From the Department of Microbiology and the
§ Department of Biochemistry and Molecular Genetics,
University of Alabama at Birmingham, Birmingham, Alabama 35294
Received for publication, December 11, 2000
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ABSTRACT |
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Enzyme activity measurement showed that
L-ascorbic acid (vitamin C (Vc)) competitively
inhibits the hyaluronan degradation by Streptococcus
pneumoniae hyaluronate lyase. The complex crystal structure of
this enzyme with Vc was determined at 2.0 Å resolution. One Vc
molecule was found to bind to the active site of the enzyme. The Vc
carboxyl group provides the negative charges that lead the molecule
into the highly positively charged cleft of the enzyme. The Vc ring
system forms hydrophobic interactions with the side chain of
Trp-292, which is one of the aromatic patch residues of this
enzyme responsible for the selection of the cleavage sites on the
substrate chain. The binding of Vc inhibits the substrate binding at
hyaluronan 1, 2, and 3 (HA1, HA2, and HA3) catalytic positions. The
high concentration of Vc in human tissues probably provides a low level
of natural resistance to the pneumococcal invasion. This is the first
time that Vc the direct inhibition on the bacterial "spreading
factor" was reported, and Vc is also the first chemical that has been
shown experimentally to have an inhibitory effect on bacterial
hyaluronate lyase. These studies also highlight the possible
structural requirement for the design of a stronger inhibitor of
bacterial hyaluronate lyase.
L-Ascorbic acid, also known as vitamin C
(Vc),1 is synthesized in
plants and in almost all animals except primates, guinea pig, Indian
fruit bat, and some insects. Vc is necessary in the diet of these
animals and usually exists in significantly large concentrations in
their tissues (1). Prolonged lack of Vc in the diet of humans results
in scurvy, which is characterized by skin lesions, blood vessel
fragility, and poor wound healing. Less severe deficiency of Vc
produces alterations in connective tissue structure and may also cause
decreased resistance to some infections (2). Vc is a multifunctional
molecule in tissues. It usually acts as an antioxidant (3), free
radical scavenger (4), neuroprotectant, and neuromodulator (5). It also
plays an important physiological function in activating peptide
hormones (2) and regulating cell division and growth (6). Vc is the single synthetic chemical that is manufactured and consumed in the
greatest quantity in the world (1). Although the importance of
Vc in the normal function of animal tissues has long been known, the
detailed molecular basis of Vc action, especially the mechanisms of its
interactions with proteins and enzymes, is still largely unknown.
Protein-ligand interaction is an important aspect of modern
biochemistry. It provides information for the understanding of the
essence of the molecular interactions, enzyme action mechanism, protein
activity control, and drug design. In regards to the important function
of Vc in so many life processes, the protein-Vc interfaces have not
been fully characterized to date. The structural basis of the
protein-Vc interface and the possible influence of Vc on enzyme
activities are certainly issues of significant interest for the
understanding of the functions and principles of Vc action. The
protein-Vc interaction was first seen in the crystal structure of
D-xylose isomerase (Protein Data Bank accession number
1xid) in which Vc was present between two tryptophan residues (7). It
clearly emphasizes the importance of hydrophobic interactions in the
protein-Vc interface. Here we present the crystal structure of
Streptococcus pneumoniae hyaluronate lyase (SpnHL)
co-crystallized with Vc, which provides another opportunity to examine
the protein-Vc interactions at the atomic level.
S. pneumoniae is a Gram-positive bacterial pathogen that
causes pneumonia, bacteremia, meningitis, sinusitis, and otitis media in humans worldwide, especially in neonates and children, and often
leads to significant rates of mortality and morbidity. It secretes
hyaluronate lyase to catalyze the degradation of hyaluronan (HA), one
of the main components of connective tissues in animals, to expose
tissue cells to bacterial toxins. Therefore, SpnHL is also being called
the "spreading factor" (8, 9). The inhibition of hyaluronate lyase
is expected to reduce the spreading of this pathogen in the most early
stages of pneumococcal invasion. The action mechanism of hyaluronan
degradation by SpnHL, which was revealed recently, provides a unique
opportunity to target hyaluronate lyase in the prevention of the
pneumococcal invasion. The Vc-complexed crystal structure of SpnHL is
an attempt on this line of research.
The enzymatic activity of SpnHL in the presence of various
concentrations of Vc was measured. The crystal structure of this enzyme
co-crystallized with Vc was determined at 2.0 Å resolution. The
structural basis of the inhibitory effect of Vc on SpnHL enzymatic activity was established.
