Báo cáo khoa học: Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes pdf

Crystal structure and enzymatic properties of a bacterial
family 19 chitinase reveal differences from plant enzymes
Ingunn A. Hoell
1
, Bjørn Dalhus
2
, Ellinor B. Heggset
1
, Stein I. Aspmo
1
and Vincent G. H. Eijsink
1
1 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, A
˚
s, Norway
2 Institute of Medical Microbiology, Section for Molecular Biology, National University Hospital, Oslo, Norway
Chitinases (EC 3.2.1.14) are glycoside hydrolases that
catalyze the hydrolysis of chitin, a carbohydrate poly-
mer of 1,4-b-linked GlcNAc. Chitin is found in the
cuticle of insect shells, in shells of crustaceans, and in
the cell walls of many fungi, making chitin the second
most abundant polysaccharide in nature after cellulose
[1,2]. Chitinases are present in a wide variety of organ-
isms, such as bacteria, viruses, higher plants and
animals [1–4]. The hydrolysis products of chitin,
chitooligosaccharides, are of interest in several biologi-
cal and biotechnological processes [1,2].
Glycoside hydrolases are divided into different fam-
ilies based on primary sequence, three-dimensional
structure, and catalytic mechanism [5,6]. Family 18
and family 19 glycoside hydrolases both contain chitin-

ases. Members of the two families have very different
three-dimensional structures and use different catalytic
mechanisms. The catalytic domains of family 18 chitin-
ases have a (b ⁄ a)
8
fold [6] and use a substrate-assisted
double-displacement mechanism, which leads to retent-
ion of the configuration of the anomeric carbon [7,8].
The catalytic domains of family 19 chitinases have
Keywords
ChiG; chitinase; family 19; Streptomyces
coelicolor A3(2); subsite structure
Correspondence
V. G. H. Eijsink, Department of Chemistry,
Biotechnology and Food Science,
Norwegian University of Life Sciences,
PO Box 5003, 1432 A
˚
s, Norway
Fax: +47 64965901
Tel: +47 64965892
E-mail:
(Received 6 June 2006, revised 1 September
2006, accepted 4 September 2006)
doi:10.1111/j.1742-4658.2006.05487.x
We describe the cloning, overexpression, purification, characterization and
crystal structure of chitinase G, a single-domain family 19 chitinase from
the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chi-
tinase G was not capable of releasing 4-methylumbelliferyl from artificial
chitooligosaccharide substrates, it was capable of degrading longer chito-

oligosaccharides at rates similar to those observed for other chitinases. The
enzyme was also capable of degrading a colored colloidal chitin substrate
(carboxymethyl-chitin–remazol–brilliant violet) and a small, presumably
amorphous, subfraction of a-chitin and b-chitin, but was not capable of
degrading crystalline chitin completely. The crystal structures of chitinase
G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M,
Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006)
J Mol Biol 358, 472–484], showed that these bacterial family 19 chitinases
lack several loops that extend the substrate-binding grooves in family 19
chitinases from plants. In accordance with these structural features,
detailed analysis of the degradation of chitooligosaccharides by chitinase G
showed that the enzyme has only four subsites () 2 to + 2), as opposed to
six () 3 to + 3) for plant enzymes. The most prominent structural differ-
ence leading to reduced size of the substrate-binding groove is the deletion
of a 13-residue loop between the two putatively catalytic glutamates. The
importance of these two residues for catalysis was confirmed by a site-
directed mutagenesis study.
Abbreviations
ChiC, chitinase C from Streptomyces griseus HUT6037; ChiF, chitinase F from Streptomyces coelicolor A3(2); ChiG, chitinase G from
Streptomyces coelicolor A3(2); CM-chitin RBV, carboxymethyl-chitin–remazol–brilliant violet; 4-MU, 4-methylumbelliferyl; TEV-protease,
tobacco etch virus NIa protease.
FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4889
high a-helical contents and share some structural simi-
larity with chitosanases and lysozyme [9,10]. Family 19
chitinases use a single-displacement mechanism, which
leads to inversion of the configuration of the anomeric
carbon [6,11]. The catalytic mechanism of family 19
chitinases has been studied in detail by modeling [12],
but experimental studies that underpin the proposed
mechanism are remarkably scarce [13]. In fact, in the

CAZY database of glycoside hydrolases [5] (http://
afmb.cnrs-mrs.fr/CAZY/), the catalytic proton donor
and the catalytic base are not annotated.
Family 19 chitinases are commonly found in many
plants, but were only recently discovered in bacteria.
The first bacterial family 19 chitinase, chitinase C
(ChiC), was found in Streptomyces griseus HUT6037
in 1996 [14]. Subsequently, several bacterial family 19
chitinases have been identified, including chitinases
from Burkholderia gladioli, Vibrio cholerae, Haemophi-
lus influenzae, and Pseudomonas aeruginosa. Although
many plant family 19 chitinases are known, crystal
structures are available for only two of these [9,15–17].
The first structure of a bacterial family 19 chitinase
has just recently been solved (Streptomyces griseus
HUT6037 ChiC; PDB accession code 1WVU) [18].
Streptomyces coelicolor A3(2) is a spore-forming
soil-borne Gram-positive bacterium that grows via a
branching mycelium, mainly by tip growth [19]. The
genome was fully sequenced in 2002, and this revealed
that the ability of S coelicolor A3(2) to exploit nutri-
ents in the soil is associated with the ability to produce
many different hydrolases, including 13 chitinases [20].
Among these chitinases we find two putative family 19
enzymes, ChiF and ChiG [21,22], which share 84%
identity to each other, and 80% and 75% identity to
the catalytic domain of ChiC from Streptomyces gri-
seus, respectively [21]. ChiF has a similar domain
structure to ChiC, consisting of a catalytic domain
and an N-terminal chitin-binding domain. ChiG, on

