From the Department of Basic and Applied Molecular
Biotechnology, Division of Food and Biological Science, Graduate
School of Agriculture, Kyoto University and the § Laboratory
of Quality Design of Exploitation, Division of Agronomy and
Horticultural Science, Graduate School of Agriculture, Kyoto
University, Uji, Kyoto 611-0011, Japan
Received for publication, August 8, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Xanthan lyase, a member of polysaccharide lyase
family 8, is a key enzyme for complete depolymerization of a bacterial
heteropolysaccharide, xanthan, in Bacillus sp. GL1. The
enzyme acts exolytically on the side chains of the polysaccharide. The
x-ray crystallographic structure of xanthan lyase was determined by the
multiple isomorphous replacement method. The crystal structures of
xanthan lyase and its complex with the product (pyruvylated mannose)
were refined at 2.3 and 2.4 Å resolution with final
R-factors of 17.5 and 16.9%, respectively. The refined
structure of the product-free enzyme comprises 752 amino acid residues,
248 water molecules, and one calcium ion. The enzyme consists of
N-terminal There is a large number of polysaccharide-degrading
enzymes.1 Generally, they can
be classified into two groups, hydrolases and lyases. The former
catalyze the hydrolysis reaction responsible for breaking glycosidic
bonds in polysaccharides. The properties of glycosyl hydrolases that
act on poly- and oligosaccharides have been well documented, and the
three-dimensional structures of many polysaccharide hydrolases, such as
amylases, chitinases, and cellulases, have already been reviewed
(1, 2).
As regards the second group, the lyases, it is known that they
recognize uronic acid residues in polysaccharides, catalyze the
To determine the structural and functional relationships exhibited by
polysaccharide lyases, we have recently been focusing on bacterial
heteropolysaccharide lyases (lyases for alginate (14), gellan (15), and
xanthan (16)) with either an endotypic or exotypic reaction mode and
with either a backbone or side chain type of cleavage site. We have
already determined the crystal structure of the endotype alginate lyase
from Sphingomonas sp. A1 (9).
Xanthan is an exopolysaccharide produced by the plant
pathogenic bacterium Xanthomonas campestris (17). This
exopolysaccharide consists of a main cellulosic chain with
trisaccharide side chains composed of one glucuronyl and two mannosyl
residues attached at the C-3 position of alternate glucosyl residues
(18) (Fig. 1A). The internal and terminal mannosyl residues
of the side chains have an O-acetyl group at the C-6
position and a pyruvate ketal at the C-4 and C-6 positions,
respectively, although the extents of acetylation and pyruvation vary
with the growth conditions and bacterial strain (19). Because the
polymer has the peculiar rheological properties of pseudoplasticity
(reversible decrease in viscosity with increase in shear rate), high
viscosity at low concentrations, and tolerance to a wide range of pH
and temperatures, it is widely utilized as a gelling and stabilizing
agent in the food, pharmaceutical, and oil industries (20).
Xanthan lyase produced by Bacillus sp. GL1 acts exolytically
on the side chains of xanthan and liberates pyruvylated mannose (PyrMan)2 through the
-helical and C-terminal
-sheet domains, which
constitute incomplete
5/
5-barrel and anti-parallel
-sheet structures, respectively. A deep cleft is located in the N-terminal
-helical domain facing the interface between the two domains. Although the overall structure of the enzyme
is basically the same as that of the family 8 lyases for hyaluronate
and chondroitin AC, significant differences were observed in the loop
structure over the cleft. The crystal structure of the xanthan lyase
complexed with pyruvylated mannose indicates that the sugar-binding
site is located in the deep cleft, where aromatic and positively
charged amino acid residues are involved in the binding. The
Arg313 and Tyr315 residues in the loop from the
N-terminal domain and the Arg612 residue in the loop from
the C-terminal domain directly bind to the pyruvate moiety of the
product through the formation of hydrogen bonds, thus determining the
substrate specificity of the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-elimination reaction, and produce unsaturated saccharides with C=C
double bonds at the nonreducing terminal uronate residues (Fig. 1).
These characteristics of lyases indicate that they share common
structural features determining their uronate recognition sites and
reaction modes (
-elimination reaction). Although structural analyses
of lyases for pectate (3-8), alginate (9), hyaluronate (10, 11), and
chondroitin (12, 13) have been made, there is little information
regarding the structural rules common to polysaccharide lyases.
-elimination reaction (Fig.
1A) (16). The enzyme is
synthesized as a precursor form (99 kDa) and is then converted into the
mature form (~75 kDa) through posttranslational excision of the
signal peptide (2 kDa) and C-terminal polypeptide (~22 kDa) (21). On
the basis of amino acid (aa) sequence similarity, the enzyme is
classified into polysaccharide lyase family 8,1 which
contains lyases for hyaluronate and chondroitin AC in addition to
xanthan lyase, although xanthan lyase does not act on hyaluronate and
chondroitin (21).
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Fig. 1.
