Download An Enzyme–Substrate Complex Defines the Cata

doi:10.1016/j.jmb.2003.12.071
J. Mol. Biol. (2004) 337, 367–386
High-resolution Crystal Structure of
Arthrobacter aurescens Chondroitin AC Lyase:
An Enzyme –Substrate Complex Defines the
Catalytic Mechanism
Vladimir V. Lunin1, Yunge Li1, Robert J. Linhardt2, Hirofumi Miyazono3
Mamoru Kyogashima3, Takuji Kaneko3, Alexander W. Bell4
and Miroslaw Cygler1*
1
Biotechnology Research
Institute, National Research
Council of Canada, and
Montréal Joint Centre for
Structural Biology, Montréal
Québec, 6100 Royalmount Ave.
Montréal, Québec, Canada
H4P 2R2
2
Department of Chemistry
Division of Medicinal
Chemistry and Department of
Chemical and Biochemical
Engineering, The University of
Iowa, 115 S. Grand Ave, PHAR
S328, Iowa City, IA
52242-1112, USA
3
Central Research Laboratories
Seikagaku Corporation, Tateno
3-1253, Higashiyamato-shi
Tokyo 207-0021, Japan
4
Montréal Proteomics Network
740 Dr Penfield Ave., Montréal
Québec, Canada H3A 1A4
*Corresponding author
Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycandegrading enzymes that act as eliminases. Chondroitin lyase AC from
Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate
and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan.
We have determined the three-dimensional crystal structure of
ArthroAC in its native form as well as in complex with its substrates
(chondroitin 4-sulfate tetrasaccharide, CStetra and hyaluronan tetrasaccharide) at resolution varying from 1.25 Å to 1.9 Å. The primary sequence of
ArthroAC has not been previously determined but it was possible to
determine the amino acid sequence of this enzyme from the highresolution electron density maps and to confirm it by mass spectrometry.
The enzyme – substrate complexes were obtained by soaking the substrate
into the crystals for varying lengths of time (30 seconds to ten hours) and
flash-cooling the crystals. The electron density map for crystals soaked in
the substrate for as short as 30 seconds showed the substrate clearly and
indicated that the ring of central glucuronic acid assumes a distorted
boat conformation. This structure strongly supports the lytic mechanism
where Tyr242 acts as a general base that abstracts the proton from the C5
position of glucuronic acid while Asn183 and His233 neutralize the charge
on the glucuronate acidic group. Comparison of this structure with that of
chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an
explanation for the exolytic and endolytic mode of action of ArthroAC
and FlavoAC, respectively.
Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved.
Keywords: chondroitin lyase; chondroitinase AC; Arthrobacter aurescens;
substrate binding; catalytic mechanism
Introduction
Glycosaminoglycans (GAGs) are the carbohydrate components of proteoglycans, which are
a major component of the extracellular matrix.1
They are highly negatively charged polysaccharSupplementary data associated with this article can be
found at doi: 10.1016/j.jmb.2003.12.071
Abbreviations used: GAG, glycosaminoglycan; MS,
mass spectrometry.
E-mail address of the corresponding author:
[email protected]
ides, composed of disaccharide repeating units of
a substituted glucosamine or galactosamine
attached through (1,4) linkage to a uronic acid
molecule. These disaccharide units are linked (1,3)
or (1,4) into a polysaccharide chain.2 The glucosamine/galactosamine units are sulfated extensively,
and their synthesis requires the concerted action
of a large number of enzymes.3,4
Glycosaminoglycans are degraded enzymatically
by two types of enzymes, hydrolases and lyases.5
Hydrolases catalyze cleavage of the glycosyloxygen bond by addition of water, producing
a saturated disaccharide. Lyases cleave the
0022-2836/$ - see front matter Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved.
368
oxygen– aglycone linkage through proton abstraction, producing an unsaturated disaccharide product with a double bond between C4 and C5. The
enzymatic mechanisms of hydrolases are well
understood and reactions proceed either according
to the retaining or inverting mechanism.6 On the
other hand, the molecular details of the enzymatic
mechanism of GAG lyases are still poorly understood. A chemically plausible mechanism for the b
elimination reaction has been proposed;7 however,
the constitution of the active site and the roles of
individual amino acids are not clear. A number of
bacterial species synthesize GAG lyases, enzymes
used to degrade and utilize glycosaminoglycans
as a source of carbon in the bacterium’s natural
environment.5,8 Polysaccharide lyases with known
three-dimensional structures fall into two architectures: the right-handed parallel b-helix (pectate/
pectin lyases, chondroitinase B, rhamnoglucuronan
lyase) and (a/a)n toroid (n ¼ 5 for Flavobacterium
heparinum chondroitin AC and chondroitin ABC
lyases, bacterial hyaluronate lyases, xanthan lyase,
and n ¼ 6 for alginate lyases). A catalytic mechanism has been proposed for pectate lyases, Ca2þdependent enzymes,9 but it remains to be seen if it
applies to other lyases having the b-helix topology.
Several plausible mechanisms have been proposed
for the lyases with the (a/a)5 toroidal fold with
a histidine or a tyrosine residue in the role of a
general base abstracting the proton from the C5
atom of glucuronic acid, and a tyrosine or an arginine residue acting as a general acid donating a proton to the bridging O4 atom.10,11 However, there is
insufficient evidence to indicate which of these
proposed mechanisms is utilized by the enzymes.
The nature of the group presumed to be necessary
to neutralize the charge of the glucuronic acid
carboxylic group is not clear, since these enzymes
do not require Ca2þ and there is no positively
charged group in the vicinity of the uronic acid, as
expected from the accepted chemical mechanism.7
Glycosaminoglycan-degrading enzymes with
defined specificity have found widespread applications as analytical tools for the analysis of the
structure of glycosaminoglycans and other polysaccharides.12 Chondroitin AC lyases are used frequently for this purpose. These enzymes cleave
the glycosidic bond on the non-reducing end of an
uronic acid and use as a substrate either chondroitin 4-sulfate or chondroitin 6-sulfate but not
dermatan sulfate. They display a varied degree
of activity toward hyaluronan.13 Enzymes from
two sources, chondroitin AC lyase from Arthrobacter
aurescens (ArthroAC) and from F. heparinum
(FlavoAC), are commercially available (Seikagaku
Corporation) and used frequently. The latter
enzyme has been cloned and overexpressed in
F. heparinum14 and in Escherichia coli.15 We previously determined the three-dimensional structure of this enzyme on its own16 and in complex
with several dermatan sulfate oligosaccharides.10
Here, we present the three-dimensional structure
of chondroitin AC lyase from A. aurescens as well
Chondroitinase AC Crystal Structure and Mechanism
as the complexes with chondroitin tetrasaccharide
(DUAp (1 ! 3)-b-D -GalpNAc4S (1 ! 4)-b-D -GlcAp
(1 ! 3)-a,b-D -GalpNAc4S, where DUAp is the
unsaturated sugar residue, 4-deoxy-a-L -threo-hex4-enopyranosyluronic acid; GlcAp, glucopyranosyluronic acid; GalpN, 2-deoxy-2-aminogalactopyranose; S, sulfate; and Ac, acetate) and
hyaluronan tetrasaccharide substrates.
Despite the purification and characterization of
ArthroAC many years ago13,17,18 and its extensive
use as an analytical tool in glycosaminoglycan
analysis, this enzyme has not been cloned and its
amino acid sequence has not been determined,
although its amino acid composition and carbohydrate content were reported.18 We obtained
crystals that diffract up to 1.25 Å resolution. The
high-resolution data led to high-quality electron
density maps of native enzyme and several complexes, and allowed us to deduce confidently the
amino acid sequence for 99% of the amino acid
residues and to propose the molecular details of
the catalytic mechanism, which is common to
FlavoAC and hyaluronate lyases. Here, we follow
the nomenclature introduced by Davies et al.19 and
designate the sugars on the reducing end of the
break with a þ sign and the sugars on the nonreducing end with a 2 sign. In this nomenclature,
the enzymes break the bond between sugars 2 1
and þ 1, the latter being an uronic acid.