the other hand, lacks this chitin-binding domain, and
consists only of a catalytic domain [22]. The chiG gene
encodes a 244 amino acid chitinase, including a 29
amino acid leader peptide sequence. The closest relat-
ive of ChiG among the two plant family 19 chitinases
with known crystal structures comes from Canavalia
ensiformis (Jack beans; 37% sequence identity). Inter-
estingly, ChiG and most other bacterial chitinases
seem to have different catalytic centers from the plant
enzymes, as there is a 13-residue deletion between the
putative catalytic residues, thus making them closer in
sequence in the former (Glu68 and Glu77 in ChiG;
Fig. 1). Inspection of the structures of the two plant
enzymes shows that this deletion is located on a loop
near the (putative) + 2 subsite [9,12]. Sequence align-
ments (Fig. 1) show several other deletions in the bac-
terial enzymes that potentially could affect interactions
with the substrate. In order to provide more insight
into family 19 chitinases in general and into the differ-
ences between plant and bacterial enzymes in partic-
ular, we have overexpressed ChiG from S. coelicolor
A3 (2) in Escherichia coli and characterized the enzyme
with respect to its catalytic properties and crystal
structure. The crystal structures of ChiG and ChiC
[18] permitted structural comparison between bacterial
and plant enzymes, which provided a structural
explanation for observed differences in enzymatic
properties. The role of the putative catalytic residues
was confirmed by site-directed mutagenesis.
Results

Enzymology
Overexpression of ChiG in E. coli BL21Star (DE3)
yielded soluble and active enzyme, which could easily
be purified by Ni-affinity chromatography (supple-
mentary Fig. S1; typical yields were in the range
2–7 mg of ChiG per liter of culture). Tests with several
substrates showed that removal of the His tag by
tobacco etch virus NIa protease (TEV-protease) did
not affect the catalytic properties of the enzyme. ChiG
did not show any activity against 4-methylumbelliferyl
(4-MU)-(GlcNAc)
2
or 4-MU-(GlcNAc)
3
, as also earlier
demonstrated by Saito et al. [21]. On the other hand,
ChiG showed activity against a-chitin and b-chitin
(supplementary Fig. S2), chitooligosaccharides (Figs 2
and 3), carboxymethyl-chitin–remazol–brilliant violet
(CM-chitin RBV) and also against chitosan (E. Hegg-
set, I. A. Hoell & K. M. Va
˚
rum, unpublished results).
Figure 2 shows the kinetics of product formation
during oligosaccharide degradation. Specific activities
(derived from initial substrate disappearance rates,
Fig. 2) were 1.13 ± 0.07 lmolÆs
)1
Æmg
)1

, 0.63 ± 0.03
lmolÆs
)1
Æmg
)1
, 0.49 ± 0.05 lmolÆs
)1
Æmg
)1
, and 0.08 ±
0.01 lmolÆs
)1
Æmg
)1
for (GlcNAc)
6
, (GlcNAc)
5
, (Glc-
NAc)
4
and (GlcNAc)
3
, respectively. Degradation of
both a-chitin and b-chitin yielded (GlcNAc)
2
and (Glc-
NAc)
3
, whereas after long incubation times (24 h)

significant amounts of GlcNAc were observed, due
to further degradation of (GlcNAc)
3
to (GlcNAc)
2
and GlcNAc (results not shown). The specific initial
activities towards a-chitin and b-chitin (judged from
a short initial linear phase in product formation)
were 0.09 ± 0.01 lmolÆs
)1
Æmg
)1
and 0.11 ± 0.03
lmolÆs
)1
Æmg
)1
, respectively. However, only a minor
fraction of the chitin was degraded at these speeds; the
reactions rapidly slowed down and a larger part of
Bacterial family 19 chitinase I. A. Hoell et al.
4890 FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS
the substrates remained undegraded, even after pro-
longed incubation. Thus, ChiG is much less effective
than e.g. the two-domain family 18 chitinase ChiA
from S. marcescens which can degrade b-chitin com-
pletely (supplementary material Fig. S2). Yields were
lowest for a-chitin, whereas ChiA is capable of almost
completely degrading this substrate too, albeit at a
slower rate [23].

Figure 2 shows that the tetramer is exclusively con-
verted to two dimers, meaning that there is only one
binding mode for this substrate. The longer oligomeric
substrates have several potentially productive binding
modes. Preferred binding modes can be analyzed by
determining anomeric ratios of products formed early
during the reaction [24–26]. The results (Fig. 3,
Table 1) show that during degradation substrates
stayed close to the expected equilibrium ratio of 60%
a-anomer and 40% b-anomer, whereas all products
had anomer ratios that were close to 80 : 20. One
would expect an 80 : 20 ratio: (a) if the enzyme is
inverting (that is, each new reducing end has an a-ano-
meric configuration, as would be expected for a family
19 enzyme [11]); and (2) if each product contains a
50 : 50 mixture of newly formed (100% a) and existing
(60% a) reducing ends. Figure 2A shows that (Glc-
NAc)
6
was hydrolyzed to (GlcNAc)
4
+ (GlcNAc)
2
and (GlcNAc)
3
+ (GlcNAc)
3
. The efficiency of the
first reaction was about double that of the second reac-
tion (see legend to Fig. 2). The 80 : 20 anomeric distri-