Structures of polysaccharides and modes of
action of polysaccharide lyases. A, xanthan;
B, hyaluronate; C, chondroitin. Thick
and dotted-line arrows indicate the cleavage sites for
polysaccharide lyases and the degradation reactions of polysaccharides,
respectively.
Moreover, xanthan lyase is peculiar in that it acts on the side chains
of a polysaccharide and releases the nonreducing terminal saccharides
of the side chains, because almost all polysaccharide lyases (including
those for pectate, alginate, hyaluronate, chondroitin, and heparin)
endolytically cleave the glycosidic bonds in the main chains of
polysaccharides. Therefore, it is thought that the structural analysis
of xanthan lyase will contribute to clarification of the structural
features that determine the uronate recognition site, the
-elimination reaction, the reaction mode (endo/exo type), and the
cleavage site (main/side chain type).
In this study, the three-dimensional structures of xanthan lyase and
its complex with the product were determined by x-ray crystallography
at 2.3 and 2.4 Å resolution, respectively. We also identified the
active cleft of the enzyme and aa residues responsible for both the
recognition of the substrate and the catalytic reaction.
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EXPERIMENTAL PROCEDURES |
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Materials-- Pyruvylated xanthan (average molecular mass, 2 × 106; pyruvylation of the terminal mannosyl residue in the side chain, ~50%) was obtained from Kohjin Co., Tokyo, Japan. Polyethylene glycol 4000 was purchased from Nacalai Tesque, Kyoto, Japan. DEAE-Toyopearl 650 M and Super Q-Toyopearl 650C were from Tosoh Co., Tokyo, Japan. Bio-Gel P2 was from Bio-Rad. The restriction endonucleases and DNA-modifying enzymes were from Takara Shuzo Co., Kyoto, and Toyobo Co., Tokyo, respectively.
Assays for Enzyme and Protein-- Xanthan lyase was assayed as described previously (16). Briefly, the enzyme was incubated in 1 ml of a reaction mixture containing 0.05% xanthan and 50 mM sodium acetate buffer, pH 5.5, and then the activity was determined by monitoring the increase in absorbance at 235 nm. One unit of the enzyme activity was defined as the amount of enzyme required to produce an increase of 1.0 in absorbance at 235 nm/min. Protein was determined by the method of Lowry et al. (22) with bovine serum albumin as a standard or by measuring the absorbance at 280 nm, assuming that E280 = 2.06 corresponded to 1 mg/ml, as calculated from the aa sequence using ProtParam (www.expasy.org/tools/protparam.html).
Purification of Xanthan Lyase-- Unless otherwise specified, all operations were carried out at 0-4 °C. Cells of Escherichia coli strain BL21(DE3)pLysS harboring a plasmid (pET17b-XL4) (21) were grown in 6 liters of LB medium (1.5 liters/flask), collected by centrifugation at 6000 × g and 4 °C for 5 min, washed with 20 mM potassium phosphate buffer (KPB), pH 7.0, and then resuspended in the same buffer. The cells were disrupted ultrasonically (Insonator model 201M, Kubota, Tokyo, Japan) at 0 °C and 9 kHz for 20 min, and the clear solution obtained upon centrifugation at 15,000 × g and 4 °C for 20 min was used as the cell extract containing the precursor form (97 kDa) of the enzyme. The cell extract, after supplementation with 1 mM phenylmethylsulfonyl fluoride and 0.1 µM pepstatin A, was fractionated with ammonium sulfate. The precipitate (0-30% saturation) was collected by centrifugation at 15,000 × g and 4 °C for 20 min, dissolved in 20 mM KPB, pH 7.0, and then applied to a DEAE-Toyopearl 650 M column (2.6 × 15 cm) equilibrated with 20 mM KPB, pH 7.0. The enzyme was eluted with a linear gradient of NaCl (0-0.7 M) in 20 mM KPB, pH 7.0 (200 ml), with 2 ml fractions collected every 2 min. The active fractions, which were eluted with 0.4 M NaCl, were combined and dialyzed against 20 mM KPB, pH 7.0. The dialysate was used as the purified precursor form (97 kDa) of the enzyme. To convert the precursor (97 kDa) autocatalytically to the mature form (~75 kDa), the purified precursor was kept at 4 °C for 1 week. After confirmation of the conversion by SDS-PAGE (23), the enzyme solution was applied to a Super Q-Toyopearl 650C column (1.7 × 5 cm) equilibrated with 20 mM KPB, pH 7.0, and eluted with a linear gradient of NaCl (0 to 0.5 M) in 20 mM KPB, pH 7.0 (50 ml). 1-ml fractions were collected every minute. The active fractions, which were eluted with about 0.2 M NaCl, were combined and dialyzed against 20 mM Tris-HCl, pH 7.5, and the dialysate was used as the purified mature form (~75 kDa) of the enzyme.