Results and Discussion
Amino acid sequence and its conservation
The molecular mass of the entire molecule was
measured by ion spray mass spectrometry. Two
species were present with molecular masses of
79,502 Da and 79,840 Da. The greater mass corresponds well to the molecular mass of 79,785 Da
(average mass) calculated from the amino acid
sequence. Based on the analysis of MS/MS spectra,
we believe that the smaller mass corresponds to
the fragment missing three N-terminal amino acid
residues. Such a good agreement between the predicted and measured molecular mass indicated
that (1) no major assignment errors were made
and (2) no glycosylation or other modification
were present (FlavoAC is glycosylated).16
Peptides extracted from an in-gel trypsin digest
of purified A. aurescens chondroitin AC lyase were
analyzed by LC-QToF mass spectrometry as
described in Materials and Methods. The resulting
peaklist of fragmentation spectra was matched inhouse against the sequence deduced from the crystal structure employing Mascot (MatrixScience)
software.20 Matched tandem MS spectra were confirmed manually and the remaining spectra were
interpreted manually. This process was repeated
several times, using the two sets of experimental
data iteratively to determine the optimal sequence
of the lyase (Supplementary Material).
The results of MS/MS analysis of 202 tandem
369
Chondroitinase AC Crystal Structure and Mechanism
mass spectra covering 88.5% of the entire
ArthroAC sequence (see Materials and Methods)
confirmed the amino acid sequence identified
from electron density maps. Moreover, this analysis of individual MS/MS fragmentation data
allowed us to make side-chain assignments for
several residues located in flexible loops for which
electron density was not easily interpretable
(shown in small letters in Figure 1).
The agreement for 18 (4 –21) residues between
the residue type derived from the electron density
maps and mass spectrometry, and that determined
by Edman degradation provides an independent
measure underscoring the low level of errors to be
expected in our assignment. While the sequence of
ArthroAC has not been determined previously, its
amino acid composition was reported nearly 30
years ago.18 These data showed good correlation
with the amino acid sequence of ArthroAC in this
work, supporting our assignments (Table 1).
The derived sequence of ArthroAC was used to
identify homologous sequences in the NCBI database with the program BLAST.21 There are , 50
such sequences for which the similarity extends
along the entire protein. They include hyaluronate,
xanthan and chondroitin AC lyases. ArthroAC
shows the highest level of sequence identity with
various hyaluronan and xanthan lyases (38%) and
a lower level of identity with chondroitin AC
lyase from F. heparinum (24%).
The structures of four proteins representative of
these sequences are known; namely, chondroitin AC
lyase 1CB8,16 Streptococcus pneumoniae hyaluronate
lyase 1EGU,11 Streptococcus agalactiae hyaluronate
Table 1. Comparison of percentage distribution of amino
acids between chemical amino acid analysis and crystallographic assignment
Residue
type
Ala
Arg
Asx
Glx
Trp
Val
Ser
Thr
His
Phe
Gly
Pro
Ile
Leu
Lys
Tyr
Met
Cysa
Sequenced from the map
(%)
From Hiyama &
Okada18
14.1
5.2
9.1
6.35
2.5
7.0
6.7
8.85
1.85
2.9
10.6
4.0
3.6
9.1
3.3
2.5
1.45
0.9
13.4
4.7
9.0
6.5
2.4
8.2
6.6
8.4
1.7
2.9
11.9
3.3
3.3
8.8
3.3
2.6
1.1
2.3
a
The significant difference in the number of cysteine residues
between our data and those reported by Hiyama & Okada while
there is very good agreement for the other amino acids, is likely
related to the fact that hydrolysis was used for the determination of most amino acids while sulfhydryl titration was used
for cysteine.
lyase 1F1S22 and Bacillus sp. xanthan lyase 1J0M.23
Structure-based alignment of their sequences is
shown in Figure 1. The residues important for the
integrity of the active site (see below) include
Asn183, His233, Tyr242, Arg296 and Glu407 of
ArthroAC and are conserved in all of the related
enzymes, and Glu412 is replaced by an aspartate
residue in one case.
The enzyme chondroitin ABC lyase I shows
good sequence similarity to the above-mentioned
enzymes only for the C-terminal domain. However, its three-dimensional structure (PDB code
1HN0) showed that the catalytic domain has (a/a)5
topology and can be structurally aligned with the
catalytic domains of the other lyases.24 While the
substrate-binding site shows little sequence conservation, the active-site residues are conserved,
suggesting the same enzymatic mechanism. There
is no equivalent, however, to the Asn183 of
ArthroAC, and there are differences in the local
structure in this region. Huang et al. suggested
that this enzyme utilizes as a replacement an arginine residue remote in the linear sequence.24 The
structure-based alignment of chondroitin ABC
lyase I with the other lyases is included in Figure 1.
Overall fold
The ArthroAC molecule has an overall a þ b
architecture and consists of two domains (Figure 2).
The N-terminal a-helical domain contains 13
a-helices, ten of which form an incomplete
double-layered (a/a)5 toroid as classified within
the SCOP database.25 There is a long, deep groove
on one side of the toroid that forms the location
of the active site and substrate-binding site. Three
a-helices at the N terminus precede the (a/a)5
toroid and constrict the cleft on one side. Residues
conserved across the sequences of proteins homologous to ArthroAC cluster in the area of this cleft.
The C-terminal domain is composed almost
entirely of antiparallel b-strands arranged into
four b-sheets. The first two sheets contain nine
b-strands, some of them rather long. The third
sheet has seven b-strands and the last one five
b-strands. There is only one short a-helix within
this domain (Figure 2). The second domain can be
subdivided into two subdomains; the first encompasses the first two large b-sheets and one short
a-helix, while the second subdomain is composed
of the third and fourth b-sheet.
Substrate-binding site
Initial experiments of soaking native crystals
of ArthroAC in a 5 mM solution of chondroitin
4-sulfate tetrasaccharide substrate for prolonged
times before collecting diffraction data showed
clear density for only a disaccharide product, indicating that, like other GAG lyases,10 ArthroAC
retains enzymatic activity in the crystals. Therefore,
we decided to investigate by X-ray diffraction
the enzyme – substrate complex as a function of
Figure 1 (legend opposite)
Chondroitinase AC Crystal Structure and Mechanism
371
Figure 2. Stereo drawing of the ribbon representation of ArthroAC showing the bound tetrasaccharide. The individual a-helical hairpins of the N-terminal (a/a)5 toroid are in different colors. The individual b-sheets of the C-terminal
domain are in discrete colors. Insertions in ArthroAC blocking the cleft are in gray. The substrate is shown in stick
representation and colored in magenta. The Figure was prepared with the programs MOLSCRIPT52 and Raster3d.53
soaking time. The soaking time ranged from 30
seconds to ten hours (Tables 2 and 3). At the end
of each soak, the crystal was immediately flashfrozen and diffraction data collected (resolution
varying between 1.25 Å and 1.6 Å, Table 3). Hyaluronan tetrasaccharide was also used as a substrate with a soaking time of two minutes and
diffraction data were collected to 1.9 Å resolution.
The structures were refined independently. In
each case, the ArthroAC molecule was refined
first, then the difference electron density map was
inspected and interpreted appropriately. The
modeled substrate/product was included in the
refinement. Several sugar units were clearly visible
in each refined model. The relative occupancy of
the 2 and þ sites along the timed snapshots were
evaluated as described in Materials and Methods.
The reaction in the present crystals occurs on the
minute timescale, indicated by well-defined electron density for the entire tetrasaccharide substrate
after 30 seconds and even longer soaks (Figure 3(a)
and (b); and Table 4). Indeed, the substrate is best
defined in this dataset, with assigned occupancies
of , 0.6. Somewhat higher occupancy for the (2 1,
2 2) subsites suggests a partial presence of the
disaccharide product in the 2 sites. For the ten
hour soak, the sugar units in subsites (2 1, 2 2) are
clearly visible in the difference electron density
map and refine with an occupancy of 1.0, while
the derived occupancy for the sugars at the (þ 1,
þ 2) subsites were 0.25. In the complex of
ArthroAC with hyaluronan tetrasaccharide, only
the sugar units in subsites (2 1, 2 2) were visible
in the electron density, corresponding to a disaccharide reaction product and in accord with higher
activity of ArthroAC toward hyaluronan.13 The
location and orientation of the hyaluronan sugars
is the same as the corresponding sugars of the
chondroitin sulfate tetrasaccharide substrate. In
the following discussion, we refer to the ArthroAC–
tetrasaccharide complex after a 30 seconds soak.
