Vitamin C Inhibits the Enzymatic Activity of Streptococcus pneumoniae Hyaluronate Lyase*

Songlin LiDagger , Kenneth B. Taylor§, Stephen J. KellyDagger , and Mark J. JedrzejasDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -domain and a C-terminal beta -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 beta -1,4-glycosidic linkage between HA1 and HA2 disaccharide units in the hyaluronan chain (8, 20).2


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Fig. 1.   2Fo-1Fc electron density map (1 sigma ) 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.


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Fig. 2.   The Vc binding position in the SpnHL structure. The positions of the N-terminal alpha -domain and C-terminal beta -domain are shown. One Vc molecule is bound to the cleft between two structural domains.

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.


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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.

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 beta -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.

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

                              
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Table I
Protein-Vc interface
Hyaluronate lyase was complexed with vitamin C.

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 beta -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.


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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.

Residues Asn-290 and Asn-580 form the narrowest part across the cleft. Asn-580 is the only residue from the beta -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.

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.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -1,4-glucuronic-beta -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.

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.


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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.

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.

    ACKNOWLEDGEMENTS

Diffraction data were collected at the Brookhaven National Laboratory, National Synchrotron Light Source at the beamline X25.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: Vc, L-ascorbic acid known as vitamin C; HA, hyaluronan; SpnHL, S. pneumoniae hyaluronate lyase.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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