Crystallization and Data Collection--
L-Ascorbic
acid (Sigma) was co-crystallized with the S. pneumoniae
hyaluronate lyase at conditions similar to the native SpnHL crystallization condition with an additional 10-100 mM
ascorbic acid. A set of data at 2.0 Å resolution was collected at 100 K using one crystal at 50 mM ascorbic acid, synchrotron
radiation, and a cryocooling technique at conditions similar to the
native crystal (10, 11). Diffraction data were processed and scaled using the HKL program package (12). The crystal belongs to the orthorhombic space group P212121
with cell parameters of a = 84.264 Å,
b = 102.666 Å, and c = 103.253 Å. The data set is 90.1% complete (60% in the last shell)
with Rsym = 0.087.
Structure Refinement and Validation--
Coordinates and
B-factors of the protein part of the native SpnHL crystal structure (8)
were used directly as a primary model in the SpnHL·Vc complex
structure refinement. All waters and solution molecules were omitted
from the model. The X-plor package (13) was used to refine the
structure against 54,217 reflections at a 2.0-45 Å resolution range
(87.8% completeness), and 1% reflections were used in
Rfree calculation to monitor the refinement
progress and the model improvement (14). Rigid body, position, and
simulated annealing (3000 K) protocols were employed. The model was
manually fitted into the electron density maps on graphics using
program O (15) in between each round of refinement calculations.
The electron densities for Vc were observed from the beginning, but the
Vc structure was only included into the structural refinement until the
R factor dropped below 25%, and water molecules were
incorporated thereafter. Only after this point, the B-factor refinement
was introduced.
Microplate Assay of SpnHL Activity--
The SpnHL enzymatic
activity was measured using a modification of previously described
protocols using either cetylpyridinium chloride (16) or cetrimide (17).
The compounds assayed for inhibitory effects on the SpnHL enzyme
activity were ascorbic acid, epinephrine, apigenin, salicylic acid, and
histamine (Sigma).
Volumes of 90 µl of inhibitor solutions at concentrations of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 mM containing 50 mM sodium acetate and 10 mM calcium chloride at
pH 6.0 (apigenin was dissolved in 10% Me2SO water
solution) were added along one row of a 96-well microtiter plate,
leaving the first row as a blank control. 10 µl of the wild-type
SpnHL at a concentration of 3.3 µg/ml was added to each well and
incubated at room temperature for 1 h. Reactions using 40 units of
bovine hyaluronidase (Sigma) at the same conditions were run in
parallel. 25 µl of 1 mg/ml HA were added to each well to start the
reaction, which was performed at room temperature for 15 min.
Undigested HA was precipitated using freshly made 50 µl of 10% (w/v)
aqueous cetylpyridinium chloride with an additional 2.0% NaOH to stop
the enzyme activity. The absorbance was measured at 630 nm for
cetylpyridinium chloride using an automated microplate reader (EL808,
Bio-Tek Instruments, Inc., Winooski, VT). A higher absorbance reading
corresponds to a higher remaining substrate HA concentration and,
therefore, lowers enzyme activity. The
A595 absorbance readings were then converted to percent inhibition by subtracting the
A595 value for the pure enzyme without inhibitor
from the reading with inhibitor divided by the difference between the
100% inhibition (average values at 12.8, 14.4, and 16.0 mM
ascorbic acid) and 0% inhibition (A595 reading
for the pure enzyme).
To determine whether this inhibition was reversible, a sample of SpnHL
enzyme activity was measured and designated as 100% activity. After
dialyzing the sample against 20 mM Vc in buffer, the enzyme
activity dropped to 0%. The sample was then dialyzed against the
reaction buffer to remove the Vc, and 94% of the enzyme activity was
recovered. Therefore, this inhibition was reversible. The slight drop
in activity was caused by the volume changes during dialysis.
The reaction initial velocity was measured in quadruplicate using
a modified microplate assay (16). Initial hyaluronan concentrations were 0.2, 0.1, 0.07, 0.05, and 0.04 mg/ml in 50 mM acetate
buffer, pH 6.0, 10 mM calcium chloride, and 8% agarose at
55 °C. 100 µl of each of the five concentrations of the
hyaluronan-agarose gel were added to the microplate columns in
quadruplicate and left at room temperature for an hour to set.