The oligosaccharide is bound within the groove
in the N-terminal domain (Figure 2) and makes
contacts with residues Asn124, Trp125, Trp126,
Arg134, Gln169, Arg174, Asn183, His233, Tyr242,
Arg296, Arg300, Asn303, Asn410 and Trp465
(Figure 3(c)). Tryptophan residues play an essential
role in substrate binding. Two of these residues,
Figure 1. Structure-based sequence alignment for ArthroAC, FlavoAC (1CB8), S. pneumoniae hyaluronate lyase
(1EGU), S. alagalactiae hyaluronate lyase (1F1S), Bacillus sp. xanthan lyase (1J0M) and P. vulgaris chondroitin ABC
lyase I (1HN0). Insertions in the ArthroAC sequence that close off one end of the substrate-binding site are boxed,
a-helices are marked in white letters on black background and b-stands are marked by black letters on gray background. The secondary structure assignments follow sPDBv.51 Residues conserved in all six proteins are marked by
an asterisk (*) above the sequence. Arrows mark residues essential for catalysis, Asn183, His233, Tyr242 and Arg296
and Glu407. Several residues that were assigned on the basis of the MS/MS data alone (disordered side-chain) are
shown in small letters. Letter ‘x’ indicates (Glu/Gln) or (Asp/Asn), which we cannot distinguish, and question
marks (?) indicate residues for which we are less certain of their amino acid type.
372
Chondroitinase AC Crystal Structure and Mechanism
Table 2. Data collection statistics for various substrate soaking times
Substrate
Soaking time
CStetra
Wavelength (Å)
a (Å
b (Å)
c (Å)
b (deg.)
Resolution range
(last shell)
Rsym (last shell)
Completeness (%)
(last shell)
I=sðIÞ (last shell)
Total reflections
Unique reflections
Redundancy
30 seconds
Two
minutes
Ten
minutes
35 minutes
Two hours
Four hours
Ten hours
HAtetra
Two
minutes
0.9798
57.9
86.9
81.5
107.0
50–1.41
(1.46–1.41)
0.081
(0.794)
99.9 (99.9)
0.9798
57.6
86.5
80.7
106.8
50–1.6
(1.66–1.6)
0.077
(0.739)
100 (100)
0.9798
57.7
86.4
80.6
106.9
50–1.5
(1.55–1.5)
0.077
(0.483)
99.9 (99.9)
0.9798
57.6
86.4
80.5
106.9
50–1.35
(1.4–1.35)
0.055
(0.567)
98.3 (96.3)
0.9798
57.6
86.5
80.6
106.9
50–1.3
(1.35–1.3)
0.057
(0.517)
96.5 (89.1)
0.9798
57.6
86.5
80.6
106.9
50 –1.35
(1.4– 1.35)
0.059
(0.530)
96.1 (93.5)
0.9798
57.6
86.3
80.5
107.0
50 –1.25
(1.29 –1.25)
0.058
(0.430)
90.8 (56.4)
0.9798
57.6
86.3
80.6
106.9
50 –1.9
(1.97 –1.9)
0.075
(0.652)
100 (99.9)
8.1 (2.0)
706,542
146,507
4.8
8.3 (2.5)
501,855
100,158
5.0
9.2 (3.4)
460,480
120,769
3.8
8.4 (2.0)
452,905
162,044
2.8
11.1 (3.5)
1,338,199
178,855
7.5
8.9 (2.9)
610,947
158,853
3.8
9.8 (2.8)
939,404
188,554
5.0
7.7 (2.8)
235,444
60,174
3.9
Table 3. Refinement statistics
Model
Resolution range
R-factor (Rfree)
No. non-hydrogen protein atoms
No. of water molecules
Average B-factor (Å2)
Protein main-chain atoms
Side-chain atoms
Water molecules
Substrate atoms
r.m.s.d. bond length (Å)
r.m.s.d bond angle (deg.)
Ramachandran plot. Residues in:
Most favorable region (%)
Disallowed regions (%)
tetra
Hg
derivative
Native
CS
30 seconds
CStetra
10 minutes
CStetra
10 hours
HAtetra
Two minutes
50–1.3
0.134 (0.155)
5687
1103
50 –1.35
0.130 (0.175)
5623
1025
50–1.45
0.138 (0.177)
5617
1049
50– 1.5
0.136 (0.170)
5627
1061
50–1.25
0.113 (0.142)
5646
1107
50–1.9
0.190 (0.251)
5629
837
14.9
16.5
28.7
14.1
16.3
29.4
0.019
1.84
0.022
1.97
15.5
17.9
31.4
24.8
0.023
1.84
15.4
16.7
30.5
21.2
0.021
1.86
13.6
15.4
29.6
15.5
0.021
2.01
21.8
22.4
31.6
22.5
0.018
1.72
90.8
0.3
90.7
0.3
90.5
0.3
89.7
0.3
89.9
0.3
88.3
0.5
Trp126 and Trp465, provide stacking interactions
with the sugar units occupying positions 2 1 and
þ 2, respectively, while Trp125 is aligned edge-on
and forms a hydrogen bond with the bridging
oxygen atom between the þ 2 and þ 1 units. The
4-O-sulfo groups of the substrate form several
interactions with the protein. The 4-O-sulfo group
of the þ 2 sugar makes H-bonds to Gln169 and,
Table 4. Relative occupancies of sugars in positions 2 2,
2 1, þ1, þ2 for different soaking times
Site occupancy
Soak time
Native
CStetra
HAtetra
30 seconds
Two minutes
Ten minutes
35 minutes
Two hours
Four hours
Ten hours
Two minutes
22
21
þ1
þ2
Phosphate
0.7
0.7
0.7
0.7
1.0
1.0
1.0
1.0
0.7
0.7
0.7
0.7
1.0
1.0
1.0
1.0
0.6
0.5
0.4
0.4
0.4
0.3
0.25
–
0.6
0.5
0.4
0.4
0.4
0.3
0.25
–
1.0
0.4
0.5
0.6
0.6
0.6
0.7
0.7
1.0
through a bridging water molecule, to Asp222 and
Gln232. The 2 1 sugar 4-O-sulfo group is positioned just above the guanidinium group of
Arg300 and, in addition, forms H-bonds to Glu412
and Asn598 through a bridging water molecule.
Both 4-O-sulfo groups contribute to substrate binding but do not add significantly to the specificity of
substrate recognition.
The chondroitin sulfate tetrasaccharide used in
these studies was obtained by the action of GAG
lyases and contained an unsaturated ring at the
non-reducing end, with a C4vC5 double bond.10
The electron density for the 2 2 sugar corresponds
very well to this unsaturated ring in E1 conformation, with the C5 having sp2 hybridization
(Figure 3a). A list of hydrogen bonds between the
tetrasaccharide and the protein side-chains is
given in Table 5.
Most interesting from the viewpoint of the
mechanism of catalysis is the conformation and
interactions with the enzyme of the þ 1 glucuronic
acid. The electron density shows that this ring
assumes a distorted boat conformation O,3B with
373
Chondroitinase AC Crystal Structure and Mechanism
Table 5. Close contacts between the substrate and the
protein in CStetra complex for the 30 second soak
experiment
Sugar number
Atom
Protein atom
Distance (Å)
22
O2
O2
O3
O6A
O6B
OD2 Asn303
NH1 Arg300
OD1 Asn303
NH1 Arg134
NH2 Arg134
2.85
3.07
3.03
3.01
2.90
21
O7
O7
SO43
SO44
NH2 Arg300
NH1 Arg300
NE Arg300
NH2 Arg300
2.92
3.01
3.27
3.46
þ1
O2
O2
O3
O4
O4
C5
O6A
O6A
O6B
ND1 Asn410
OD1 Asn124
ND2 Asn124
NH2 Arg296
OH Tyr242
OH Tyr242
NE2 His233
ND2 Asn183
OD1 Asn183
3.20
2.77
2.91
3.02
2.88
2.75
2.76
3.11
2.62
þ2
O1
O3
O7
SO41
SO43
NH2 Arg174
NE1 Trp125
NH1 Arg174
NE2 His233
NE2 Gln169
3.22
3.32
3.07
2.85
2.34
the hydroxyl groups equatorial and the C5
carboxylate group pseudoaxial (Figure 3(b)). This
carboxylate group is placed exactly opposite the
conserved side-chain of Asn183, whose OD1 and
ND2 atoms are clearly distinguished by their peak
height in the electron density map (Supplementary
Material). The distance between the amide ND2
atom and the carboxyl O6A is 3.1 Å, and that
between carbonyl OD1 and carboxyl O6B is 2.6 Å.
This short O6B· · ·OD1 distance suggests the existence of a hydrogen bond between them,26 which
in turn would indicate that the glucuronic acid
carboxylate group is protonated and therefore in a
neutral state. The O6A, in addition to accepting a
hydrogen bond from ND2 of Asn183, also forms a
second, 2.8 Å long hydrogen bond with the NE2
atom of His233. The geometry of hydrogen bonds
involving the carboxylate group is very close to
ideal, the C6 –O6A/B-donor angles are in the
range 115 – 1248 and the –COO group and the
three hydrogen bond donor atoms are nearly
coplanar (Figure 3(d)). His233 donates, in addition,
a second hydrogen bond from the ND1 atom to
the side-chain of the conserved Glu407; therefore,
this histidine residue must be protonated. The
hydroxyl groups at C2 and C3 of the þ 1 unit are
held firmly through several hydrogen bonds.
