hevamine cluster along
with concanavalin B, another GH18 member with no
enzymatic function. A large number of the related
sequences are chitinases, but the evolutionary tree also
includes receptor-like kinase (Chrk1) and individual
sequences with no catalytic residue. Based on the pre-
sent work, we suggest that the proteins emerging from
cluster (a) also have xylanase inhibitor activity. The
tree clearly shows that the xylanase inhibitors appeared
after the emergence of the various subfamilies of
chitinases from their common ancestor. In this respect,
the xylanase inhibitors are a relatively new invention,

and so far no protein has been reported to display
both xylanase inhibition and chitinase activities.
The assignment of sequences to large disparate
multifunctional glycosidase families such as GH18 is
useful to unravel ancient evolutionary events, but is of
limited use for ORF function prediction based solely
on sequence similarity. The present study adds to the
growing body of evidence that sequence similarity
alone would result in a wrong (and self-propagating)
functional assignment, and that biochemical characteri-
zation is required to establish protein function. The
GH18 xylanase inhibitors are an example of evolution-
ary inventions from pre-existing proteins. The xylanase
inhibitor and its chitinase ancestors are believed to be
produced by the plant as part of their defence system
against fungi. It is tempting to speculate that the novel
function emerged based on a class of proteins whose
synthesis was already triggered by fungal attack, and
that evolution has kept the existing signal recognition
and expression-regulation pathways [24]. Additional
biochemical and structural characterization of plant
GH18 ‘chitinase’ sequences will clarify some features
of the evolution of this family of chitinases.
Experimental procedures
Materials and strains
The cDNA clone encoding a putative rice class III chitinase
(DNA Data Bank of Japan; AN: D55712 corresponding to
the protein AN: BAA23810.1 in the GenPept databank)
was a kind gift of T. Sasaki (National Institute of Agrobio-
logical Resources, Tsukba, Japan) [13]. The full-length

cDNA encoding XIP-I (GenPept databank; AN:
CAD19479.1) was from in house collection [7]. The P. pas-
toris clone encoding a basic active rice chitinase (GenBank;
AN: AB003195) was a kind gift from S M. Park (Basic
Y189
C164
C195
W205
L143
L158
R149
GH10
GH11
A
B
Fig. 4. (A) Close-up view of the interaction between XIP-I (in purple)
or hevamine (in blue) and GH10 A. nidulans xylanase. The hevamine
structure (pdb code 2HVM) was superimposed onto the XIP-I struc-
ture in complex with A. nidulans xylanase (pdb code 1TA3) using
TURBO-FRODO [33]. The loop between residues 195 and 205 in
XIP-I (in purple) is in close contact with the GH10 xylanase. The cor-
responding loop in hevamine (in blue) is four residues longer. This
insertion may induce steric clashes with the enzyme. (B) Close-up
view of the interaction between XIP-I (in purple) or hevamine (in
blue) and GH11 P. funiculosum xylanase. The hevamine structure
(pdb code 2HVM) was superimposed onto the XIP-I structure in
complex with P. funiculosum xylanase (pdb code 1TE1) using
TURBO-
FRODO [33]. The Arg149 located in the P-shape loop from residues
143–158 (in purple) plays an important role for the inhibitory activity

of XIP-I. Such interaction is not possible in the case of hevamine
because the corresponding loop (in blue) is two residues smaller.
A. Durand et al. A novel GH18 xylanase inhibitor identified in rice
FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1751
Science Research Institute, Chonbuk National University,
Korea) [12]. The GH11 xylanases from P. funiculosum
(XYNC) and A. niger were from in house [25,26]. The
GH10 xylanases from B. subtilis and A. aculeatus, A. niger,
C. japonicus, and A. nidulans, were from L. Saulnier
(INRA, Nantes, France), K. Gebruers (Laboratory of Food
Chemistry, University of Leuven, Belgium), H. Gilbert
(University of Newcastle, United Kingdom), and P. Man-
zanares (Instituto de Agroquı
´
mica y Technologı
´
a de Ali-
mentos, Valencia, Spain), respectively. The native XIP-I
was purified from wheat as described previously [3,6]. Low
viscosity arabinoxylan, T. longibrachiatum M3 and rumen
microorganism (M6) xylanases were from Megazyme Inter-
national Ireland Ltd (Co. Wicklow, Ireland). The P. pastor-
is expression kit containing the pPICZaA vector and the
anti-[His(C-term)-HRP] Ig were from Invitrogen (San
Diego, CA, USA). The HisTrap purification kit was from
Amersham Pharmacia Biotech (Uppsala, Sweden). PmeI
was from New England Biolabs (Beverly, MA, USA) and
the other restriction endonucleases and DNA modifying
enzymes from Promega (Madison WI, USA). Escheri-
chia coli DH5a (supE44, hsdR17, recA1, endA1, gyrA96,