bution in the tetramer and dimer fractions (Fig. 3,
Table 1) shows that the first reaction equally often
results from cleavage between sugars 4 and 5 (new
reducing end on the tetramer product) as from clea-
vage between sugars 2 and 3 (new reducing end on the
dimer product). Thus, the hexamer is degraded
through three types of productive binding modes with
approximately similar frequencies, leading to cleavage
after sugars 2, 3 or 4. Hydrolysis of (GlcNAc)
5
initially
Fig. 1. Structure-based multiple sequence alignment of all family 19 chitinases with known structure. The figure shows two plant enzymes,
from Hordeum vulgare (barley [9]) and Canavalia ensiformis (Jack bean [17]), and two bacterial enzymes, chitinase C (ChiC) (PDB accession
code 1WVU) from Streptomyces griseus HUT 6037 (catalytic domain only [18]) and chitinase G (ChiG). The alignment was made using the
protein structure comparison service SSM at the European Bioinformatics Institute ( [45]). Four major dele-
tions in the bacterial enzymes are indicated by A, B, C and C-term, and the 161–166 loop (numbering of the barley enzyme; see text) is indi-
cated by dots above the sequence. Residues involved in disulfide bridge formation are marked with closed or open bullets, for conserved
and nonconserved bridges, respectively. The closed triangles indicate two conserved glutamate residues involved in catalysis. Fully con-
served secondary structure assignments are indicated with h for a-helix and s for b-strand. The consensus helix comprising residues 169–
177 in ChiG is extended towards Cys183 in the other three enzymes.
I. A. Hoell et al. Bacterial family 19 chitinase
FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4891
yielded equal amounts of (GlcNAc)
3
and (GlcNAc)
2
;
the 80 : 20 anomeric ratio of the products indicates
that cleavage after sugar 2 or sugar 3 occurs approxi-
mately equally often.

Structure
The overall structure of ChiG (Fig. 4, supplementary
Fig. S3) is similar to that of the family 19 chitinase
from barley, the best studied of the plant chitinases
[9,12,13,27,28] and essentially identical to that of the
catalytic domain of ChiC from S. griseus HUT6037
([18]; rms 0.84 A
˚
). The only notable difference between
the two bacterial enzymes occurs in the 178–183
region: the consensus helix comprising residues 169–
177 in ChiG (Fig. 1) is extended towards Cys183 in
ChiC (and in the plant enzymes).
Compared to the barley enzyme, ChiC and ChiG
lack three loops (A, B and C) and a C-terminal exten-
sion (Figs 1 and 4B). In addition, one other loop,
comprising residues 161–166 in the barley enzyme, has
shifted its position by up to 9 A
˚
(Fig. 4B). Two of the
three disulfide bridges found in the plant enzymes
are conserved (in ChiG: Cys87–Cys95 and Cys183–
Cys215), whereas the third bridge is lacking, due to
the deletion of the A-loop (Fig. 1). The enzyme has a
deep groove that is likely to bind the substrate [6,12]
and that contains the putative catalytic residues Glu68
and Glu77 (Figs 1 and 4). For an inverting enzyme,
one would expect the distance between the carboxyl
oxygens of these two glutamates to be about 10 A
˚

[6].
In ChiG, this distance is 9.5 A
˚
for the closest pair of
oxygens.
The four major deletions in ChiG (loops A, B and
C and the C-terminus) as well as the one major struc-
tural difference (161–166 loop) compared to the plant
enzymes can be divided into two subsets of interrelated
changes, with each subset affecting one side of the sub-
strate-binding groove of the enzyme (Fig. 4B,C). On
the side where the nonreducing end of the substrate
Fig. 2. Time course of the degradation of chitooligosaccharides by chitinase G (ChiG). (A) Hexamer. (B) Pentamer. (C) Tetramer. (D) Trimer.
The concentrations of the various oligosaccharides are indicated by
(hexamer), } (pentamer), h (tetramer), n (trimer), X (dimer) and s
(monomer). All reactions were run under identical conditions, except for the reaction with trimer, in which the enzyme concentration was
increased 50-fold. In (A), note that a single cleavage can produce two trimers or one dimer + one tetramer; the graph thus shows that the
reaction producing tetramer + dimer happens about twice as often as the trimer-producing reaction. In the reactions depicted in (A) and (B),
monomers were only detected after prolonged incubation, i.e. after depletion of the original substrate. With the tetramer (C), monomers
were never detected.
Bacterial family 19 chitinase I. A. Hoell et al.
4892 FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS
binds, loop C and the C-terminal extension affect the
position of the 161–166 loop, which contains two polar
side chains (Gln162 and Lys165) thought to be import-
ant for sugar binding in subsites –3 and –4 (in the
barley enzyme [12,27]). In ChiG, in the absence of the
C-loop and the C-terminal extension, the 161–166 loop
has moved toward the catalytic center. In addition, the
Gln and Lys residues have been replaced by Thr