Atoms O2 and O3 are H-bonded to OD1 and ND2
of Asn124, respectively, while O2 is H-bonded
also to ND2 of Asn410 (Figure 3(c)).
Two other residues make crucial contacts with
the substrate. The hydroxyl O atom of Tyr242 is
within H-bonding distance of the bridging oxygen
atom between þ 1 and 2 1 sugars (2.9 Å), and is
3.3 Å from O5 of the þ 1 sugar ring. This hydroxyl
group is also only 2.8 Å from the C5 of the glucuronic acid, with the O atom nearly along the
derived direction of the C5 – H5 bond (Figure
3(d)). The Arg296 side-chain also forms a hydrogen
bond to the bridging oxygen atom between þ 1 and
2 1 sugars and is 3.0 Å from the Tyr242 hydroxyl
group. On the other hand, the distance from the
potential base His233 NE2 to the C5 of the þ 1
glucuronic acid is relatively long at 4.0 Å.
The electron density corresponding to the
carboxylate group of the glucuronic acid in the þ 1
site showed not two but three bulges, with the
third one having somewhat lower density. This
shape was common to the density observed in all
datasets and its position coincided with the phosphate group in the native structure. We have
modeled a phosphate group with partial occupancy in the same location (Table 4), assuming
that it is present there when the glucuronic acid
does not occupy this site. The total occupancy of
the þ 1 site, that is glucuronic acid plus phosphate,
equals 1.
A strong peak in the electron density map was
found in the proximity of the tetrasaccharide. It is
surrounded by six oxygen atoms in tetragonal
bipyramidal coordination, with distances to the
equatorial oxygen atoms of 2.2– 2.4 Å and to the
axial oxygen atoms of 2.8 Å. This peak was interpreted as a sodium ion. The equatorial ligands are
the carbonyl groups of His233 and Trp465 and
two water molecules, while the axial ligands are
the OG1 of Thr235 and a water molecule.
Substrate specificity
The initial characterization of ArthroAC showed
that the enzyme degrades chondroitin 4-sulfate,
chondroitin 6-sulfate and hyaluronan.13 Our results
with soaking various oligosaccharides indicated
that ArthroAC displays higher activity toward
hyaluronan than to chondroitin sulfate. We have
determined the kinetic parameters of ArthroAC
with GAG obtained from whale cartilage (CS-A,
predominantly chondroitin 4-sulfate), shark cartilage (CS-C, predominantly chondroitin 6-sulfate),
shark fin (CS-D, a mixture of chondroitin 4-sulfate,
6-sulfate and -4,6-disulfate), low molecular mass
hyaluronan and high molecular mass hyaluronan
(Table 6). Indeed, the Vmax for hyaluronan is twice
as high as that for chondroitin sulfate, while the
KM calculated on a per monomer basis is about
three times lower. These values point to a rather
small contribution made by the sulfate groups to
the total binding energy of the substrate.
Comparison with other GAG lyases
ArthroAC is very similar in overall structure to
other GAG lyases with the (a/a)5 fold. The closest
similarity is with S. pneumonia hyaluronate lyase
(SpHL, PDB code 1EGU). These two structures
superimpose with root-mean-squares (rms) deviation of 1.3 Å for 632 Ca atoms out of 750 residues
374
Chondroitinase AC Crystal Structure and Mechanism
Figure 3 (legend opposite)
(Figure 4). The superposition with FlavoAC results
in an rms deviation of 1.4 Å for 468 Ca atoms. The
similarity extends as well to the general mode of
substrate binding. Comparison with the SpHL
Y408F mutant (inactive, equivalent to Y242 of
ArthroAC) complexed with hyaluronan oligosaccharide,27 FlavoAC complexed with the dermatan sulfate hexasaccharide, and its inactive Y234F
Chondroitinase AC Crystal Structure and Mechanism
375
Figure 3. (a) Electron density for the tetrasaccharide of chondroitin 4-sulfate substrate in the omit map calculated
without the substrate present with the data for the 30 seconds soak of ArthroAC in 5 mM tetrasaccharide solution
and contoured at the 3s level. The phosphate group with partial occupancy is shown. An arrow marks the bond that
is cleaved by the enzyme. (b) Close-up of the same map for the þ 1 glucuronic acid showing the distorted boat conformation; (c) stereo view of the substrate-binding site. The tetrasaccharide is shown in thick semitransparent lines,
hydrogen bonds are shown with broken lines; (d) close-up of the active site showing residues involved in catalysis
and the hydrogen bonding network connecting these residues. Magenta dotted line marks the close contact between
the Tyr hydroxyl group and the C5 atom of glucuronic acid.
mutant (ArthroAC Y242 equivalent) complexed
with chondroitin tetrasaccharide10 shows that the
mode of oligosaccharide binding is nearly identical, with the largest difference restricted to the
glucuronic acid at the þ 1 site (Figure 4(b)). The
side-chains making crucial contacts with the
oligosaccharide are conserved in their type and
position. In the previously observed complexes
with Tyr-to-Phe mutant enzymes, the þ 1 glucuronic acid ring is in a chair conformation, with the
carboxylic group at C5 in an equatorial position.
That differs from the conformation of the glucuronate sugar observed in the wild-type ArthroAC –
substrate complex, where the ring forms a distorted boat with a pseudoaxial carboxylate group.
A detailed comparison of these structures
reveals subtle differences in the hydrogen bonding
network involving active-site residues. In ArthroAC,
the hydrogen bonds between the C5 carboxylic
group of the þ 1 sugar and Asn183 and His233
have nearly ideal geometry. The corresponding
H-bonds in the FlavoAC(Y234F) and SpHL(Y408F)
376
Chondroitinase AC Crystal Structure and Mechanism
Table 6. Kinetic parameters of ArthroAC
CS-A
CS-C
CS-D
V (DABS/s)
Km (mg/ml)
Ma (Da)
Km (mM)
Mb (Da)
Km (nM)
1.42 £ 10 2 3
0.196
505.2313
0.387
19,000
10.304
1.74 £ 10 2 3
6.10 £ 10 2 4
0.196
0.188
511.1607
532.166
0.383
0.353
43,000
30,000
4.554
6.256
Disaccharide composition (%)c
GAG source
DDi-0S
DDi-6S
DDi-4S
DDi-diSD
DDi-diSE
DDi-triS
Whale cartilage
1.6
19.3
76.2
2.7
0.3
–
Shark cartilage
1.7
72.9
15.4
9.3
0.6
–
a
b
c
LMHA (50K)
HA (1000K)
2.57 £ 10 2 3
0.052
401.3
0.130
50,000
1.046
2.78 £ 10 2 3
0.082
401.3
0.205
1,000,000
0.082
Shark fin
0.6
43.9
26.9
21.3
7
0.3
Disaccharides composition.
GPC-HPLC (CS STD).
Analysis of unsaturated disaccharides from glycosaminoglycan by HPLC.
complexes have less favorable geometry (Figure
4(b)). This asparagine residue in SpHL is modeled
with the opposite orientation of the amide group
to that in the other two enzymes. In the FlavoAC
(Y234F)-chondroitin sulfate tetrasaccharide complex, the glucuronate sugar remains in a chair conformation with all substituents to the ring being
equatorial, but rotates so that the carboxylic group
occupies the space vacated by the missing
hydroxyl group of Tyr234.10 Thus, even small
changes in the active site affect the mode of binding of the substrate and may result in a nonproductive binding.
Catalytic mechanism
The enzymatic reaction carried out by GAG
lyases is thought to proceed via abstraction of the
C5 proton by a general base followed by proton
donation by a general acid or a water molecule to
the bridging O4, with concomitant b-elimination
of the leaving group.7 Recent kinetic analysis of
the FlavoAC using a well defined synthetic substrate agrees with the predicted stepwise, as
opposed to concerted, mechanism.28 A proposal
for the mechanism of polysaccharide lyases formulated by Gacesa included the neutralization of the
acidic group by a positively charged group to shift
the equilibrium toward the enolate tautomeric
form.7 Unlike polysaccharide lyases that adopt
the b-helix fold, the structures of FlavoAC and
hyaluronate lyases showed an absence of such a
positively charged group in the vicinity of the
acidic group of uronate. Instead, an asparagine
side-chain was found to face and form a H-bond
with the acidic group, leaving the issue of neutralization of the acidic group an open question. The
structures of the complexes presented here suggest
that Asn183 OD1 forms a strong hydrogen bond
with this carboxylic group, substantially increasing
its pKa and promoting its protonation.29,30 This
asparagine residue is aided by His233, which also
forms a H-bond to the carboxylate group of uronic
acid and is protonated in the complex, as judged
from it being hydrogen bonded to two acidic
groups (Figure 3(d)).