Preparation of PyrMan-- Xanthan (0.5%) dissolved in 50 mM sodium acetate (pH 5.5) (200 ml) was treated with the purified xanthan lyase (10 mg). The solution was mixed with ethanol (400 ml) and then centrifuged at 15,000 × g and 4 °C for 20 min. The supernatant was concentrated to 1.5 ml through evaporation and then applied to a Bio-Gel P2 column (0.9 by 122 cm) equilibrated with distilled water. The sugar eluted was determined to be PyrMan by confirming the release of mannose and pyruvate on hydrolysis with trifluoroacetic acid, as described previously (16). The fractions containing PyrMan were collected, freeze-dried, and dissolved in distilled water. The purity and content of PyrMan were determined by TLC analysis as described previously (16).
Crystallization and X-ray Diffraction--
The mature form
(~75 kDa) of xanthan lyase was crystallized by the hanging-drop vapor
diffusion method. The solution for a crystallization drop was prepared
on a siliconized coverslip by mixing 3 µl of protein solution (7.18 mg of protein/ml) with 3 µl of mother liquor comprising 23%
polyethylene glycol 4000, 0.2 M ammonium formate, and 0.1 M sodium Bicine buffer, pH 9.0. The crystals were soaked in
several heavy atom derivative solutions comprising 2 mM
NaAuCl4, 0.2 mM AgNO3, 1 mM Ac2UO2, 2 mM
SmCl3, 1 mM GdCl3, 1 mM
HoCl3, and 1 mM CdCl2 for 1 or
2 h at 20 °C. Crystals were also soaked in a sugar solution
containing 75 mM PyrMan. All heavy atom and sugar solutions
were prepared with a modified mother liquor consisting of 23%
polyethylene glycol 4000, 0.2 M ammonium formate, and 0.1 M Tris-HCl buffer, pH 7.4. Diffraction data for the native
and derivative crystals were collected with a Bruker Hi-Star multiwire
area detector at 20 °C, using CuK radiation generated by a MAC
Science M18XHF rotating anode generator, and were processed with the
SADIE and SAINT software packages (Bruker, Karlsruhe, Germany) (Table
I).
Structure Determination and Refinement--
The crystal
structure of xanthan lyase was solved by the multiple isomorphous
replacement (m.i.r.) method. The major sites of heavy atoms were
determined by interpretation of the peaks in difference Patterson maps
obtained at 3.0 Å resolution. Additional heavy atom sites were
determined from the peaks in difference Fourier maps. Phase refinement
was performed with the program package PHASES (24). The results of
heavy atom refinement and phasing by m.i.r. at 3.0 Å resolution are
presented in Table II. The phase was improved greatly and the
figure-of-merit increased to 0.854 after solvent flattening with PHASES
(25). The model was built using the program TURBO-FRODO (AFMB-CNRS,
Marseille, France) on a Silicon Graphics Octane computer.
Simulated annealing refinement was carried out with this model using
50-2.3 Å resolution data obtained with CNS (26). The model was heated
to 2500 K and then slowly cooled to 300 K (time-step, 0.5 fs; decrease
in temperature, 25 K; number of steps at each temperature, 50), and then 150 cycles of Powell minimization were carried out.
Fo Fc and
2 Fo
Fc maps
were used to locate the correct model. Several rounds of positional and
B-factor refinement followed by manual model building were
carried out to improve the model by increasing the data to 2.3 Å resolution. Water molecules were incorporated where the difference in
density was more than 3.0
above the mean and the
2 Fo
Fc map
showed a density of more than 1.0
. The final R-factor
was 17.5% for 30,582 data points in the 50.0-2.3 Å resolution range
(83.9% completeness). The R-free value calculated for the
randomly separated 10% data was 24.0%.
A crystal soaked with PyrMan was isomorphous with the crystal used for
the native set. A Fo Fc map (contoured at 3.0
) at 2.4 Å resolution was obtained using the reflection data for the sugar-soaked
crystal, and the phase was calculated from the final model of
xanthan lyase.
The stereo quality of the model was assessed using the programs
PROCHECK (27) and WHAT-CHECK (28). Ribbon plots were prepared using the
programs MOLSCRIPT (29), BOBSCRIPT (30), RASTER3D (31), and GRASP (32).
The coordinates of lyases for hyaluronate and chondroitin AC were taken
from the RCSB Protein Data Bank (33). These molecular models were
superimposed by means of fitting the programs RIGID and TOP included in
TURBO-FRODO and CCP4, respectively.
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RESULTS AND DISCUSSION |
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Crystallization and Structure Determination-- The mature form (about 75 kDa) of xanthan lyase of Bacillus sp. GL1 was purified from recombinant E. coli cells harboring plasmid pET17b-XL4 (21). A crystal of xanthan lyase (0.3 × 0.2 × 0.05 mm) was obtained by the hanging-drop vapor diffusion method. The space group was determined to be P212121 (orthorhombic) with unit cell dimensions of a = 54.3 Å, b = 91.4 Å, and c = 160.7 Å; the solvent content was 50.2% assuming one molecule/asymmetric unit. The results of the x-ray data collection are summarized in Table I. The structure of the enzyme was determined by the m.i.r. method. Table II shows the refinement statistics for the heavy atoms at 3.0 Å resolution. The protein model was built after solvent flattening of the m.i.r. phase with the PHASES program (24), and the model was refined by means of simulated annealing and the restrained least-squares method using CNS (26), as shown in Table I.