Solutions of SpnHL (0.33 µg/ml) with 0, 1, 5, and 10 mM
Vc in 10 mM calcium chloride and 50 mM acetate
buffer, pH 6.0, were preincubated at 37 °C for 1 h, and 100 µl of each of the enzyme preparations were added to the first four
lines of each column of the microplate. Reaction buffer was added to
the remaining four lines of the microplate as control. The plates were
then incubated at 37 °C for 14 h. The enzyme samples were
removed, and each well was washed three times with the buffer. Each
well was then filled with 100 µl of 2% aqueous cetrimide
(hexadecyltrimethylammonium bromide) and incubated at 37 °C for
2 h. The absorbance was measured at 405 nm using an automated
microplate reader (EL808).
Data Deposition--
Structural factors and coordinates of
SpnHL·Vc complex have been deposited in the Protein Data Bank with
accession number 1f9g.
Overall Structure of SpnHL·Vc Complex--
The crystal
structure of S. pneumoniae hyaluronate lyase in complex with
Vc (SpnHL·Vc complex) was determined at 2.0 Å resolution. In total,
725 of 731 residues were modeled, and 303 waters were incorporated. One
Vc molecule was clearly seen in the electron density map (Fig.
1). The final crystallographic
R factor was 0.208, and Rfree = 0.253. The protein part of the complex structure contained an
N-terminal
The protein part of the SpnHL·Vc complex structure has only slight
changes when compared with the native SpnHL crystal structure. The root
mean square deviation is only 0.538 Å for all protein atoms. In the
cleft region, all corresponding atoms in the protein-Vc complex
structure are located at the outer side, leaving the cleft about 0.2 Å wider than it is in the native SpnHL structure (8). The position of Vc
relative to the active site residues is shown in Fig.
3a.
The aromatic patch of the enzyme active center is composed of
three aromatic residues, Trp-291, Trp-292, and Phe-343 (8) (Fig.
3a). The side chains of Trp-292 and Phe-343 form hydrophobic interactions with the hydrophobic patches on the hyaluronan chain. Through this matching, the cleavage sites are selected. Trp-292 hydrophobically interacts with a HA2 disaccharide unit and accurately anchors HA2 into catalytic position. In the hyaluronan proton acceptance and donation degradation model,2 the
enzyme catalyzed the degradation of the The Binding of Vc to SpnHL--
The bound Vc forms 25 interactions
with 7 residues of the enzyme (Table I).
These residues are also shown to interact with the substrate (8). The
relative positions of these residues to the bound Vc are shown in Fig.
3b. 5 of the 7 interface residues, Arg-243, Asn-290,
Trp-292, Tyr-408, and Asn-580, have been studied extensively in our
previous biochemical and structural studies and have been shown
to play important roles in the normal function of this enzyme (8,
20).3
Trp-292 forms eight interactions with Vc, which accounts for the most
interactions among all these seven residues in the protein-Vc interface. The indole group of Trp-292 is in parallel to the
five-member ring of Vc (Fig. 3a). This structural
arrangement provides the main hydrophobic interaction that stabilizes
the Vc molecule inside the cleft. Trp-292 is one of the aromatic patch
residues and is responsible for the selection of the cleavage sites on
the substrate chain. Therefore, the binding of Vc probably competes
with the binding of hyaluronan substrate at the HA2 position, which is located at the middle of the cleft.
Tyr-408 is one of the three key catalytic residues in the hyaluronan
degradation. It donates one proton to the glycosidic oxygen to break
the
Residues Asn-290 and Asn-580 form the narrowest part across the cleft.
Asn-580 is the only residue from the
The cleft is highly charged positively by the accumulation of lysine
and arginine residues inside the cleft. There are nine conserved
arginine residues present in the cleft. Three of them are involved in
the interaction with Vc. Arg-462 and Arg-466 are involved extensively
in Vc binding (Table I). Arg-243 forms one salt bridge with the Vc
carboxyl group. When the substrate is bound into the cleft, Arg-243
interacts with HA2 and HA3 disaccharide units and with the oxygen atom
of the second glycosidic linkage, which is suspected to be the next
glycosidic linkage to be degraded. The mutation R243V surprisingly
decreases the enzyme activity by 33% (8). In our previous studies, we
proposed that Arg-243 plays an important role in the substrate
translocation after the initial glycosidic bond is degraded
(9).2 The glycosidic linkage in contact with Arg-243 is the
next linkage to be degraded. The interaction with Arg-243 suggests that
the Vc binding also inhibits substrate binding at HA3 position. It was
recently reported that one arginine residue is believed to be involved
in two Vc binding sites in the Vc-peroxidase complex structure (22).