On the basis of structural evidence from GAG
lyase –oligosaccharide complexes, several proposals have been put forward as to the identity of
the general base and general acid participating in
the reaction. Jedrzejas and co-workers proposed
that the proton is abstracted by a nearby histidine
residue (His233 in the present structure), and that
another proton is donated to O4 by a tyrosine residue (Tyr242 here).11,27,31 They support this proposed role of the histidine residue as the general
base by the close distance between the histidine
NE1 and C5 in their structures. Huang et al. considered three possible mechanistic scenarios, ultimately favoring one in which a tyrosine residue
initially functioned as a general base and subsequently as a general acid.10 In a structurally related
but sequence-distant alginate lyase also, the central
catalytic role was assigned to a tyrosine residue.32,33
The kinetic characterization of FlavoAC with a
synthetic substrate strongly favors tyrosine as the
proton acceptor.28
We have carefully re-analyzed the available
structural data to assess the role of histidine
(His233 in ArthroAC and its equivalent) in catalysis. The first suggestion for the role of histidine as
a general base was derived from the structure of
hyaluronan lyase and its complexes with a disaccharide product,11,34 in which the þ 1 site was not
occupied. These authors modeled the þ 1 sugar
and estimated the NE1· · ·C5 distance to be , 4 Å.
This value corresponds to the N· · ·C van der
Waals distance and seems to be too long for the
proposed proton-abstracting role of the histidine.
A direct observation of the enzyme – substrate
complex was accomplished for FlavoAC(Y234F)10
and for SpHL(Y408F).27 In the case of FlavoAC
(Y234F), it was concluded that the mode of substrate binding in the þ 1 site is influenced by the
Chondroitinase AC Crystal Structure and Mechanism
377
Figure 4. (a) Stereo view of the superposition of the Ca traces of ArthroAC (blue) and SpHL (1EGU) (red); (b) overlay
of oligosaccharide substrates from ArthroAC (blue), FlavoAC(Y234F) (1HMW, magenta) and SpHL(Y408F) (1LXK,
green) based on the superposition of the backbone of active site Asn, His, Tyr (Phe) and Arg residues. The Figure
was prepared with programs sPDBv51 and POV-Raye (http://www.povray.org/).
mutation and does not reflect the reaction intermediate (the C5 carboxylic group occupies the
volume vacated by the missing Tyr hydroxyl
group in the Phe mutant) therefore precluding the
distinction between the possible mechanisms. In
the case of SpHL(Y408F), the distance between
His399 and the C5 of the uronic acid is 3.73 Å, but
the C5 proton lies almost along the line of CD2 –
NE2 bond, very poor geometry indeed for proton
abstraction. If the Phe408 is replaced in this model
by the original Tyr, its hydroxyl O atom would be
only , 3.0 Å from C5 and would have a much
better geometry for interacting with the C5 proton.
Hence, we find these results equally inconclusive
concerning the assignment of His as the general
base.
A structure of the crystals of S. agalactiae hyaluronate lyase soaked for several days in 10 –
50 mM hexasaccharide substrate was reported
recently.31 In the model, the enzyme assumes a
more open conformation and Tyr408 and His399
are further away from the carbohydrate. Specifically, the distance of 5.7 Å between the NE1 atom
of His399 and the C5 atom of the þ 1 sugar does
not allow the conclusion to be drawn that the His
plays the role of general base. As well, the temperature factors in this structure (PDB code 1LXM)
for most of the atoms of the modeled substrate are
equal to 100 Å2, significantly higher than the average value of , 35 Å2 for the surrounding atoms,
suggesting poor order of the substrate. The difference electron density map (not reported in the
378
Chondroitinase AC Crystal Structure and Mechanism
Figure 5. Proposed catalytic mechanism of (a/a)5 GAG lyases. Panel 1, Tetrasaccharide bound in the substrate-binding site. The hydrogen bonding network involving the active site and substrate-binding residues is shown schematically. The tryptophan residues stack against sugars in 21 and þ2 subsites. His233 is protonated and OD1 of Asn183
forms a strong hydrogen bond with the protonated carboxylic group of glucuronic acid. Deprotonated Tyr242 abstracts
the C5 proton. Panel 2, Tyr242 accepts the proton from C5 atom leading to carbanion formation. Now Tyr242 forms
hydrogen bond with bridging O4. Panel 3, The proton from Tyr242 is transferred to the O1 of galactosamine in the
2 1 subsite with concomitant break of C4 –O4 bond and formation of unsaturated ring in þ 1 subsite. Panel 4, Movement of Trp465 triggers the release of disaccharide product from (þ1, þ 2) subsites, reorganization of the active site
and release of product from (22, 2 1) subsites. The enzyme is ready for the next catalytic cycle.
original paper) calculated from the deposited
structure factors and coordinates, with the substrate excluded from calculations, showed weak
and scattered density that in our view cannot be
modeled reliably as a single carbohydrate molecule
(Supplementary Material). The role of His399 as a
general base is further put in doubt by the fact
that His399Ala mutant of SpHL retains a significant fraction (6%) of the wild-type enzyme
activity.11
The present series of timed snapshots of
structures of complexes of the wild-type ArthroAC
with chondroitin 4-sulfate tetrasaccharide substrate
provides very strong support for Tyr242, rather
than His233, playing the role of a general base in
the first step of catalysis. The proposed reaction
mechanism is shown in Figure 5. Substrate binding
to ArthroAC is associated with a deformation of
the glucuronate sugar ring at the þ 1 site from a
chair to a distorted boat and protonation of its
acidic group. Such a change to a higher-energy conformation of the sugar ring is not unusual and has
been observed in other carbohydrate-processing
enzymes.35 – 40 The distortion of the ring brings the
pseudoaxial acidic group coplanar with the amide
group of Asn183, and the presence of an additional
proton leads to the formation of two hydrogen
bonds, one of them being a strong O· · ·H· · ·O
379
Chondroitinase AC Crystal Structure and Mechanism
hydrogen bond. The high-resolution structure of
the complex shows that the Oh atom of Tyr242 is
positioned 2.8 Å from the C5 atom of the þ 1 glucuronic acid, along the direction of the C5 – H
bond. This Oh atom is at the same time within
hydrogen bonding distance of the O atom bridging
þ 1 and 2 1 sugars and of Arg296. We postulate
that Tyr242 becomes deprotonated upon substrate
binding and that the Oh takes up the proton from
the C5 atom of glucuronic acid (Figure 5, panel 2),
which is then transferred to the bridging O atom
2.9 Å away, with a concomitant break of the O4 –
C4 bond (Figure 5, panel 3). A similar mechanism
utilizing a deprotonated tyrosine residue was proposed for alginate lyase A1-III,33 which is structurally similar to ArthroAC despite little sequence
similarity.
What then is the role of His233? This side-chain,
and its equivalents in other lyases, is in the
proximity of the þ 1 glucuronic acid, but with the
NE2 atom at a distance of , 4 Å or more from C5
of the þ 1 sugar, typical for van der Waals interactions. The geometry of the H· · ·NE2 is also such
that the direction of the expected lone pair on NE2
is , 608 away from the direction to the C5 proton.
This geometry is observed consistently in the structures of the FlavoAC and SpH carbohydrate complexes. Finally, NE2 of His233 is hydrogen bonded
to the carboxylate group of the þ 1 glucuronic acid
and, thus, must be protonated. Considering these
facts collectively, it is unlikely that His233 plays
the role of general base to remove the C5 proton.
We postulate that this side-chain has two functions:
(1) it helps properly orient the carboxylate group of
the glucuronic acid through the NE2· · ·O6A hydrogen bond; (2) being protonated in the complex and
in conjunction with Arg296, it lowers the pKa of
Tyr242 leading to the deprotonation of its hydroxyl
group and priming it for the role as a general base
(Figure 5, panel 1). This second function of His233
is directly related to the nearby presence of
Glu407, which engages the histidine ND2 proton
in a hydrogen bond and through electrostatic interactions aids in histidine protonation. Glu407 forms
at the same time a salt-bridge with Arg296, completing a tetrad of hydrogen bonded residues
(Tyr242, His233, Arg296, Glu407) forming the
active site.