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Quality of the Refined Model--
The refined model of xanthan
lyase comprises 752 aa residues, 248 water molecules, and one calcium
ion. The N- and C-terminal aa residues of the mature form produced from
the preproform through posttranslational processing were confirmed to
be Ser26 and Gly777, respectively, by electron
density mapping. All of the polypeptide chain sequences could be well
traced, and the electron density of the main and side chains was
generally very well defined in the 2 Fo Fc map. The final overall
R-factor for the refined model was 0.175, with 30,582 unique
reflections within the 50.0-2.3 Å resolution range. The final free
R-factor calculated with the randomly selected 10% data was
0.240. The final root-mean-square (r.m.s.) deviations from the standard
geometry were 0.0060 Å for bond lengths and 1.29o for bond
angles. Based on the theoretical curves in the plot calculated
according to Luzzati (34), the absolute positional error was estimated
to be close to 0.25 Å of 5.0-2.3 Å resolution. Judging from the
results of Ramachandran plot analysis, in which the stereochemical
correctness of the backbone structure is indicated by the (
,
)
torsion angles (35), most of the non-glycine residues (86.1%) lay
within the most favored regions, and the other residues (13.6%) fell
in the additional and generously allowed regions, except for the
Thr247 and Asp695 residues. The
Thr247 and Asp695 residues are in
-turns. In
particular, the latter turn, containing the Asp695 residue,
is similar to a
-hairpin consisting of four amino acid residues with
the conformation of
-
-
-
in a Ramachandran plot (36),
because the Ala694, Asp695, Leu696,
and Ile697 residues fell in or near the
,
,
, and
regions of the plot, respectively. Furthermore, there is one
cis-peptide between the Ala753 and Pro754 residues.
Overall Structure of Xanthan Lyase--
Figs.
2 and 3 depict a ribbon model of the
overall structure and topology of the secondary structure elements of
xanthan lyase, respectively. The enzyme
has approximate dimensions of 100 × 70 × 50 Å and is
composed of two globular domains (N- and C-terminal domains) that form
- and
-structures, respectively. The N-terminal domain comprises
the 352 aa residues from Ser26 to Asp377 and is
composed predominantly of 13
-helices, 10 of which form an
/
barrel structure. The C-terminal domain comprises the 389 aa residues
from Leu389 to Gly777 and one calcium ion and
consists of 30
-strands arranged in five anti-parallel
-sheets. A
peptide linker composed of the 11 aa residues from Asp378
to Asn388 connects the N- and C-terminal domains. In the
structure of xanthan lyase, 25.7% of all aa residues are in
-helices, 26.2% in
-strands, and the remaining 48.1% in turns
and coils.
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N-terminal -Helical Domain--
The N-terminal domain is
composed primarily of two short
-strands and 13
-helices. The
latter contribute to the formation of an
/
-barrel structure with
a deep cleft, which is considered to be an active site (Figs. 2 and
4A). The 13 major
-helices, numbered sequentially from HA1 to HA13 (HA1, aa residues 28-41; HA2,
51-69; HA3, 90-106; HA4, 118-131; HA5, 153-165; HA6, 173-184; HA7,
192-208; HA8, 212-221; HA9, 255-270; HA10, 284-291; HA11, 318-333;
HA12, 338-354; and HA13, 366-376), vary in length between 8 and 19 aa
residues and consist of 187 aa residues (Fig. 3). These
-helices are
arranged as follows: four (HA5, HA6, HA8, and HA13) with three turns;
seven (HA1, HA3, HA4, HA7, HA9, HA11, and HA12) with four turns; one
(HA2) with five turns; and one (HA10) with two turns.
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There are 12 loops (from LA1 to LA12) connecting an -helix to the
following
-helix in the N-terminal domain (Fig. 3). The loop (LA8)
between HA8 and HA9 includes two short
-strands (S1, aa residues
235-238, and S2, 243-245). Therefore, in the N-terminal domain,
52.3% of all aa residues are in
-helices, 2.0% in
-strands, and
the remaining 45.7% in turns and coils.
The N-terminal -helical domain includes an incomplete
5/
5-barrel formed by five inner and five
outer
-helices, and the 10
-helices (from HA3 to HA12)
constituting the
5/
5-barrel are located
within the core of the domain (Fig. 4A). These 10 helices
are connected by short and long loops in a nearest neighbor, up-and-down pattern. This arrangement is described as a "twisted
/
-barrel" with five inner
-helices (HA3, HA5, HA7, HA9, and HA11), which are oriented in roughly the same direction, and five outer
-helices (HA4, HA6, HA8, HA10, and HA12) running in the opposite direction.