thi-1, relA1) was used for DNA subcloning. Chitin azure,
dinitrosalicylic acid (DNS), 4-nitrophenyl b-d-N,N¢,N¢¢-tri-
acetylchitotriose and Streptomyces griseus chitinase were
from Sigma Chemical Co. (St. Louis, MO, USA). Oligo-
nucleotides were synthesized as high purity salt-free oligos
by Sigma-Genosys Ltd. (Pampisford, UK).
Cloning and expression in P. pastoris
The cDNAs encoding XIP-I and the putative rice xylanase
inhibitor (pRIXI) were amplified by PCR using the fol-
lowing primers: 5¢-CCG
GAATTCGCGGGGGGAAAG-3¢
(forward primer) and 5¢-GC
TCTAGAGCGGCGTAGTAC
TT-3¢ (reverse primer) for XIP-I and 5¢-CCG
GAATTCGG
CCCGGCGATG-3¢ (forward primer) and 5¢-GC
TCTAGA
GCAGCCCAGTACTT-3¢ (reverse primer) for pRIXI.
EcoRI and XbaI restriction sites (underlined) were intro-
duced in 5¢ and 3¢ of these primers, respectively, to facilitate
subsequent cloning steps. DNA amplification was carried out
through 25 cycles of denaturation (1 min at 94 ° C), annealing
(30 s at 61 °C) and extension (1.5 min at 72 °C) in a DNA
thermocycler (Perkin Elmer GeneAmp PCR system 2400)
using Pfu polymerase (Stratagene, UK) and Vent polymerase
New England Biolabs (Beverly, MA, USA) for amplification
of RIXI and XIP-I cDNAs, respectively. The gel-purified
fragments and pPICZaA vector were digested with EcoRI
and XbaI. The digested cDNA fragments were purified and
ligated into pPICZaA vector at 16 °C overnight using T4

DNA ligase. After transformation into E. coli DH5a by
heat-shock and plating the cells on low salt LB agar contain-
ing 25 lgÆmL
)1
zeocin (Invitrogen), positive clones were se-
quenced to check the integrity of the insert. The recombinant
plasmids (10 lg) were linearized by PmeI and used to trans-
form P. pastoris strain (his4) GS115 [27] using an adaptation
of the spheroplast method [28]. Transformed colonies were
selected on RDB plates containing zeocin (100 lgÆmL
)1
) and
histidin (4 mgÆmL
)1
). After incubation at 30 °C for 4–5 days,
50 clones of each transformation were screened on minimum
methanol medium (MM) agar plates and on minimal
dextrose medium (MD) agar plates at 30 °C. After 3 days,
the transformants growing on MD medium and slowly on
MM medium were screened for protein secretion. Single
zeocin-resistant colonies were grown in rich nonbuffered gly-
cerol complex medium (10 mL) at 30 °C with shaking
(250 r.p.m.). After 48 h, the cells were centrifuged and
resuspended in the induction medium (nonbuffered rich
methanol complex medium). The secretion of the recombin-
ant proteins in the supernatants was analysed after 48 and
96 h of methanol induction on 12% SDS ⁄ PAGE gels and
the proteins revealed using Coomassie blue staining or
Fig. 5. Unrooted phylogenetic tree for plant
members of family GH18. The scale bar ind-

icates the number of substitutions per posi-
tion following alignment with
MUSCLE [34]
and bootstrap analysis by
CLUSTAL W [35].
The tree was displayed with
TREEVIEW [36].
The thick lines identify the various clusters
discussed in the text. The accession num-
bers (GenBank or SwissProt) of the isolated
sequences are given on the figure. Acces-
sion numbers of representative members of
the various subfamilies: subfamily 1, CA-
A07608; subfamily 2, AAO15366; subfamily
3, O81862; subfamily 4, Q41675.
A novel GH18 xylanase inhibitor identified in rice A. Durand et al.
1752 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS
transferred onto nitrocellulose membranes for immuno-reve-
lation. Clones secreting the highest level of the recombinant
proteins were chosen for large-scale expression.
Protein production and purification
For large-scale production of the recombinant proteins,
P. pastoris transformants were grown in 200 mL of rich
nonbuffered glycerol complex medium in 1 L flasks at
30 °C with shaking (170 r.p.m.) for 2 days. After centrifu-
gation (2500 g, 10 min, room temperature), the pellet was
resuspended in the induction medium (40 mL). The cells
were shaken in 250 mL flask for 72 h at 30 °C and
170 r.p.m. Purification was performed using a one-step
nickel affinity-chromatography using the HisTrap kit. Fol-