The conservation in FlavoAC and hyaluronate
lyases of all Asn183, His233, Tyr242, Arg300 and
Glu407, residues critical for the above described
mechanism supports the view that this is a common catalytic mechanism for this class of enzymes.
A possible mechanism of product release was
illuminated by the structure of thimerosal-soaked
ArthroAC solved here. Of the three bound heavyatoms, two bound to surface-exposed cysteine residues. The third bound to Cys408, which in the
native structure is covered by the 460 –469 loop
and inaccessible to the solvent, suggesting that
this loop is flexible enough to allow access for a
relatively large thimerosal molecule. At the tip of
this loop is Trp465, which provides stacking inter-
actions to the sugar unit of the substrate bound in
the þ 2 site (Figure 3(c)). This loop in ArthroACHg assumes an open conformation, indicating its
intrinsic mobility even in the crystal environment.
When Trp465 is sequestered from the substratebinding cleft, the side-chains of Arg296 and
His233 extend into the volume previously occupied by Trp465 (Figure 6). His233 moves away
from Glu407, forming hydrogen bonds with water
molecules, and is most likely neutral. The saltbridge between Glu412 and Arg296 is broken, but
the side-chain of Glu407 follows Arg296 and
forms a stronger salt-bridge, with two H-bonds.
Movement of this loop after the reaction is completed would substantially decrease the binding of
the product in (þ 1, þ 2) sites and aid in its release
from the enzyme. A rearrangement of residues in
the active site together with their hydrogen bonding network and neutralization of His233 would
lead to reprotonation of Tyr242 making it ready
for the next catalytic cycle (Figure 5, panel 4). We
propose that the deprotonation of Tyr242 and protonation of His233 is concomitant with substrate
binding and triggered by the interaction of the
carboxylate group on the þ 1 sugar with Asn183
and His233.
The access of the substrate to the active site
necessitates either local movements of two to
three loops closing off the binding site,10 or a
hinge movement between the domains leading to
a global movement of the N and C-terminal
domains creating an opening to the active site.27,33
Structural rationale for the exolytic versus
endolytic mode of action
Enzymatic characterization of ArthroAC showed
that it acts as an exolyase, releasing disaccharides
from the glycosaminoglycan substrate,41 while
FlavoAC acts as an endolyase.42 Comparison of
the structures of these two enzymes provides a
rationale for the observed differences in the
mode of action. ArthroAC has two insertions in
the N-terminal domain relative to the FlavoAC: ,15
residues (Arg23– Ser38) and 25 residues (Thr343 –
Gly366). These two segments form a-helicescontaining loops near the N and C termini of the
domain, which come together, closing off a large
part of the cleft running along the side of the (a/a)5
toroidal N-terminal domain. These loops form a
wall that converts the open cleft into a deep cavity
and precludes binding of an extended oligosaccharide. The size of the cavity restricts the binding
of the carbohydrate on the non-reducing end and
can accommodate approximately, two to three
sugar molecules (Figure 7(a)), which correlates
with the exolytic activity of this enzyme. Binding
of a longer carbohydrate would require substantial
rearrangement of these two loops and apparently
does not occur frequently. The open cleft of the
FlavoAC does not impose such a constraint and
allows binding to the middle of a long carbohydrate (Figure 7(b)).
380
Chondroitinase AC Crystal Structure and Mechanism
Figure 6. Stereo view of the conformation of the 460– 469 loop in native and complexed ArthroAC (blue) and in
ArthroAC-Hg (magenta). The location of the thimerosal Hg atom near the Cys408 in the open conformation is shown
as a magenta ball. The side-chains of His233, Arg296, Glu407 and Trp465 are shown explicitly with the hydrogen
bonds marked in broken lines. The þ2 and þ 1 sugars are also shown.
Materials and Methods
Protein purification
The protein was purified from its natural host at
Seikagaku Corporation. To stimulate expression of chondroitin AC lyase II, A. aurescens was grown under the
following culture conditions: medium (32.5 l) containing
0.4% (w/v) peptone (Kyokuto Pharmaceutical Industry
Co. Ltd, Tokyo), 0.4% (w/v) Ehrlich’s fish extract
(Kyokuto Pharmaceutical Industry Co. Ltd, Tokyo) and
0.75% (w/v) chondroitin sulfate C (Seikagaku Co.,
Tokyo); initial pH 6.2; aeration rate 1 vessel volume per
minute; agitation speed 220 rpm; cultivation time 24
hours. The enzyme was purified essentially as
described13 but with some modifications. Briefly, after
bacterial cells were pelleted by centrifugation at 15,000g
for 15 minutes, solid ammonium sulfate was added to
the supernatant fluid up to 75% saturation. At 4 8C, 75 g
of the protein precipitate (50,000 units) was dialyzed
against 20 mM sodium acetate (pH 5.2) and loaded onto
an SP-Sepharose (Amersham Bioscience Corp, Piscataway, NJ) column (2.6 cm £ 70 cm) pre-equilibrated
with the same buffer. The enzyme was eluted with a
linear gradient from 20 mM to 300 mM sodium acetate
buffer (pH 5.2). Enzyme activity and protein amounts
were monitored as described.13 This chromatography
procedure was repeated three times and the enzymecontaining fractions collected and concentrated by
ultrafiltration (Ultrafilter Type P0200, cut-off 20,000 Da,
Advantec, Tokyo Japan) under N2 : Finally, the enzyme
was loaded onto a Sephacryl S-200 HR gel-filtration
column (Amersham Bioscience Corp, Piscataway, NJ)
equilibrated with 0.01 M sodium acetate buffer (pH 5.6)
and eluted with the same buffer. The fractions showing
the highest specific activity and high purity by SDSPAGE were pooled and used in crystallization
experiments.
Oligosaccharide preparation
Chondrotin 4-sulfate tetrasaccharide (CStetra) and hyaluronan tetrasaccharide were prepared and characterized
as described.10 Briefly, chondroitin 4-sulfate from bovine
trachea and dermatan sulfate from porcine intestinal
mucosa were subjected to controlled depolymerization
using chondroitin ABC lyase and the reactions were terminated prior to completion by boiling for five minutes.
Each oligosaccharide mixture was separated on a BioGel P6 column and fractions consisting of tetrasaccharides and hexasaccharides were collected. These mixtures
were further fractionated by strong anion-exchange
HPLC, single oligosaccharides were obtained, and their
purity confirmed by capillary electrophoresis and their
structures by MS and NMR analyses. The structure of
CStetra was DUAp (1 ! 3)-b-D -GalpNAc4S (1 ! 4)-b-D GlcAp (1 ! 3)-a,b-D -GalpNAc4S, where DUAp is the
unsaturated sugar residue, 4-deoxy-a-L -threo-hex-4-is
Chondroitinase AC Crystal Structure and Mechanism
381
Figure 7. Stereo view of the molecular surface within the extended substrate binding site of (a) ArthroAC– tetrasaccharide complex. The insertions in the sequence capping the substrate binding site are shown in green. (b) FlavoAC,
with bound tetrasaccharides. The Figure was prepared with the program GRASP.54
enopyranosyluronic acid; GlcAp is glucopyranosyluronic acid; GalpN is 2-deoxy-2-aminogalactopyranose;
S is sulfate; and Ac is acetate.
Protein crystallization and data collection
Needle-shaped crystals of ArthroAC were reported
many years ago;13 however, they were not characterized.
We obtained crystals by the hanging-drop, vapor-diffusion method in drops containing 2 ml of protein (10 mg/
ml) and 2 ml of reservoir solution (23% (w/v) PEG8000,
0.1 M sodium phosphate buffer (pH 6.4), 0.4 M
ammonium acetate, 10% (v/v) glycerol) suspended
over 1 ml of reservoir solution. Small crystals appeared
within a few days. Larger crystals were obtained by
macroseeding with precipitant concentration lowered to
21% (w/v).
382
Chondroitinase AC Crystal Structure and Mechanism
The crystals belong to the monoclinic space group P21
with cell dimensions a ¼ 57:6 Å, b ¼ 85:5 Å, c ¼ 80:5 Å,
b ¼ 106.98 and contain one molecule in the asymmetric
unit. Prior to data collection the crystals were immersed
for ten seconds in a cryoprotectant solution containing
22.5% (w/v) PEG8000, 0.1 M sodium phosphate buffer
(pH 6.4), 0.4 M ammonium acetate and 20% (v/v)
glycerol, mounted in a nylon loop and flash-cooled in a
cold stream of N2 gas to 100 K. These crystals diffracted
to 1.3 Å resolution at the X8C beamline at Brookhaven
National Laboratory.