(Thr153 and Thr157). Thus, ChiG seems to have a
reduced ability to bind sugars in the ) 3 and ) 4 posi-
tions.
On the side where the reducing end of the substrate
binds, the interacting loops A and B in the barley
enzyme extend the substrate-binding surface beyond
subsite + 2, primarily through the exposed Trp72. The
importance of tryptophans in positions such as Trp72
for the efficiency of chitinolytic enzymes is well estab-
lished [29]. There is another Trp at position 82 in loop
B, which is shielded from solvent in the barley enzyme
(Fig. 4C), but which is more exposed in the absence of
Trp72, as in the jack bean enzyme. ChiG lacks loops
A and B, and thus seems to have reduced ability to
bind sugars beyond subsite + 2. In addition, Thr69,
thought to be important for sugar binding in subsite
+ 2 of the barley enzyme, is not conserved and is
replaced by Gly in ChiG.
No structural data were obtained for the 11 N-ter-
minal residues of ChiG. Compared to the barley and
the jack bean enzymes, ChiG contains an N-terminal
extension of eight and seven residues respectively
(Fig. 1). The N-termini of the plant enzymes and the
first residue in the ChiG structure (Phe12) (Fig. 4A),
are located on the opposite side of the enzyme to the
catalytic center and the substrate-binding groove. The
same applies to the structurally observed N-terminus
of the catalytic domain of ChiC, which corresponds to
residue 8 in ChiG [18]. In ChiC, this N-terminus is
part of a linker (with unknown structure) that connects

an N-terminal chitin-binding domain to the catalytic
domain. All in all, it is highly unlikely that the N-ter-
minal extensions in ChiG directly affect catalytic archi-
tecture.
Mutagenesis of the catalytic center
Figure 1 shows that two glutamates thought to make
up the catalytic center in the barley family 19 chitinase
[9,13] are conserved in ChiG, despite the large deletion
in between these two residues. The role of these gluta-
mates was demonstrated by site-directed mutagenesis.
Table 2 shows that all mutants had greatly reduced
catalytic activity. Some detectable activity was still left
upon mutation of Glu77 (2000–6000-fold reduction in
activity), whereas mutation of Glu68 reduced activity
to below the level that could be detected with our
assays (> 24 000-fold reduction in activity).
Discussion
Whereas family 19 chitinases are widespread in higher
plants, their occurrence in prokaryotes has only
recently been discovered [14,22,26,30]. Judged by avail-
Fig. 3. HPLC analysis of reaction mixtures under conditions pre-
venting anomeric equilibrium. The top panel represents a standard
mixture of GlcNAc oligomers showing the standard 60 : 40 ratio
between the a-anomer and the b-anomer at equilibrium. The other
panels show the results of partial hydrolysis of (GlcNAc)
6
and (Glc-
NAc)
5
by chitinase G (ChiG). In these panels, substrates display the

60 : 40 ratio, whereas the ratios for the products are close to
80 : 20. See text and Table 1 for details. The small peaks close to
the tetramer position in the chromatogram for (GlcNAc)
5
and close
to the pentamer position in the chromatogram for (GlcNAc)
6
were
also present in control samples and are not due to enzyme action.
Table 1. Anomeric configuration in the reaction mixtures depicted
in Fig. 3.
Hydrolysis of (GlcNAc)
6
Hydrolysis of (GlcNAc)
5
a-Anomer
(%)
b-Anomer
(%)
a-Anomer
(%)
b-Anomer
(%)
Products
(GlcNAc)
2
78 22 81 19
(GlcNAc)
3
79 21 79 21

(GlcNAc)
4
80 20 – –
Substrate 62 38 62 38
I. A. Hoell et al. Bacterial family 19 chitinase
FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4893
able sequences, some bacterial family 19 chitinases
have catalytic domains that are at least as large as
those of the plant enzymes and that may contain at
least six subsites [26,30]. However, the catalytic
domains of ChiG, ChiC and most other known bacter-
ial family 19 chitinases are smaller than those of the
plant enzymes. Unfortunately, there is no direct struc-
tural information concerning the interaction between
A
B
C
Fig. 4. Structure of chitinase G (ChiG) and comparison with the barley chitinase. (A) Cartoon showing the overall fold of ChiG with trans-
parent surface. The side chains of the catalytic residues, Glu68 and Glu77 are shown in red. (B) Structural superposition of ChiG and the
barley enzyme. The picture shows a cartoon of the barley enzyme (PDB accession code 2bba; cyan) and the surface of ChiG, with the
view being rotated 90° relative to (A) (the view is into the substrate-binding groove). Important structural elements are labeled (see text
for details), and the catalytic glutamates are shown in red. (C) Differences between the barley enzyme (cyan, left) and ChiG (blue, right)
in the substrate-binding cleft. The side chains of the catalytic residues are shown in green. The side chains of residues that are deleted
(Trp72, Trp82), mutated (Thr69 ⁄ Gly70) or mutated and relocated (Lys165 ⁄ Thr157 and Gln162 ⁄ Thr153) in ChiG are shown in red. The side
chains of four fully conserved residues in subsites ) 2 (right side) to + 2 (left side) are shown in purple. Note that the ) 1 and ) 2 sub-
sites partly consist of backbone atoms [12]; these are structurally well conserved, but not shown in the picture. The pictures were made
with
PYMOL [47].
Bacterial family 19 chitinase I. A. Hoell et al.
4894 FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS

family 19 chitinases and their substrates. Soaking
experiments were not successful and nor were cocrys-
tallization experiments with the inactive mutant E68Q
[9] (B. Dalhus, S. I. Aspmo & I. A. Hoell, unpublished
results). However, the interaction between the barley
chitinase and (GlcNAc)
6
has been studied in great
detail by computational techniques, exploiting the (lim-
ited) structural similarity between family 19 chitinases
and lysozyme [9,12,27,31]; (the crystal structure of a
lysozyme–(GlcNAc)
3
complex was used for modeling
purposes). By analogy to lysozyme, these studies
assumed the presence of six subsites, running from ) 4
to + 2 [subsites are numbered according to standard
nomenclature; cleavage occurs between the sugar units
bound in subsites ) 1 and + 1 [32]; (note that in the
older literature, these subsites are referred to as A
() 4) to F (+ 2)]. Judging by the structure of the bar-
ley enzyme, one would assume that there is affinity
for the substrate beyond the + 2 subsite, primarily
because of the prominent Trp residue at position 72,
approximately 15 A
˚
from the catalytic center. This Trp
would be able to interact with sugars bound at posi-
tions + 3 and + 4. Indeed, analysis of the hydrolysis
of (GlcNAc)