C-terminal -Sheet Domain--
The C-terminal domain consists
predominantly of 30
-strands (SA1, aa residues 389-396; SA2,
398-402; SA3, 407-411; SA4, 437-441; SB1, 465-467; SC1, 482-487;
SC2, 491-499; SC3, 506-513; SC4, 518-526; SB2, 533-541; SC8,
550-552; SC9, 554-557; SB5, 563-567; SC7, 571-575; SC6, 585-589;
SB4, 593-604; SB3, 620-632; SC5, 638-646; SD1, 662-667; SD2,
671-676; SD3, 681-686; SE1, 692-694; SE2, 697-699; SD4, 703-710; SD5, 714-720; SE3, 729-734; SD6, 740-743; SE5, 747-751; SE4,
756-761; and SD7, 769-775) (Figs. 2, 3, and 4B). The
-strands vary in length between 3 and 13 aa residues and consist of
190 aa residues.
There are 29 loops (from LB1 to LB29) connecting a -strand to the
following
-strand in the C-terminal domain (Fig. 3). The loop (LB18)
between SC5 and SD1 includes one
-helix (HB1, aa residues 650-658)
with two turns. Therefore, in the C-terminal domain, 48.8% of all aa
residues are in
-strands, 2.3% in
-helices, and the remaining
48.9% in turns and coils.
The C-terminal domain contains a calcium ion (Fig. 2). The site is
located within a loop (LB8) and a -strand (SD2) (Figs. 3 and
4B). The six oxygen atoms, OD1 of Asp515, OD2 of
Asp516, OE1 and OE2 of Glu517, OE1 of
Glu676, and O of WAT951, are coordinated to the
calcium ion, and the coordination geometry comprises a distorted
octahedron (Fig. 5). The distance between the calcium ion and the oxygen atoms ranges from 1.92 to 2.73 Å (average, 2.31 Å).
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In the C-terminal domain, five anti-parallel -sheets (sheets A-E)
are formed by the 30
-strands, all of which are anti-parallel in the
-sheets (Fig. 3). The
-sheets are composed of four to nine
-strands (sheet A, SA1-4; sheet B, SB1-5; sheet C, SC1-9; sheet
D, SD1-7; and sheet E, SE1-5). Sheet A is parallel to the small
-sheet consisting of S1 and S2 in the N-terminal
-helical domain.
As a result, the C-terminal
-sheet domain shows a five-layered
-sheet sandwich structure (Fig. 4B).
Structural Comparison of Polysaccharide Lyases--
On the basis
of their sequence similarity, polysaccharide lyases are classified into
12 families. The three-dimensional structures of lyases belonging to
families 1, 3, 5, 6, and 8 have been determined and divided into three
groups (parallel -helix,
/
-barrel, and
/
-barrel + anti-parallel
-sheets).1 Xanthan lyase belongs to
polysaccharide lyase family 8, together with lyases for hyaluronate and
chondroitin AC, although the sequence identity of xanthan lyase with
the other lyases is less than 30% (Fig.
6).
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The overall structure of xanthan lyase is similar to that of the family
8 lyases for hyaluronate (10, 11) and chondroitin AC (13), which
consist of N-terminal -helical and C-terminal
-sheet domains. The
crystal structures of xanthan lyase and the other enzymes were
superimposed by means of a fitting program, RIGID, included in
TURBO-FRODO (Fig. 7). The aa sequences of
the lyases for xanthan, hyaluronate, and chondroitin AC were aligned through analyses of the aa similarity and three-dimensional structures (Fig. 6). The r.m.s. deviations of C
atoms
between xanthan lyase and the other enzymes were determined by means of
a fitting program, TOP, included in CCP4 (37) (Table
III). The overall structure of xanthan
lyase is more similar to that of hyaluronate lyase than of
chondroitin AC lyase, and among the family 8 lyases, the geometries of
the
-domains are more well conserved than those of the
-domains.
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However, the following structural differences were observed among the
lyases for xanthan, hyaluronate, and chondroitin AC. Hyaluronate lyase
of Streptococcus agalactiae has a small N-terminal -domain composed of seven
-strands preceding the
-helical
domain (11). It should be noted that in the case of hyaluronate lyase of Streptococcus pneumoniae, the presence of an
N-terminal
-domain has not been reported due to the crystal
structure of the truncated enzyme (10). The C-terminal
-sheet domain
of xanthan lyase is slightly larger than those of the lyases for
hyaluronate and chondroitin AC; i.e. xanthan lyase has 30
-strands in the C-terminal domain, whereas the C-terminal domains of
the lyases for hyaluronate and chondroitin AC contain 24 and 28
-strands, respectively (10, 13). The
-helical domain of xanthan
lyase is similar to that of hyaluronate lyases but differs from that of
chondroitin AC lyase, as the latter has 12
-helices in its
N-terminal domain (Fig. 6).