Crystals of the protein complexed with thimerosal
were obtained by an overnight soak of native crystals in
the cryoprotectant solution containing 2 mM Hg salt.
These crystals were non-isomorphous with the native
crystals and had cell dimensions: a ¼ 57:4 Å, b ¼ 85:3 Å,
c ¼ 82:2 Å, b ¼ 105.88. Multiwavelength anomalous diffraction (MAD) data at three wavelengths were collected
(Table 7).
Enzyme –substrate complexes
To obtain complexes of ArthroAC with its substrates
we resorted to a series of soaks of the native crystals in
cryoprotectant solution containing the substrate for
times ranging from 30 seconds to ten hours (Table 2).
Native crystals were soaked in the cryo-protecting solution containing 5 mM CStetra or HAtetra for a specified
length of time, then flash-frozen in a cold N2 gas stream
(100 K) on the detector and used immediately for data
collection. There was no significant change in cell dimensions as compared to the crystals of native ArthroAC
(Table 2).
Diffraction data were collected at the X8C beamline,
NSLS, Brookhaven National Laboratory, using the
Quantum-4 CCD detector. The highest-resolution data,
1.25 Å, were obtained from the native crystal soaked for
ten hours in 5 mM CStetra. Data processing and scaling
was performed with HKL2000.43 Data collection statistics
are shown in Tables 2 and 7.
Structure determination and refinement
The structure of ArthroAC was solved from the Hg-
Table 7. MAD data collection statistics
Wavelength (Å)
a (Å)
b (Å)
c (Å)
b (deg.)
Resolution
range (Å) (last
shell)
Rsym (last shell)
Completeness
(%) (last shell)
I=sðIÞ (last
shell)
Total reflections
Unique reflections
Redundancy
Hg—
peak
Hg—
inflection
Hg—
remote
1.005133
1.009078
57.3
85.2
82.1
105.8
40–1.3
(1.35–
1.30)
0.067
(0.325)
98.8
(89.0)
14.7 (6.8)
0.997068
40– 1.3
(1.35–
1.30)
0.066
(0.274)
98.4
(85.0)
14.9
(5.3)
876,240
182,880
4.8
Native
0.950000
57.6
86.5
80.5
106.9
50–1.3
(1.35–
1.30)
0.086
(0.540)
96.4
(93.4)
8.6 (3.4)
884,015
183,424
50–1.4
(1.45–
1.40)
0.067
(0.221)
99.0
(90.7)
13.7
(4.5)
721,975
147,941
1,294,307
178,066
4.8
4.9
7.3
containing thimerosal derivative of ArthroAC. Analysis
of the MAD datasets using the program SOLVE44
revealed three Hg sites in the asymmetric unit. These
sites were used to calculate experimental phases to a
resolution of 1.3 Å and resulted in an overall figure of
merit (FOM) of 0.33 – 1.3 Å. Electron density modification
performed with the program RESOLVE45 assuming a
solvent content of 0.4 led to a significant increase of
the FOM (0.45 at 1.3 Å resolution) and substantially
improved the electron density map. Approximately,
80% of the protein main chain was built automatically
using the program RESOLVE. Additional fragments of
the main chain and many side-chains were built manually using the program O.46 Since the primary sequence
of the protein was unknown, the amino acid type for
each residue was selected to fit the experimental electron
density map. At 1.3 Å resolution, most of the assignments were unambiguous and nearly the entire chain
was traced in the initial map. This initial sequence
assignment was adjusted during the progress of refinement as the electron density features improved. Initial
refinement was performed with the program CNS, version 1.147 and the model was rebuilt manually using the
program O. Subsequently, refinement was continued
using the program REFMAC5, version 5.1.08.48 During
refinement of this model, 1% of the reflections were set
aside for the calculation of Rfree : Water molecules were
initially added automatically with the program CNS
and subsequently updated and corrected by visual
inspection of the difference map. The final model of the
thimerosal-soaked crystal has been refined to an R-factor
of 0.134 and Rfree of 0.156 at 1.3 Å resolution (Table 3).
The current model contains 754 residues (Pro4 – Arg757).
The final amino acid sequence of the model was derived
from the combination of electron density maps and mass
spectrometry data.
Refinement of native protein and enzyme –
substrate complexes
The model of Hg-bound ArthroAC was taken as a
starting point for refinement of the crystal structure of
the native protein. Refinement was performed with the
program REFMAC5. For monitoring of Rfree during
refinement, 1% of reflections were set aside. The electron
density map showed that one loop had a substantially
different conformation and was rebuilt manually. This
model was refined at 1.35 Å resolution to a final R-factor
of 0.130 and Rfree of 0.175. The model contains residues
4 – 757, 1025 water molecules, one sodium ion and one
phosphate ion (present at 0.1 M concentration in the
mother liquor).
This model, in turn, was used to determine the structures of all the complexes of chondroitin AC lyase with
chondroitin 4-sulfate tetrasaccharide (CStetra) with different soaking times (30 seconds, two minutes, ten minutes,
35 minutes, two hours, four hours, ten hours) and of the
HAtetra complex (two minutes soaking time). It was clear
from the height of peaks in the difference electron density map that the occupancies of sugars in sites (22, 2 1)
and (þ 1, þ 2) differed systematically from structure to
structure, in a manner consistent with the enzyme being
active in the crystal. In such a case, the initial concentration of tetrasaccharide would decrease with time,
while that of a disaccharide product increases. At any
time, active sites of some enzyme molecules might
be temporarily empty. Previous structural studies on
chondroitin lyase showed that disaccharides were found
383
Chondroitinase AC Crystal Structure and Mechanism
predominantly in subsites (2 2, 21), and not in (þ 1,
þ2).10 Therefore, at any given time there is a mixture of
tetrasaccharides bound to sites (2 2, 2 1, þ1, þ2) and
disaccharides bound predominantly to sites (22, 2 1).
For that reason, the occupancies of sites (22, 2 1) are
expected to be higher than for sites (þ1, þ 2). To obtain
an estimate of the relative occupancies at the 2 and þ
sites, we assumed that, since the substrate is bound
tightly to the enzyme through numerous hydrogen
bonds, stacking and van der Waals interactions, the temperature factors of the substrate are similar to those of
the residues with which it interacts. We have adjusted
independently the occupancies of sites (22, 21) and
(þ1, þ2) in steps of 0.1 until the average temperature
factor of the substrate was close to that of the surrounding atoms (Table 4). The derived occupancy values provide information about the relative rather than absolute
occupancies. In the HAtetra complex, only two sugar
rings were visible in the difference electron density
map, in positions (22, 2 1) (reaction product). Full
statistics of refinements are summarized in Table 3.
PROCHECK49 showed that all models have good geometry with no outliers.
Protein Data Bank accession numbers
Coordinates of the native chondroitinase AC
(ArthroACnat), the mercury derivative (ArthroACHg),
the two minutes soaked complex with hyaluronan
(ArthroACHA), and 30 seconds, ten minutes and ten
hours soaked complexes with CStetra (ArthroACCStetra) are
deposited in the Protein Data Bank, RCSB, with accession codes 1RW9, 1RWA, 1RWC, 1RWF, 1RWG, 1RWH.
Amino acid sequence assignment
As mentioned above, the amino acid sequence of the
protein has not been previously determined. The high
resolution of the diffraction data and the parallel
independent refinement of several structures (native, thimerosal derivative and several enzyme – substrate complexes) allowed us to deduce the sequence directly from
the electron density maps. This sequence was confirmed
and corrected in several places by mass spectrometry
analysis. For each of the five best datasets at resolutions
1.25– 1.5 Å, the refinements converged at very low Rfactors of 0.114 – 0.138 (R-free of 0.142– 0.178), indicating
highly refined and reliable structures.
Electron density-based assignments
At 1.3 Å resolution the residue types Trp, Tyr, His,
Arg, Lys, Pro, Ile, Leu are defined unambiguously by
their shape (Supplementary Material). The three highest
peaks in the MAD-derived experimental map corresponded to Hg atoms and each was associated with a
cysteine residue. The subsequent 12 highest peaks occur
in the middle of the side-chain electron densities and
were associated either with a cysteine or a methionine
residue, based on the shape of the electron density. The
next group of peaks corresponded to main chain or
side-chain oxygen atoms. During the refinement, three
additional residues on the protein surface were assigned
as methionine based on the refined shape of the electron
density. A total of seven cysteine and 11 methionine
residues were identified. The distinction between Val
and Thr could be made based on the height of the electron density for CG2 versus CG1/OG1, often supported
by the presence of hydrogen bond donors or acceptors
within 3 Å. The assignment of Phe was rather straightforward. The remaining amino acid types, Ser, Ala and
Gly, were assigned based on the shape of the electron
density.