The presence of one or more arginine residues is probably one of the
characteristic features in the protein-Vc interface.
The 25 interactions in the SpnHL·Vc interface (Table I) can be
classified into two groups, hydrophobic and ionic interactions. Trp-292
contributes mostly to the hydrophobic interactions with Vc. Three
arginine residues, Arg-243, Arg-462, and Arg-466, form several salt
bridges, whereas Tyr-408, Asn-290, and Asn-580 form hydrogen bonds with
the ligand. In comparison with the protein-Vc interface observed in the
D-xylose isomerase in which hydrophobic interactions play
the dominant role, hydrophobic and ionic interactions are almost
equally important in the SpnHL·Vc interface. Therefore, the
SpnHL·Vc interface represents a novel type of protein-Vc interface.
The protein-Vc interactions cause minor structural changes at both
parts of the interface. In comparison with the native SpnHL structure,
the side chain displacement of these interface residues is very small.
On the contrary, Vc itself has relatively significant structural
changes compared with its native crystal structure (23) used for
modeling and refinement. The carboxyl group is forced to bend toward
the plane of the Vc ring (Fig. 4). The negative charges caused by the
carboxyl group at physiological condition are apparently important in
leading the Vc molecule into the highly positively charged cleft. The
actual binding geometry shows that the five-member ring of the Vc
molecule provides the most hydrophobic binding interface, whereas the
carboxyl group interferes with the Trp-292 indole group, which is not
in favor of the Vc binding.
Inhibitory Effect of Vc on SpnHL Enzyme Activity--
Vitamin C,
salicylate, and flavonoids have been reported to have certain
inhibitory effects on the enzyme activity of hyaluronidases (18), which
are a group of hydrolases employed by mammals for the hyaluronan
degradation. Bacteria usually produce hyaluronate lyases to degrade
hyaluronan. The search for the inhibitors of bacterial hyaluronate
lyases was started from these chemicals. Our activity measurements
clearly showed that Vc inhibited the hyaluronan degradation by SpnHL.
Vc is structurally similar to one of the sugar units of hyaluronan, the
main substrate of SpnHL. Hyaluronan is composed of linear repeats of
the disaccharide unit
The effects of these compounds on the enzyme activity of SpnHL and
bovine hyaluronidase were investigated and measured by the microplate
enzyme activity essay. The results showed that none of these compounds
had any significant influence on the activity of bovine hyaluronidase
at our experimental condition (data not shown). However, Vc inhibited
the SpnHL activity (Fig. 5). At our
experimental condition, the IC50 of this inhibition was
approximately 5.8 mM.
The initial velocity of the degradation at various concentrations of
hyaluronan was measured in the presence of three concentrations of Vc
and in its absence. The results were fitted to both the competitive
model and the noncompetitive model by nonlinear regression. The fitting
attempts with the noncompetitive model repeatedly resulted in an
unreasonably high value (Ki > 50,000 mM) for the Ki parameter, which is
not present in the competitive model. Because this parameter
indicates that the concentration of Vc is required for the binding to
the enzyme-substrate complex, it is apparent from this analysis that Vc
binds only to the free enzyme (Ki = 53 mM), not to the enzyme-substrate complex in the
experiments described in this work. Therefore, the substrate competes
successfully for the binding of Vc, and this inhibition is competitive.
Physiological Significance--
The degradation of
hyaluronan in the host connective tissues is an important step in the
pneumococcal invasion. The bacterial strains that produce more
hyaluronate lyase were shown to be more virulent than those strains
producing less hyaluronate lyase (24). S. pneumoniae strains
with hyaluronate lyase and cell toxin pneumolysin double mutations
showed significant additive attenuation in virulence (25). Therefore,
the inhibition of hyaluronate lyase activity is probably important in
the control of the pneumococcal invasion. And because animals usually
use hydrolases to degrade hyaluronan, pneumococcal hyaluronate lyase
becomes a potential target for developing a novel antibacterial agent.
Vc is the first chemical shown to have inhibition effect on the
activity of this pneumococcal enzyme.