6
by barley chitinase [28] and by a highly
similar chitinase from rice [25] led to the conclusion
that these enzymes do have a + 3 subsite with consid-
erable affinity for a sugar moiety.
All residues thought to be involved in sugar binding
at the ) 2 to + 2 subsites in the barley enzyme are
fully or, at least functionally, conserved in ChiG
(Fig. 4C), except for Thr69 in the + 2 subsite, which is
replaced by a glycine. Beyond this central region,
ChiG clearly differs from the barley enzyme, as a
consequence of the loop deletions and the resulting
conformational change in the 161–166 loop. The dele-
tion of the Trp72-containing loop (loop B in Fig. 4B)
removes putative subsites + 3 and + 4, whereas the
conformational change of and the mutations in the
161–166 loop remove putative subsites ) 4 and ) 3
[12,27]. Thus, in ChiG, the substrate-binding
groove ⁄ surface is less extended and does not seem to
contain more than the four central subsites. The pres-
ence of only four subsites was confirmed by studies on
the degradation of pentamers and hexamers (Fig. 2,
Table 1). For example, productive binding of the hex-
amer by ChiG occurs in three different binding modes
(in ‘subsites’ ) 4 to +2, ) 3 to +3 and ) 2 to +4)
with almost identical frequencies. This shows that
there is little binding affinity in subsites beyond ) 2
and + 2. The barley and rice enzymes show clearly dif-
ferent product profiles, primarily due to the presence
of a + 3 subsite [25,28]. Most interestingly, whereas

ChiG hydrolyzed tetrameric, pentameric and hexamer-
ic substrates with rather similar rates (varying less than
2.5-fold), the efficiency of the barley enzyme is strongly
dependent on substrate length. Studies by Hollis et al.
[27] showed that the barley enzyme degrades the hex-
amer about 200 times faster than the tetramer. This
confirms that the barley enzyme and ChiG have dif-
ferent catalytic properties, in accordance with the
observed structural differences. Using structural infor-
mation only, Kezuka et al. [18] have hypothesized that
ChiC has six subsites, namely subsites ) 4 to + 2, as in
hen egg-white lysozyme. This hypothesis is not con-
firmed by the present analysis of enzymatic properties
of ChiG, or by our analysis of the ChiG and ChiC
structures.
Despite the deletion of the B-loop, the two putative
catalytic residues Glu68 and Glu77 are structurally
well conserved between ChiG and the plant enzymes.
Andersen et al. [13] have previously shown that the
corresponding residues in the barley enzyme, Glu67
and Glu89, are essential for catalysis. The mutagenesis
studies presented here show that Glu68 and Glu77 are
essential for catalysis by ChiG. Mutation of Glu68 to
Gln resulted in total inactivation, whereas mutation of
Glu77 did not. This is in accordance with the notion
that Glu68 is the catalytic acid, whereas Glu77 is the
catalytic base [6,13].
The activity of ChiG towards chitooligosaccharides
was found to be comparable to that of other chitinas-
es, including, for example, well-known family 18

exochitinases and endochitinases from Serratia marces-
cens [23]. ChiG showed relatively high initial activity
towards chitin, but the overall ability to degrade the
polymer was limited, as compared to, for example
multidomain family 18 chitinases from S. marcescens
[33] (supplementary Fig. S2). Thus, while ChiG is
rather active towards soluble substrates and some
(amorphous) regions of insoluble chitin, the enzyme is
not very efficient in degrading crystalline polymeric
substrates. Other bacterial family 19 chitinases such as
Table 2. Specific activity of site-directed mutants of ChiG towards
(GlcNAc)
6
.
Enzyme
Specific activity
towards (GlcNAc)
6
(lmolÆmg
)1
Æs
)1
)
Relative
specific
activity
Wild-type ChiG 1.13 ± 0.07 1
E68Q Not detectable < 4.2 · 10
)5a
E68A Not detectable < 4.2 · 10

)5a
E77Q 0.00057 5.0 · 10
)4
E77A 0.00019 1.7 · 10
)4
a
Estimated on the basis of the approximate detection limit of the
assay.
I. A. Hoell et al. Bacterial family 19 chitinase
FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4895
ChiC and ChiF contain an additional substrate-bind-
ing domain, which could make these enzymes more
efficient with crystalline substrates. However, deletion
of this domain had only a modest effect on enzyme
efficiency with crystalline chitin [34]. Like ChiG, both
ChiC and ChiF display relatively high activities
towards noncrystalline chitin forms, and low activities
towards crystalline chitin [22,35]. It has previously
been shown that chitinases with high activity towards
crystalline chitin, such as Bacillus circulans chitinase
A1 and S. marcescens chitinase A, have extended sub-
strate-binding grooves (at least six subsites). Notably,
these grooves contain a stretch of linearly aligned aro-
matic residues that play an important role in guiding a
chitin chain from the crystalline chitin surface to the
catalytic center [29]. Our finding that the bacterial
enzymes have only four subsites and the absence of
aromatic residues in these subsites may explain why
ChiG and related enzymes have low activity towards
crystalline chitin. The open active site of ChiG sug-