Although the C-terminal domain of chondroitin AC lyase contains a
calcium ion (13), the localization and coordination of the calcium ion
in xanthan lyase are different from those in the AC lyase in that the
calcium ion of the AC lyase coordinates with seven oxygen atoms in two
water molecules and in the side chains of four aa residues
(Glu405, Asp407, Asp416, and
Tyr417) in strand 5 corresponding to SB1 of xanthan
lyase. Furthermore, structural differences between xanthan lyase and
the other enzymes were found in the loops over the deep cleft formed in
the N-terminal domain. In particular, loop LB16 of xanthan lyase
connecting SB4 and SB3 in the C-terminal domain protrudes from the
C-terminal domain and covers the cleft in the N-terminal domain (Fig.
2). The extreme protrusion of the loop is caused by the
Arg612-Thr615 residues; there are several gaps
in the corresponding sites of the lyases for hyaluronate and
chondroitin AC (Fig. 6). The significance of the loop is described below.
The topology of the secondary structure elements of the N-terminal
-helical domain of xanthan lyase resembles that of alginate lyase,
A1-III (polysaccharide lyase family 5), with an
6/
5-barrel structure, which we have
determined to be an example of an endotype polysaccharide lyase (9).
The
/
-barrel structure is found in sugar-related enzymes such as
glucoamylases (38, 39) endoglucanase (40, 41), endo/exocellulase (42),
N-acyl-D-glucosamine 2-epimerase (43),
-1,2-mannosidase (44), and maltose phosphorylase (45), as well as
polysaccharide lyases. These enzymes form the
/
-toroid family in
the SCOP data base (scop.berkeley.edu/data/scop.b.b.bbc.html). Although
these enzymes catalyze different reactions (hydrolysis, epimerization,
and
-elimination), their
/
-barrel structures possibly are
responsible for the binding of polysaccharides in the hydrolytic and
eliminative depolymerization reactions and for the production of common
intermediates in the epimerization and
-elimination reactions. In
fact, the occurrence of a common step in the catalytic reactions of two
types of alginate-modifying enzymes, lyases and epimerases, has been
reported (46).
Structure of Xanthan Lyase Complexed with PyrMan-- Although almost all polysaccharide lyases analyzed thus far, including those for hyaluronate and chondroitin AC, attack the main chains of polysaccharides in an endolytic manner and release the oligosaccharides from the polysaccharides, xanthan lyase is characteristic in that it attacks the side chains of a polysaccharide exolytically. To identify the substrate-binding site, and to clarify the structural features causing the different substrate specificities of xanthan lyase and the other lyases, the crystal structure of the enzyme complexed with PyrMan (product) was determined at 2.4 Å resolution.
PyrMan was soaked in a native crystal of xanthan lyase. Diffraction data for the crystal of xanthan lyase complexed with the product up to 2.40 Å resolution were collected and refined to 2.4 Å resolution with CNS (26) using the refined structure of the native enzyme as a primary model. The results of the x-ray data collection and refinement are summarized in Table I.
The refined model of the enzyme-product complex consists of 752 aa
residues (Ser26-Gly777), 261 water molecules,
one calcium ion, and one PyrMan. A stereodiagram of a ribbon
presentation of the complex is shown in Fig.
8A. The final overall
R-factor for the refined model was calculated as 0.169 (free
R-factor, 0.242) using the data from 50 to 2.4 Å resolution (26,294 reflections).
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The difference Fourier map contoured at the 3 level calculated with
the 10-2.4 Å resolution data exhibited the highest densities in the
product region (Fig. 8B). The average B-factor of
PyrMan was 20.6 Å2. The nomenclature for the sugar-binding
site in the enzyme proposed by Davies et al. (47) was used
here, and the sugars are numbered starting from the cleavage site, with
positive numbers increasing toward the reducing terminus. Because
xanthan lyase cleaves the glycosidic bond between PyrMan and GlcUA
residues (Fig. 1), the position of PyrMan is designated as
"
1."
Xanthan lyase and its complex with PyrMan were superimposed by means of
a fitting program included in TURBO-FRODO. The r.m.s. deviation was
0.305 Å for the 752 common C atoms. There was no
significant conformational change between the protein structures with
and without PyrMan. PyrMan in the complex structure is bound in the
deep cleft formed on the N-terminal domain of the enzyme facing the
interface between the N- and C-terminal domains (Fig. 8A),
indicating that the active center (substrate-binding site) is located
in the cleft. Some aromatic and positively charged aa residues are
arranged in the active cleft, suggesting that these aa residues are
responsible for the binding and depolymerization of acidic
polysaccharide xanthan (Fig. 9). This
feature is common to family 8 polysaccharide lyases, which depolymerize
acidic polysaccharides (10, 13).
|
Structure of the Active Cleft--
Several aa residues and water
molecules have been shown to be crucial for the binding of PyrMan (Fig.