The human adult minimum daily requirement for Vc is ~10
mg. Vc exists in human tissues at a level of 0.2 to >10 mM
concentrations and has an unusually varied distribution compared with
other vitamins (26, 27). Large concentrations of Vc were detected in
the adrenal gland and the aqueous humor of the eye. Human corneal epithelium normally contains approximately 1.33 mg of Vc/g of wet
weight tissue (27), which corresponds to approximately 7.5 mM Vc. In the activated human neutrophils, internal Vc
concentrations as high as 14 mM were detected when external
vitamin was kept at physiological concentrations (28). Therefore, the
inhibitory effect of Vc on the SpnHL enzymatic activity may have a
physiological meaning. It has long been known that the deficiency of Vc
may cause decreased resistance to some bacterial infections. One
explanation to this belief is that animal cells, such as neutrophils,
generate oxidants to kill bacteria using Vc to quench and control the
extra oxidants released (28). The inhibitory effect of Vc on SpnHL activity provides an additional possible explanation to the Vc function
as an antibacterial agent. The large concentration of Vc in human
tissues makes the tissue environment more unfavorable to the
pneumococcal invasion, thereby providing a low level of natural
resistance to such bacterial invasion. The infections and diseases
caused by pneumococci are thus significantly reduced. Therefore, Vc is
probably a natural constituent of the biochemical defense system
against the pneumococcal invasion in the host tissues. Pneumococcal
invasions usually occur in tissues with relatively low concentrations
of Vc. The normal Vc concentration in plasma is around 0.1-0.2
mM and about 10 times higher in the lungs, brain, kidneys,
lymph glands, and small intestinal mucosa (19), which is about 10 times
less than the highest Vc concentrations detected in human tissues.
The SpnHL·Vc complex structure also provides some clues for the
design of a more efficient hyaluronate lyase inhibitor. Based on the
interface characteristics, it can be expected that a stronger hyaluronate lyase inhibitor should have a larger ring system to benefit
the hydrophobic binding to the Trp-292 indole group. At least one
negative charge provider, a carboxyl group, for example, is required in
the inhibitor structure to provide the negative charges to lead the
inhibitor into the cleft region. In summary, a stronger inhibitor can
be expected to have an increased area of hydrophobic interactions and
to have more properly placed negatively charged substituents such as
carboxyl groups.
The molecular properties of Vc are closely related to its
structural characteristics. The widely studied free radical
scavenger and antioxidant properties of Vc are directly related to
the active redox chemical characteristics of this molecule. Our
studies emphasized the significance of these structural
similarities of Vc, a sugar derivative, to polysaccharides. This
structural similarity confers on Vc the capacity of protecting
hyaluronan, the main component of connective tissues, from being
degraded by bacterial hyaluronate lyases. Any destructive factors of
polysaccharides, oxidants or hyaluronate lyases, may be buffered by the
existence of the large amount of Vc in tissues. The Vc structural
similarities to sugars, its interaction patterns with proteins revealed
from the SpnHL·Vc complex structure, and the importance of both
hydrophobic and ionic contacts in the protein-Vc interface might lead
to the reevaluation of the structure and function relationships of
Vc.
Conclusions and General Implications--
Vc may compress or
retard bacterial invasion by directly inhibiting the bacterial
spreading factor, such as hyaluronate lyase, through binding to the
enzyme active site and competing with the binding of the hyaluronan
substrate. All seven protein interface residues interacting with Vc are
strictly conserved among all known bacterial hyaluronate lyases (8).
The studies on the SpnHL·Vc interface are thus significantly relevant
to all these bacterial hyaluronate lyases. For example,
Streptococcus agalactiae hyaluronate lyase crystal structure
was recently determined (21).2 Its active center
construction and geometry is nearly the same as it is in SpnHL.
Therefore, the results shown may be applicable to Streptococcus
agalactiae hyaluronate lyase, which means that Vc might also
provide the host with the ability to resist the S. agalactiae invasion to a certain extent.