gests that ChiG binds polymers in an endo-fashion.
This was confirmed by the observation that hydrolysis
of chitin led to significant production of trimers during
degradation of both a-chitin and b-chitin (exoenzymes
tend to almost exclusively produce dimers). Studies on
the degradation of colloidal chitin by ChiC led to a
similar conclusion [14].
Most probably, family 19 chitinases were transferred
from plants to bacteria by horizontal gene transfer [22].
In plants, family 19 chitinases are thought to form part
of a defense mechanism against chitin-containing fun-
gal pathogens [36]. The family 19 chitinases are thought
to attack the hyphal tips, which are believed to consist
of newly synthesized chitin that is not firmly crystal-
lized [35]. This is in accordance with the observation
that family 19 chitinases generally have relatively low
activities towards the more crystalline forms of chitin.
Only a few chitinolytic bacteria possess family 19
chitinases, and these also display antifungal activity
[22,35]. In bacteria, chitinases, primarily belonging to
family 18, are usually thought to be produced for the
exploitation of chitinous substrates as a source of nutri-
tion. Production of multiple enzymes with varying
properties (endo-action or exo-action, processive or
not, presence of additional substrate-binding domains,
preference for soluble or insoluble substrates) is benefi-
cial, because this enables the bacterium to use parallel
and potentially synergistic strategies during chitin
breakdown. Chitin occurs in a variety of forms and co-
polymeric structures [37], which may require different

chitinases for effective degradation. It remains to be
seen whether the two family 19 chitinases of S. coelicol-
or simply add to the bacterium’s enzymatic repertoire
for effective chitin turnover, or play a specific role in
some form of interaction with fungi.
Experimental procedures
DNA techniques
The chiG gene was amplified from genomic DNA (ATCC
BAA-471D) from S. coelicolor A3(2) with: primer Chi-
Gul_S.coeli-F, 5¢-GCATCGTCTCACATGGAGAAGTCC
GACACCCGGA-3¢ (BsmBI restriction site is in bold type);
and primer ChiG_S.coeli-R, 5¢-GCATGGTACCCTAAC
AGCTCAGGTT-3¢ (KpnI restriction site is in bold type).
PCR reactions were conducted with Phusion DNA polym-
erase (Finnzymes, Espoo, Finland) in a PTC-100 Program-
mable Thermal Cycler (MJ Research, Inc., Waltham, MA,
USA). The amplification protocol consisted of an initial
denaturation cycle of 30 s at 98 °C, followed by 30 cycles
of 10 s at 98 °C, 30 s at 58 °C, and 30 s at 72 °C, followed
by a final step of 10 min at 72 °C. Amplified fragments
were ligated into vector pCR
Ò
4Blunt-TOPO
Ò
Zero Blunt
TOPO (Invitrogen, Carlsbad, CA, USA). The gene frag-
ments were excised from the TOPO vectors for insertion in
an expression vector, using BsmBI and KpnI for cloning
into NcoI–KpnI-digested pETM11 vector (Gu
¨

nter Stier,
EMBL, Heidelberg, Germany). The pETM11 vector con-
tains an N-terminal His6 tag followed by a TEV-protease
cleavage site. The final constructs were transformed into
E. coli BL21Star (DE) (Invitrogen).
ChiG mutants (E68Q, E68A, E77Q and E77A) were
made with the QuickChange Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA, USA), essentially as described
by the manufacturer. DNA sequencing was performed
using a BigDye Terminator v3.1 Cycle Sequencing Kit
(Perkin Elmer ⁄ Applied Biosystems, Foster City, CA, USA)
and an ABI PRISM 3100 Genetic Analyser (Perkin
Elmer ⁄ Applied Biosystems).
Production and purification of recombinant
protein
One hundred and fifty milliliters of E. coli BL21Star
(DE3) transformants containing the pETM11–chiG con-
struct were grown at 37 °C in LB medium with
100 lgÆmL
)1
kanamycin at 225 r.p.m., to a cell density of
0.6 at 600 nm. Isopropyl- b -d-thiogalactopyranoside was
added to a final concentration of 0.4 mm, and the cells
were further incubated for 4 h at 30 °C, and harvested
by centrifugation (9 820 g, 8 min at 4 °C, Beckman Coul-
ter Avanti J-25, Rotor JA14). The cell pellet was lysed
by making a periplasmatic extract. First, the cell pellet
was resuspended in 15 mL of ice-cold spheroplast buffer
(10 mL of 1 m Tris ⁄ HCl, pH 8.0, 17.1 g of sucrose,
100 lL of 0.5 m EDTA, pH 8.0, and 200 lL of phenyl-

Bacterial family 19 chitinase I. A. Hoell et al.
4896 FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS
methanesulfonyl fluoride and incubated on ice for 5 min.
The cells were then harvested by centrifugation (7 741 g,
8 min at 4 °C, Beckman Coulter Avanti JA25-5), the
supernatant was removed, and the pellet was incubated
for 10 min at room temperature. The pellet was then re-
suspended in 12.5 mL of ice-cold, sterile water, and incu-
bated on ice for 45 s before supplementing with 625 lL
of 20 mm MgCl
2
. After centrifugation (7741 g, 8 min at
4 °C, Beckman Coulter Avanti JA25-5), the supernatant
was pressed through a 0.20 lm sterile filter, supplied with
20 lLof50mm phenylmethanesulfonyl fluoride per
10 mL of extract, and stored at 4 °C. It has previously
been shown that these extracts are good, relatively ‘clean’
starting points for purification of intracellularly produced
chitinases [38].
ChiG was purified on an Ni-NTA column (Qiagen,
Venlo, The Netherlands) using a flow rate of 2 mLÆmin
)1
.
The column was equilibrated in 100 mm Tris ⁄ HCl buffer
(pH 8.0), containing 20 mm imidazole. After the protein
sample was loaded, the column was washed with the start-
ing buffer. The His-tagged protein was then eluted with
100 mm Tris ⁄ HCl buffer (pH 8.0), containing 100 mm imi-
dazole. The purified protein was dialyzed against 20 mm
Tris ⁄ HCl (pH 8.0) and stored at 4 °C.