10A). The data listed in
Table IV represent the interaction of the
enzyme with the bound PyrMan molecule in the complex. There are six
direct hydrogen bonds between the protein and PyrMan atoms (Table
IV). In particular, the carboxyl group of the pyruvate moiety in
PyrMan is directly bound to the Arg313, Tyr315,
and Arg612 residues through the formation of four hydrogen
bonds. This conformation accounts for why the xanthan lyase of
Bacillus sp. GL1 specifically liberates the nonreducing
terminal saccharide, PyrMan, from the side chains of xanthan and is
inactive on nonpyruvylated xanthan (16). The Arg313 and
Tyr315 residues are located in the LA10 loop of the
N-terminal domain, whereas the Arg612 residue is in the
LB16 loop of the C-terminal domain. As we described above, there are
differences between xanthan lyase and family 8 lyases as regards the
loop structure over the active cleft, and there are no direct hydrogen
bonds between aa residue atoms in the C-terminal domain and sugar atoms
at position 1 are present in lyases for hyaluronate (11, 48)
and chondroitin AC (49) complexed with the substrate or product.
Therefore, the extreme protrusion of the LB16 loop of xanthan lyase
from the C-terminal domain is responsible for the recognition and
binding of PyrMan. Apart from their interaction with the carboxyl group
of PyrMan, the Tyr255 and Arg313 residues
directly interact with the pyranose ring of PyrMan through the
formation of hydrogen bonds. The side chain (OH) of the
Tyr255 residue forms a hydrogen bond with an oxygen atom
(O-1) of PyrMan, which forms a glycosidic bond with the GlcUA residue
before the enzyme reaction, suggesting that the Tyr255
residue plays an important role in the catalytic reaction. In addition to direct hydrogen bonds, there are six hydrogen bonds between water molecules and PyrMan atoms (Table IV).
Furthermore, there are six water-mediated hydrogen bonds between
the protein and PyrMan atoms:
O-1=WAT812=Tyr255OH (2.9 Å),
O-2=WAT818=Ser258 OG (3.0 Å),
O-2=WAT859=Glu310 OE2 (2.8 Å),
O-3=WAT818=Glu310 OE2 (2.8 Å),
O-6=WAT1066=Arg612 NH2 (3.1 Å), and
O-8=WAT1073=Tyr315 OH (3.2 Å).
|
|
Carbon-carbon (C-C) contacts were also observed between the protein
(Trp148, Trp197, Tyr255, and
Arg313 residues) and PyrMan atoms (Table IV). The
Trp148 residue is parallel to the pyranose ring of the
mannose moiety of PyrMan, indicating that the residue undergoes a
stacked interaction with the sugar ring (Fig. 10A). This
stacking is thought to be common to family 8 lyases, because the
Trp127 and Trp292 residues of chondroitin AC
and hyaluronate lyases, respectively, corresponding to the
Trp148 residue of xanthan lyase, show a stacked interaction
with the sugar positioned at 1 (48, 49) (Fig. 10, B and
C).
The arrangements of aa residues at the 1 position of the family 8 lyases were compared (Fig. 10, B and C). The
active-site architecture has been reported to be well conserved in
lyases for hyaluronate and chondroitin AC (48). The geometry of the Arg313 residue of xanthan lyase is similar to that of the
Arg466 and Arg292 residues of lyases for
hyaluronate and chondroitin AC, respectively, which are essential for
the direct binding of sugars and which correspond to the
Arg313 residue of xanthan lyase (Figs. 6 and 10,
B and C). Recently, the Arg292
residue of chondroitin AC lyase responsible for the binding of GlcNAc
positioned at
1 was found to be involved in the subsequent, processive, stepwise, and exolytic cleavage reaction of the enzyme (50). On the other hand, the following differences among the lyases for
xanthan, hyaluronate, and chondroitin AC were observed in the
arrangement of aa residues. In the case of xanthan lyase, the
Arg313 and Tyr315 residues in the N-terminal
domain and the Arg612 residue in the C-terminal domain
directly bind to the carboxyl group of PyrMan. However, no direct
interaction of lyases for hyaluronate and chondroitin AC with the sugar
positioned at
1 involves residues in the C-terminal domain (11, 48,
49), although the Asn374, Glu376,
Ser552, and His553 residues of the C-terminal
domain of chondroitin AC lyase associate with the sugar via water
molecules (49). No aa residues corresponding to the Tyr315
and Arg612 residues of xanthan lyase are conserved in
lyases for hyaluronate and chondroitin AC (Fig. 6). The
Arg462 residue of hyaluronate lyase directly binds to O-4
of GlcNAc positioned at
1, whereas the Arg309 residue of
xanthan lyase, corresponding to the Arg462 residue of
hyaluronate lyase, undergoes no interactions with PyrMan. Therefore,
these differences in the arrangement of aa residues at the
1 position
determine the substrate specificities of the family 8 lyases, because
the substrates of lyases for xanthan, hyaluronate, and chondroitin AC
have PyrMan, GlcNAc, and GalNAc residues, respectively, attached to the
common GlcUA residues (Fig. 1).