The electron density was convincing for Asx and Glx.
Initially, all Asx were refined as Asp and all Glx as Glu.
When the R-factor dropped below 0.2 we reassigned
these residues to either Gln (Asn) or Glu (Asp) based
on: (1) the height of peaks corresponding to the sidechain atoms; (2) the hydrogen bonding network and the
directionality of hydrogen bonds; (3) sequence conservation in related protein sequences. All these assignments
were re-evaluated during each rebuilding cycle and crosschecked between all independently refined structures.
Mass spectrometry-based assignments
Mass spectrometry was applied to determine the consistency between the measured molecular mass of the
entire molecule and trypsin-derived peptides and the
predicted molecular mass based on the established
amino acid sequence. The molecular mass of the entire
protein was determined on an LC/MS Agilent 1100
mass spectrometer. For MS/MS analysis the protein was
in-gel digested with trypsin. Peptides were separated on
a CapLC HPLC (Waters, Milford, USA) with 0.1% (v/v)
aqueous formic acid and 0.21% (v/v) formic acid in
acetonitrile used for the gradient composition. A volume
of 20 ml of in-gel digest sample was injected onto the
trapping column at a flow-rate of 15 ml/minute and
then washed for five minutes with 0.1% (v/v) aqueous
formic acid. The peptides from the trapping column
were directed to the PicoFrite analytical column (New
Objective, MA) filled with 10 cm of C18 BioBasice packing (5 mm, 300 Å, 75 mm ID £ 10 cm). The spraying tip
of the PicoFrite column was positioned near the
sampling cone of the mass spectrometer and the capillary voltage adjusted to achieve the best plume possible.
The analysis was done on a QTOF-2 mass spectrometer
(Micromass, UK) upgraded with EPCAS electronics. The
instrument was set in data directed analysis (DDA)
mode, where an MS survey scan from 350 to 1600 m/z
was recorded in one second, then the strongest ion was
selected for MS/MS (50 –2000 m/z) for duration of one
second. The DDA was set to select doubly and triply
charged ions. The instrument then switched back to the
MS survey to select the second most intense ion for the
next MS/MS spectrum. The mass spectrometric analysis
was designed to collect a maximum of different ions to
get a maximum of protein coverage. The interscan time
was set to 0.1 second.
The optimized lyase sequence of 757 amino acid residues when matched with the experimental 317 tandem
mass spectra resulted in the assignment of 157 of the
317 tandem MS spectra that identified 37 tryptic fragments (645 residues) that covered 85.2% of the sequence.
Mascot search parameters were restricted to oxidation of
methionine, histidine and/or tryptophan, alkylation of
cysteine by iodoacetamide or in-gel during electrophoresis by acrylamide, and the deamidation of asparagine/
glutamine or the carbamidomethyl derivative of cysteine, with a parent ion mass tolerance of 0.5 Da and
allowing for one missed cleavage. Relaxing the stringency of the search to allow for non-specific proteolysis
by trypsin (and maintaining the parameters above)
resulted in the identification of a further 28 (non-tryptic)
fragments defined by 35 tandem MS spectra with a
384
concomitant increase in coverage by 25 amino acid residues to 88.5%, while further decreasing the stringency
of the matching by increasing the tolerance on the parent
peptide mass to 2 Da resulted in the assignment of
another ten spectra to the sequence. In total, 202 of the
tandem MS spectra were matched to the best sequence
in this manner. The quality of the remaining MS/MS
spectra was insufficient for confirmation of sequence
although sequence tags50 of low confidence could be generated and about 42 of the spectra were weak, sparse and
possibly correspond to background noise. An accounting
of the peptides as predicted from the X-ray data but not
observed by tandem MS indicated that several short
tryptic peptides (residue numbers 44 – 50, 51 – 57, 175–
176, 295– 296, 297– 300, 342– 343, 356– 360, 385– 388,
496– 501, 502, 586– 588, 639, 640– 645, 646– 650, 662– 664,
752– 754, 755– 756, 757) that account for 64 residues
were most likely lost during the loading/washing of the
reversed-phase LC column, whereas the rather large
tryptic fragment (residues 503–525) that is well defined
by the X-ray data may have remained in the gel during
the robotic in-gel digestion/extraction and/or remained
bound to the reversed-phase column throughout the
LC-QToF MS analysis. Results of Mascot analysis and
samples of MS/MS spectra for several peptides are
deposited as Supplementary Material.
An example of the experimental and the refined electron density map for the region around Asn183 is
shown in Supplementary Material. Finally, the sequence
of ArthroAC derived by combining information from
electron density maps and mass spectrometry was
aligned with related enzymes based on their structural
superposition (Figure 1).
At completion of this process we were confident of the
identity of 746 out of 754 modeled residues (98.9%). For
three additional residues (Asn/Asp and Gln/Glu) we
are less certain of the assignment and it is based primarily on differences in the B-factors and potential formation
of hydrogen bonds. Finally, five residues located in flexible loops exposed to solvent and with B-factors of more
than 30 Å2 (average B for the protein is , 15 Å2) and for
which no fragment was found in MS/MS data could
not be identified unambiguously. These residues are distant from the substrate binding site (marked in Figure 1).
At the end of the structure determination process we
have determined the N-terminal amino acid sequence of
21 residues of a dissolved crystal by Edman degradation.
The first three N-terminal residues are disordered in the
structure, the identity of the subsequent 18 residues
agrees with the electron-density-based assignments.
Kinetic analysis
Glycosaminoglycans CS-A (19,000 Da), CS-C (43,000 Da),
CS-D (30,000 Da), low molecular mass hyaluronic acid
(50,000 Da) and hyaluronic acid (1,000,000 Da) were
obtained from Seikagaku Corporation (Tokyo, Japan).
Arthro AC was dissolved at concentration of 50 mU/ml
in 0.1% (w/v) BSA (solution A). Each chondroitin sulfate
and lower molecular mass of hyaluronic acid was dissolved in distilled water at concentration of 10 mg/ml,
whereas hyaluronic acid was dissolved in it at concentration of 2 mg/ml, respectively. From 10 ml to 200 ml of
solution containing each GAG thus prepared was added
to 160 ml of 0.4 M sodium acetate buffer (pH 6.0) and
the appropriate amount of distilled water was added to
each solution to adjust the final volume to 700 ml (solution B). Each solution B was incubated at 37 8C. After 30
Chondroitinase AC Crystal Structure and Mechanism
seconds, 100 ml of solution A was added to solution B
and the increased absorbance (ABS) at 232 nm due to
the generation of unsaturated bonds in disaccharides
caused by Arthro AC was monitored at every 30 seconds
by spectrophotometer (UV 160A, Shimadzu, Kyoto,
Japan). Reaction velocities (v) at different concentration
of the GAGs were plotted as DABS=Dt: Since all GAGs
used in the experiment were catalyzed to disaccharide
units, we calculated substrate concentrations of the
GAGs as molar basis of disaccharide units contained in
each GAG. For example, in the case of the CS-A, it was
composed of 95.5% of mono-sulfated disaccharide unit
(503.3 Da), 3.0% of di-sulfated disaccharide unit (605.3 Da)
and 1.6% of non-sulfated saccharide unit (401.3 Da).
Therefore, mean molecular mass of the CS-A as disaccharide unit can be calculated to be 505.2. Accordingly,
mean molecular mass of the CS-C, the CS-D and both
hyaluronic acid from the disaccharide-unit basis were
calculated as 511.2 Da, 532.2 Da and 401.3 Da, respectively. By use of substrate concentration based on this
calculation, a Lineweaver –Burke plot was obtained, and
V and KM of every GAG were determined.
Acknowledgements
We thank Drs Allan Matte, Joseph D. Schrag and
J. Sivaraman for help with data collection,
Ms France Dumas for amino acid sequencing,
Ms Christine Munger, Dr Daniel Boismenu,
Mr Sajid Karsan and Montreal Proteomics Network
for mass spectrometry analysis. This work was
supported, in part, by CIHR grant 200009MOP84373-M-CFAA-26164 to M.C. and NIH grants
GM38060 and HL62244 to R.J.L.
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Edited by I. Wilson
(Received 23 September 2003; received in revised form
19 December 2003; accepted 29 December 2003)
Supplementary Material comprising Figures
showing electron density maps at various contour
levels and Tables and plots displaying peptide
identification by mass spectrometry is available on
Science Direct