Removal of the (His)
6
tag was preformed by mixing
0.1 mg of (His)
6
–ChiG with 75 lLof10· TEV-protease
buffer (0.5 m Tris ⁄ HCl, pH 8.0, and 5 mm EDTA), 1 mm
dithiothreitol, 0.005 mg of TEV-protease and dH
2
Oupto
750 lL. This mixture was incubated at 37 °C for 3 h. After
incubation, the mixture was dialyzed against 100 mm
Tris ⁄ HCl (pH 8.0) and 20 mm imidazole overnight. The
dialyzed mixture was then applied onto an Ni-NTA col-
umn, as described above. The flow-through fraction, now
containing the ChiG protein with no (His)
6
tag, was dia-
lysed against 20 mm Tris ⁄ HCl (pH 8.0) and stored at 4 °C.
The protein produced via this procedure contains a three-
residue N-terminal extension (Gly-Ala-Met) compared to
the mature wild-type enzyme.
Structure determination and bioinformatics
High-quality diffracting crystals of ChiG were obtained
by the vapor diffusion method in hanging drops. Prior
to crystallization, ChiG was concentrated by using a
Centricon Plus-20 Centrifugal Filter Device as described
by the manufacturer (Millipore, Billerica, MA, USA) in
20 mm Tris ⁄ HCl (pH 8.0) to a final concentration of
10 mgÆmL

)1
. Equal volumes of the protein solution were
mixed with the reservoir solution containing 13% (w ⁄ v)
PEG8000 and 110 mm zinc acetate in 80 mm sodium
cacodylate buffer (pH 6.5), and equilibrated against the
reservoir solution at room temperature. Crystals, in the
shape of thin plates, grew to a final size of about
0.2 mm within a week. Crystals were mounted in nylon
loops and flash-frozen in liquid nitrogen following a
short (< 10 s) soak in mother liquor containing addi-
tional PEG400 to a final concentration of 15%.
Complete X-ray data were collected at beamline ID14-
EH3 at the ESRF in Grenoble, equipped with an ADSC
Q4R detector. Diffraction images were processed with mos-
flm [39] and scaled and merged with scala in CCP4
[40,41]. The crystals belong to space group P2
1
with cell
dimensions a ¼ 48.67 A
˚
, b ¼ 74.38 A
˚
, c ¼ 64.18 A
˚
and
b ¼ 108.6°, and diffracted to at least 1.5 A
˚
resolution.
Crystal data and data collection statistics are summarized
in Table 3.

Calculation of the Matthews coefficient suggested two
molecules in the asymmetric unit. The structure was solved
by molecular replacement using cns [42]. The search model
was a polyalanine chain comprising residues 90–294 of the
ChiC structure (1WVU). Two solutions in the cross-rota-
tion function were readily identified, and a subsequent
translation search gave the positions in the unit cell. Side
chains were progressively added, guided by information
Table 3. Crystal parameters, data collection and refinement statis-
tics for Streptomyces coelicolor chitinase G (ChiG).
Crystal parameters
Crystal dimensions (mm) 0.2 · 0.2 · 0.05
Space group P2
1
Unit cell dimensions a ¼ 48.67 A
˚
, b ¼ 74.38 A
˚
,
c ¼ 64.18 A
˚
, b ¼ 108.6°
Data collection
Source ⁄ beamline ESRF ⁄ ID14EH3
Wavelength (A
˚
) 0.980
Resolution (A
˚
) 74.3–1.50

u-range, Du (°) 180, 1.0
Temperature (K) 100
R
merge
(%)
a
7.6 (48.1)
No. of reflections 250 071 (36 192)
Unique reflections 68 373 (9865)
Multiplicity 3.7 (3.7)
Mean I ⁄ rI 13.2 (3.0)
Refinement
Resolution range (A
˚
) 74.3–1.50
Completeness (%)
b
98.5 (97.3)
R
work
(%) 18.5 (24.3)
R
free
(%)
c
20.7 (26.9)
rms deviation from ideal geometry
Bond lengths (A
˚
) 0.010

Bond angles (°) 1.1
Ramachandran distribution (%)
d
Most favorable regions 97.5
Allowed regions 2.0
Disallowed regions 0.5
a
Values in parentheses refer to the outermost shell of data (1.58–
1.50 A
˚
).
b
Values in parentheses refer to the outermost shell of
data (1.54–1.50 A
˚
).
c
Five per cent of data, randomly distributed
over the full resolution range, were flagged as belonging to the
R
free
cross-validation set, not used in the refinement.
d
According
to
COOT [43].
I. A. Hoell et al. Bacterial family 19 chitinase
FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS 4897
from both the r
A