This is the first time that the direct action of Vc on a
bacterial spreading factor has been observed. The structural basis of
this inhibition is due to the structural similarity of Vc to the
glucuronate residues in hyaluronan, the substrate of hyaluronate lyases. The inhibitory effect, confirmed by our enzyme activity measurements and the SpnHL·Vc complex structure studies, shows that
Vc is probably directly involved in the inhibition of bacterial invasion in addition to its antioxidant and free radical scavenger properties.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-domain and a C-terminal
-domain connected by a
10-residue linker. The active site of this enzyme lies in the middle of
the molecule in which a predominant cleft was formed between these two
structural domains (Fig. 2). The cleft
was about 30 × 10 Å in dimension, enough to accommodate three
disaccharide units of the hyaluronan substrate chain
simultaneously, which were named HA1, HA2, and HA3, respectively, from
the reducing end to the nonreducing end of the hyaluronan
chain.2 The active site is
located at one end of the cleft, corresponding to the reducing end of
the bound hyaluronan chain, and is composed of two parts, an aromatic
patch responsible for the cleavage site selection on the substrate
chain and a catalytic group responsible for the cleavage of the
-1,4-glycosidic linkage between HA1 and HA2 disaccharide units in
the hyaluronan chain (8, 20).2
View larger version (54K):
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Fig. 1.
2Fo 1Fc
electron density map (1
) for the refined Vc
bound to the active site of SpnHL. One hydrogen of a water
molecule that bonded to a Vc oxygen atom can be identified.
View larger version (61K):
[in a new window]
Fig. 2.
The Vc binding position in the SpnHL
structure. The positions of the N-terminal -domain and
C-terminal
-domain are shown. One Vc molecule is bound to the cleft
between two structural domains.
View larger version (33K):
[in a new window]
Fig. 3.
The environment of Vc in the cleft of the
S. pneumoniae hyaluronate lyase. A,
the relative position of Vc to the active center residues.
B, all residues interacting with Vc.
-1,4-glycosidic linkage
between HA1 and HA2 and produced 4,5-unsaturated HA1. Vc in the
SpnHL·Vc complex structure was found to bind to Trp-292 (Fig.
3a) indole group and occupy the HA2 position.
Protein-Vc interface
-1,4-glycosidic linkage between HA1 and HA2 disaccharide units
(8). In the complex structure, Tyr-408 forms one salt bridge with the
Vc O-1 oxygen (refer to Fig. 4 for Vc atom labeling). Therefore, both the aromatic patch and the catalytic group of the active center of SpnHL are involved in the binding of Vc.
The binding of Vc blocks both the aromatic patch and the catalytic
group.
View larger version (36K):
[in a new window]
Fig. 4.
Structural comparison of protein-bound Vc
overlapped with the Vc crystal structure. Hydrogen atoms are not
shown. Vc atoms were labeled as conventional (23). SpnHL-bound
Vc is shown in green, whereas the Vc crystal structure is in
lavender. A large atomic displacement in the carboxyl group
atoms can be seen, whereas the ring atoms are less displaced.
-domain that is involved in the
SpnHL-substrate and the SpnHL·Vc interface. The mutation N580G causes
a small increase (about 15%) in the enzyme activity because the wider
cleft opening
allows for easier substrate entry (8).3 SpnHL·Vc complex
structure showed that Vc is also in contact with Asn-580 and
Asn-290.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,4-glucuronic-
-1,3-glucosamine. One of
the main components of hyaluronan, glucuronic acid, is also the
precursor in the Vc biosynthesis. Therefore, Vc can be regarded as a
substrate analogue of hyaluronate lyase.
View larger version (8K):
[in a new window]
Fig. 5.
Enzyme activity of SpnHL in the presence of
ascorbic acid. The final enzyme concentration was 0.33 mg/ml. The
final Vc concentrations were 0.8-16 mM. Each
point represents the averaged value of seven parallel
measurements. Vc-inhibited SpnHL activity is shown in a
dose-dependent manner with 50% inhibition
(IC50) at approximately 5.8 mM.
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ACKNOWLEDGEMENTS |
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Diffraction data were collected at the Brookhaven National Laboratory, National Synchrotron Light Source at the beamline X25.
![]() |
FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AI 44079 (to M. J. J.).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 the structure factors (code 1f9g) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed: Dept. of Microbiology, 933 19th St. South, 545 CHSB-19, UAB, Birmingham, AL 35294-2041. Tel.: 205-975-7627; Fax: 205-975-5424; E-mail: jedrzejas@uab.edu.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M011102200
2 S. Li and M. J. Jedrzejas, submitted for publication.
3 S. J. Kelly, K. B. Taylor, S. Li, and M. J. Jedrzejas, Glycobiology, in press.
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ABBREVIATIONS |
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The abbreviations used are: Vc, L-ascorbic acid known as vitamin C; HA, hyaluronan; SpnHL, S. pneumoniae hyaluronate lyase.
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REFERENCES |
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