The Tyr255 residue interacts directly in the active cleft
with the O-1 oxygen atom of PyrMan involved in the formation of the glycosidic bond between the 1 and +1 sugars. This finding indicates that the residue is responsible for the catalytic reaction. Several structural studies on catalytic residues in the clefts of lyases for
hyaluronate and chondroitin AC have been reported (11, 48, 49). More
recently, the Asn349, His399, and
Tyr408 residues of hyaluronate lyase from S. pneumoniae were shown to participate in the catalytic reaction
through x-ray crystallographic analysis of a mutant enzyme (Y408F)
complexed with hyaluronate tetra- and hexasaccharides (51).
Asn349 interacts with the carboxyl group of the GlcUA
residue, His399 functions as a base and withdraws a proton
from the C-5 carbon of the GlcUA residue, and Tyr408,
acting as an acid, donates a proton to the glycosidic oxygen to be
cleaved. These three residues are conserved in xanthan lyase of
Bacillus sp. GL1, and the Asn194,
His246, and Tyr255 residues of the xanthan
lyase correspond to the respective residues of hyaluronate lyase (Fig.
6). The His225 and Tyr234 residues of
chondroitin AC lyase corresponding to the His399 and
Tyr408 residues of hyaluronate lyase have also been
reported as crucial for the catalytic reaction (49). The arrangement of
these residues is highly conserved (Fig. 10, B and
C) in the family 8 lyases; xanthan lyases (N194A, H246A, and
Y255F) in which the Asn194, His246, and
Tyr255 residues were substituted with Ala, Ala, and Phe,
respectively, exhibited little enzymatic
activity.3 These results
suggest that the Asn194, His246, and
Tyr255 residues play a crucial role in the
-elimination
reaction of xanthan lyase, as seen for the lyases for hyaluronate and
chondroitin AC. To clarify the reaction mechanism of the exotype
xanthan lyase in more detail and to establish common structural rules
for polysaccharide lyases, we have attempted to determine the
structures of the wild type and mutant enzymes complexed with
xanthan-branched pentasaccharide as a substrate (52).
Conclusions and Implications--
To the best of our knowledge,
this is the first report on the determination of the crystal structure
of an exotype polysaccharide lyase that can act on the side chains of a
polysaccharide. The enzyme consists of N-terminal -helical and
C-terminal
-sheet domains and has a deep cleft in the N-terminal
domain facing the interface between the N- and C-terminal domains.
Because the basic frames of lyases for xanthan, hyaluronate, and
chondroitin AC are similar to each other, all of the polysaccharide
lyases belonging to family 8 are considered to share a common structure
consisting of N-terminal
-helical and C-terminal
-sheet domains.
Furthermore, their active sites are all located in a deep cleft.
Based on the crystal structure of the enzyme complexed with PyrMan, the deep cleft in the enzyme was revealed to be responsible for the recognition of the substrate and the catalytic reaction. The enzyme specifically binds to the nonreducing terminal PyrMan of xanthan side chains in the cleft, as the aromatic and positively charged aa residues in the active cleft directly interact with the carboxyl group of PyrMan through the formation of hydrogen bonds. The arrangement of aa residues of xanthan lyase in the recognition site for PyrMan attached to the GlcUA residue differs from that in lyases for hyaluronate and chondroitin AC in the corresponding sites for GlcNAc and GalNAc, respectively, attached to the GlcUA residue. These differences in the aa arrangement are thought to determine the substrate specificity.
As seen for lyases for hyaluronate and chondroitin AC, the
Asn194, His246, and Tyr255 residues
in the active cleft of xanthan lyase are thought to be involved in the
catalytic reaction. As regards family 5 alginate lyase A1-III, similar
to the case of the N-terminal -helical domain of family 8 lyases, we
have clarified that the activated Tyr246 residue homologous
to the Tyr255 residue of xanthan lyase is bifunctional as a
base and an acid (53). Therefore, the Tyr residue is considered
important for the catalytic reactions of these polysaccharide lyases
including an
/
-barrel structure.
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ACKNOWLEDGEMENT |
---|
Computation time was provided by the Supercomputer Laboratory at the Institute for Chemical Research, Kyoto University.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Bio-oriented Technology Research Advancement Institution (BRAIN) of Japan and by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to K. M., B. M., and W. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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 of xanthan lyase and its complex with PyrMan (codes 1J0M and 1J0N, respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
81-774-38-3766; Fax: 81-774-38-3767; E-mail:
murata@food2.food.kyoto-u.ac.jp.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M208100200
1 B. Henrissat, P. Coutinho, and E. Deleury, afmb.cnrs-mrs.fr/~cazy/CAZY/index.html.
3 W. Hashimoto, H. Nankai, B. Mikami, and K. Murata, unpublished results.
![]() |
ABBREVIATIONS |
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The abbreviations used are: PyrMan, pyruvylated mannose; Glc, D-glucose; GlcUA, D-glucuronic acid; Man, D-mannose; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; m.i.r., multiple isomorphous replacement; aa, amino acid; r.m.s., root-mean-square; WAT, water molecule; Ac, acetate; KPB, potassium phosphate buffer; Bicine, N,N-bis(2-hydroxyethyl)glycine; CNS, crystallography NMR software; PDB, protein data bank.
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