-weighted 2F
o
–F
c
and F
o
–F
c
maps, during
several cycles of modeling using coot [43], following refine-
ment with refmac5 [44]. Water molecules were appended
using the ‘add water’ function of coot. Four peaks close to
His67 ⁄ Glu182 and Asp184 in molecule A and His67 ⁄
Glu182 and Asp137 in chain B, originally modeled as
waters, were replaced by zinc ions, based on the refined
B-values and residual peak heights in the F
o
–F
c
map. These
zinc ions originate from the crystallization buffer. The main
chain was readily traced from residue Phe12 all the way to
the C-terminal Cys215. A few side chains at the protein sur-
face are flexible, with no distinct conformation. Refinement
of ChiG with zero occupancy for residues 170–183 con-
firmed the (slightly) different conformation for ChiG in the
178–183 region, as judged by inspection of the difference
Fourier electron density map. The final model comprises
408 residues in two chains, four zinc ions and 324 water
molecules.

Coordinates and structure factors have been deposited in
the Protein Data Bank, accession code 2CJL.
To create the alignment of Fig. 1, structural superposition
with other family 19 chitinases was performed using the pro-
tein structure comparison service SSM at the European
Bioinformatics Institute ( />[45].
Enzymology
Protein concentrations were determined according to Brad-
ford with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercu-
les, CA, USA) with BSA as a standard.
Analyses of the specific activity against chitooligosaccha-
rides were performed in 100 lL reaction mixtures con-
taining 200 lm (GlcNAc)
3
, (GlcNAc)
4
, (GlcNAc)
5
,or
(GlcNAc)
6
(Sigma, St Louis, MO, USA), 0.1 mgÆmL
)1
BSA
and 0.25 nm purified ChiG in 50 mm sodium acetate buffer
(pH 6.0). In the case of the (GlcNAc)
3
substrate, the
enzyme concentration was 12.5 nm. In the case of ChiG
mutants, the enzyme concentration was varied between

250 nm and 500 nm (see below for details). All the reaction
mixtures were incubated at 37 °C for several hours, with
regular sampling. Sixty microliter samples of the reaction
mixture were transferred to new tubes containing 60 lLof
70% acetonitrile, to stop the reaction, and stored at
) 20 °C until they were analyzed by HPLC at room tem-
perature. All reactions were analyzed in triplicate.
HPLC analysis of 20 lL portions of the stored reaction
mixtures was performed on a Gilson HPLC System (Gil-
son, Inc., Middleton, WI, USA), equipped with a Tosoh
TSK-Gel amide-80 column (0.46 internal diameter · 25 cm)
(Tosoh Bioscience, Montgomeryville, PA, USA), and a 234
autoinjector (Gilson). The liquid phase consisted of 70%
(v ⁄ v) acetonitrile, the flow rate was 0.70 mLÆmin
)1
, and
eluted oligosaccharides were monitored by recording
absorption at 210 nm.
In cases where analysis of the anomeric configuration of
the newly formed degradation products was desirable, reac-
tions were performed with higher enzyme concentrations
(20 nm) and very short incubation times (approximately
15 s). To stabilize the anomeric ratio as fast as possible and
to avoid reaching the anomeric equilibrium, reactions were
stopped by freezing on liquid nitrogen and samples were
stored at ) 80 °C until analyzed. Ten microliter samples of
the reaction mixtures were injected with a Gilson 234 auto-
injector immediately after thawing (that is, samples were
not ‘stored’ in the autoinjector).
Analyses of the degradation of a-chitin and b-chitin were

conducted by incubating 100 lL solutions containing
1mgÆmL
)1
of b-chitin (squid pen b-chitin, 3 lm in size; Sei-
kagaku, Tokyo, Japan) or 1 mgÆmL
)1
a-chitin (crab-shell
a-chitin, Sigma) and 20 n m purified ChiG in 50 mm sodium
acetate buffer (pH 6.0) at 37 °C and 230 r.p.m. for periods
varying from 0 min (just after addition of enzyme) to 24 h.
Reactions were stopped by adding one volume of 70%
(v ⁄ v) acetonitrile, and samples were stored at ) 80 °C until
they were injected with a 234 autoinjector (Gilson).
Activity tests with a colloidal chitin substrate, CM-chitin
RBV (LOEWE Biochemica GmbH, Mu
¨
nchen), and with
4-MU-(GlcNAc)
2
(Sigma) or 4-MU-(GlcNAc)
3
(Sigma)
were performed as described earlier [46].
Acknowledgements
We thank Gu
¨
nter Stier, EMBL Heidelberg, for provi-
ding us with vector pETM11, Gustav Vaaje-Kolstad
for help with the chitin degradation experiments and
helpful discussions, and May Bente Brurberg and

Svein Horn for helpful discussions. The authors
acknowledge the beamline staff at ID14-EH3 for tech-
nical assistance, and the ESRF and the Norwegian
Research Council (project Sygor) for financial support.
This work was funded by a grant from the Norwegian
Research Council (no. 140 ⁄ 140497).
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Supplementary material
The following supplementary material is available
online:
Fig. S1. SDS ⁄ PAGE analysis of purified chitinase G
(ChiG).
Fig. S2. Degradation of b-chitin.
Fig. S3. Close-up view of the r
A
-weighted 2F
o
–F
c
electron density map in a representative part of the

structure of chitinase G (ChiG).
This material is available as part of the online article
from
Bacterial family 19 chitinase I. A. Hoell et al.
4900 FEBS Journal 273 (2006) 4889–4900 ª 2006 The Authors Journal compilation ª 2006 FEBS