lowing centrifugation (2500 g, 10 min, 4 °C), the pH of the
supernatant was adjusted to pH 7.1–7.2 with 1 m sodium
phosphate buffer pH 7.4 and filtered through 0.45-lm filter.
The HiTrap chelating HP column was charged with Ni
2+
ions according to the manufacturer instructions and equili-
brated with phosphate buffer pH 7.4 (10 mL) containing
0.5 m NaCl and 10 mm imidazole using a peristaltic pump.
The sample was loaded at a 1.5 mLÆmin
)1
flow rate. The
histidine-tagged proteins were eluted using phosphate buffer
containing increasing concentrations of imidazole (20, 50,
100, 300 and 500 mm). The A
280
of the collected fractions
was measured and the proteins analysed by SDS ⁄ PAGE.
Fractions containing pure protein were pooled and dialysed
overnight against McIlvaine buffer pH 5.5 at 4 °C.
Purification of the recombinant basic chitinase (GenPept
databank; AN: BAA22266.1) was performed as described
previously [12] with the exception of the last chromatogra-
phy step on con-A agarose.
Enzyme assays
Xylanase inhibition activity was measured using the dinitro-
salicylic acid (DNS) assay [29] in McIlvaine’s buffer
(pH 5.5) at 30 °C for varying times depending on the
enzyme used. One unit of xylanase activity was defined as
the amount of protein that released 1 lmol xylose per min
at 30 °C and pH 5.5. Enzyme sample (2–4 lL) was added

to 10 mgÆmL
)1
low viscosity arabinoxylan (145 lL) dis-
solved in McIlvaine’s buffer pH 5.5. The reaction was ter-
minated by the addition of (DNS) reagent (150 lL), and
the samples were boiled for 5 min. After centrifugation at
13000 g for 5 min, the supernatant (200 lL) was transferred
to a microtitre plate and the absorbance at 550 nm meas-
ured relative to a xylose standard curve (0–180 lgÆmL
)1
).
For determination of the inhibition parameters, the activ-
ity of the enzymes was measured at 30 °C, in McIlvaine’s
buffer pH 5.5 using low viscosity arabinoxylan substrate.
The E : I
50
value corresponded to the molar ratio of enzyme–
inhibitor required to inhibit xylanase activity by 50%. The
inhibitor was preincubated with substrate for 10 min at
30 °C. The reaction was initiated by the addition of the
enzymes: A. niger xylanase (53 pmol), XYNC (30 pmol) and
T. longibrachiatum M3 xylanase (40 pmol) and carried out at
30 °C for 10 min for A. niger xylanase or for 5 min for
XYNC and T. longibrachiatum M3 xylanases. The E : I
50
was calculated with the sigma plot program.
The chitinase activity assay was performed at two differ-
ent pH using McIlvaine buffer pH 5.5 or 100 mm Tris ⁄ HCl
pH 8.0 and with two different size substrates. The assay
using insoluble chitin azure was performed as previously

described [6]. 4-Nitrophenyl b-d-N,N¢,N¢¢-triacetylchitotri-
ose [p-nitrophenol-(GlcNAc)
3
] was used following the
method previously described [30]. Briefly, the purified pro-
teins were incubated at 37 °C with 5 lL of substrate
(3 mgÆmL
)1
dissolved in sterile demineralized water) and
the volume adjusted to 70 lL with buffer. After 2.5 h of
incubation, the reaction was terminated by adding 0.4 m
Na
2
CO
3
(50 lL) and the absorbance was measured at
410 nm. The amount of p-nitrophenol released was deter-
mined from a standard curve.
Protein assays and protein sequencing
Total protein concentration was calculated using an extinc-
tion coefficient at 280 nm of 73090 m
)1
Æcm
)1
for recombin-
ant XIP-I and 62990 m
)1
Æcm
)1
for recombinant pRIXI

based on the amino acid composition derived from the pri-
mary structure ( />N-Terminal sequencing was performed at the Protein and
Nucleic Acid Chemistry Facility, University of Cambridge
using an ABI 491 Procise sequencer.
Electrospray mass spectrometry (ESI-MS)
Electrospray mass spectra were performed at the Depart-
ment of Chemistry, University of Cambridge, on an ABI
QSTAR pulsar mass spectrometer (Applied Biosystems)
equipped with a nanospray ion source. The sample (10 lm)
was put into the nanospray needle and 1000 V was applied
to start spraying. The declustering potential was 30. The
scans were summed and the raw data was analysed using
the instrument’s analyst software.
Gel electrophoresis and immunoblotting
SDS ⁄ PAGE was performed using 12% homogeneous
Novex Tris ⁄ Glycine gels (Invitrogen) according to the
manufacturer’s instructions using Mark12 unstained stand-
ard as markers (Invitrogen). The samples were reduced with
dithiothreitol and boiled before loading on the gels.
For immunodetection, proteins were transferred onto
nitrocellulose membrane by semidry blotting (Bio-Rad).
The blots were probed with 1 : 5000 dilution of the anti-
[His(C-term)-HRP] Ig, after the washing steps they were
A. Durand et al. A novel GH18 xylanase inhibitor identified in rice
FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1753
developed using enhanced chemiluminescent detection rea-
gents (ECL Plus Detection Kit, Amersham Pharmacia Bio-
tech, Uppsala, Sweden). The blots were probed with a
1 : 5000 dilution of polyclonal antiserum raised in rabbits
against XIP-I purified from wheat [14]. Immunoreactive

proteins were visualized using a horseradish peroxidase
anti-rabbit secondary Ig (Sigma, 1 : 2000) together with the
chemiluminescent detection as above.
Isoelectric focusing gels were run using the Bio-Rad sys-
tem and performed using the Novex IEF gel from Invitro-
gen following the instructions manual.
Electrophoretic titration
Titration curves of inhibitors alone or in complex with dif-
ferent xylanases were performed using the Phast system
(Amersham Pharmacia Biotech, Uppsala, Sweden) as descri-
bed previously [3,31]. Prior to loading, inhibitors (1–2 lL)
were preincubated with xylanases (0.5–1.8 lL) in McIlva-
ine’s buffer pH 5.5 for 10 min at room temperature in a
total volume of 4 lL (E : I molar ratio, 1 : 1 using 116 pmol
of each protein).
Surface plasmon resonance
BIAcore X system, Hepes-buffered saline buffer [10 mm
Hepes (pH 7.4) ⁄ 0.15 m NaCl ⁄ 3.4 mm of EDTA ⁄ 0.005% of
surfactant P20], CM5 sensor chips and amine coupling kit
were from BIAcore AB (Uppsala, Sweden). RIXI (1 lm)
and rXIP-I (0.9 lm )in10mm sodium acetate buffer
(pH 5.5) were immobilized by the amine coupling method
[32] at a flow rate of 10 lLÆmin
)1
, using Hepes-buffered
saline as running buffer. Briefly, equal volumes of N-hy-
droxysuccinimide (0.06 m in water) and N-ethyl-N¢-(3- di-
ethylaminopropyl)carbodiimide (0.2 m in water) were mixed
and injected on to a CM5 sensor chip to activate the carb-
oxymethylated dextran surface. The volume used was adjus-

ted to achieve immobilization levels of inhibitors giving
2000–3000 resonance units (RU); 1 RU is defined as 1 pg
of bound protein per mm
2
. After injection of inhibitor
(40 lL), the residual N-hydroxysuccinimide esters were
deactivated by the injection of 35 lL of ethanolamine (1 m,
pH 8.5). Flow cell 2 was used to immobilize inhibitors, and
control flow cell 1 was treated identically but without inhib-
itor. XYNC (80 lL, 14 lm)in10mm sodium acetate
(pH 5.5) was injected at a flow rate of 50 lLÆmin
)1
, using
10 mm sodium acetate (pH 5.5) as running buffer.
Acknowledgements
We thank Dr Takuji Sasaki (Japan) for the kind gift of
the cDNA clone of the putative rice class III chitinase,
Dr Seung-Moon Park (Korea) for providing the Pichia
pastoris strain expressing the basic active chitinase and
Dr Caroline Furniss, Dr Tariq Tahir, Dr Kurt Gebr-
uers, Professor Harry Gilbert, Dr Paloma Manzanares
and Dr Luc Saulnier for providing xylanases.
This study has been carried out with the financial sup-
port from the Commission of the European Communi-
ties, under the specific programme for RTD and
Demonstration on ‘Quality of Life and management of
living resources’, Key Action 1-Food, Nutrition and
Health, Contract: QLK1-2000–00811 GEMINI ‘Solving
the problem of glycosidase inhibitors in food
processing’.

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