Vitamin C inhibits the enzymatic activity of Streptococcus pneumoniae hyaluronate lyase · 2001-01-12 · Summary Enzyme activity measurement showed that L-ascorbic acid (vitamin

Vitamin C inhibits the enzymatic activity of Streptococcus

pneumoniae hyaluronate lyase

Songlin Li1, Kenneth B. Taylor2, Stephen J. Kelly1, and Mark J. Jedrzejas1*

1Department of Microbiology, 2Department of Biochemistry and Molecular Genetics

University of Alabama at Birmingham, Birmingham, AL 35294, USA

Running title: Hyaluronate lyase complexed with vitamin C

* To whom correspondence should be addressed at

Department of Microbiology

933 19th Street South, 545 CHSB-19

The University of Alabama at Birmingham

Birmingham, Alabama 35294-2041, USA

Tel./fax: (205)975-7627/(205)975-5424

email: [email protected]

Hyaluronate lyase complexed with vitamin C

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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 12, 2001 as Manuscript M011102200 by guest on M

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Summary

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 (PDB ID code: 1f9g). 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 Trp292 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 HA1, HA2, and

HA3 catalytic positions. The high concentration of Vc in human tissues likely provides a

low level of natural resistance to the pneumococcal invasion. This is the first time that Vc

inhibition directly on the bacterial “spreading factor” was reported and Vc is also the

first chemical that had experimentally shown to have 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.

Keywords: Streptococcus pneumoniae, hyaluronate lyase, vitamin C, enzymatic activity

inhibition, antibacterial activity, protein-inhibitor complex


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Introduction

L-ascorbic acid, also known as vitamin C (Vc1), is synthesized in plants and

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, characterized by the 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 multi-functional molecule

in tissues. It usually acts as 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 manufactured and consumed in the greatest amount 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, are 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 of the drug design. In regards to

the important function of Vc in so many life processes, the protein-Vc interfaces have

________________________1 Vc: vitamin C, L-ascorbic acid. SpnHL: Streptococcus pneumoniae hyaluronate lyase. GbsHL: S. agalactiae hyaluronate lyase.


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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 (PDB ID

code: 1xid) where Vc is 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)

cocrystallized with Vc which provides another opportunity to examine the protein-Vc

interactions at the atomic level.

S. pneumoniae is a Gram-positive bacterial pathogen which causes pneumonia,

bacteremia, meningitis, sinusitis, and otitis media in humans world wide, 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 “spreading factor” (8,9). The inhibition of

hyaluronate lyase is expected to reduce the spreading of this pathogen in the most early

stages of the pneumococcal invasion. The action mechanism of hyaluronan degradation

by SpnHL 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 at the presence of various concentrations of Vc

were measured. The crystal structure of this enzyme cocrystallized with Vc was

determined at 2.0 Å resolution. The structural basis of the inhibitory effect of Vc on


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SpnHL enzymatic activity was established.

Experimental Procedures

Crystallization and data collection L-Ascorbic acid (Sigma) was cocrystallized with

the S. pneumoniae hyaluronate lyase at conditions similar to the native SpnHL

crystallization condition with additional 10 to 100 mM ascorbic acid. A dataset at 2.0 Å

resolution was collected at 100 K using one crystal at 50 mM ascorbic acid, synchrotron

radiation and cryocooling technique at conditions similar to the native crystal (10, 11).

Diffraction data were processed and scaled using HKL program package (12). The crystal

belongs to the orthorhombic space group P212121 with cell parameter a = 84.264 Å, b =

102.666 Å, 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 primary model in the

SpnHL-Vc complex structure refinement. All waters and solution molecules were

omitted from the model. X-plor package (13) was used to refine the structure against

54,217 reflections at 2.0 to 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) between each round of refinement calculations. The


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electron densities for Vc were observed from the beginning, but Vc structure was only

included into the structural refinement until the R factor dropped below 25% and water

molecules were incorporated thereafter. And only after this point the B-factors

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). 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% DMSO water solution) were added along one row

of a 96 well microtiter plate, leaving the first row as 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 hour. 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 proceeded at room temperature for 15 minutes. Undigested

HA was precipitated using freshly made 50 µl of 10 % (w/v) aqueous cetylpyridinium

chloride with 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, lower

enzyme activity. The O.D. 595 absorbance readings were then converted to percent


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inhibition by subtracting the O.D. 595 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 mM, 14.4 mM, and 16.0 mM ascorbic acid) and 0% inhibition (O.D. 595

reading for the pure enzyme).

To determine if this inhibition is 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 is reversible. The slightly 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 at pH 6.0 and 10 mM calcium chloride and 8%

agarose at 55 ∞C. 100 µl of each of the 5 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, 10 mM Vc in 10 mM

calcium chloride and 50 mM acetate buffer at pH 6.0 were pre-incubated at 37 ∞C for an

hour and 100 µl of each 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 hours. 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 two hours. The absorbance was measured at 405 nm


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using an automated microplate reader (EL808; Bio-Tek Instruments, Inc., Winooski,

VT).

Data deposition Structural factors and coordinates of SpnHL-Vc complex have been

deposited in the Brookhaven Protein Data Bank with accession number 1f9g.

Results

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 out of 731 residues were modeled and 303 waters were

incorporated. One Vc molecule was clearly seen in the electron density map (Figure 1).

The final crystallographic R factor is 0.208 and Rfree 0.253. The protein part of the

complex structure contains an N-terminal α-domain and a C-terminal β-domain

connected by a ten residue linker. The active site of this enzyme lies in the middle of the

molecule where a predominant cleft is formed between these two structural domains

(Figure 2). The cleft is 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 non-reducing end of the

hyaluronan chain (19). 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 β-1,4 glycosidic linkage between HA1


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and HA2 disaccharide units in the hyaluronan chain (8, 19, 20).

The protein part of the SpnHL-Vc complex structure has only slight changes

when compared to the native SpnHL crystal structure. The r.m.s. 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

was shown in Figure 3a.

The aromatic patch of the enzyme active center is composed of three aromatic

residues, Trp291, Trp292, and Phe343 (8) (Figure 3a). The side chains of Trp292 and

Phe343 form hydrophobic interactions with the hydrophobic patches on the hyaluronan

chain. Through this matching, the cleavage sites are selected. Trp292 hydrophobically

interacts with HA2 disaccharide unit and accurately anchor HA2 into catalytic position.

In the hyaluronan PAD degradation model (19), the enzyme catalyses the degradation of

the β-1,4 glycosidic linkage between HA1 and HA2 and produces 4,5-unsaturated HA1.

Vc in the SpnHL-Vc complex structure is found to bind to Trp292 (Figure 3a) indole

group and occupies the HA2 position.

The binding of Vc to SpnHL The bound Vc forms 25 interactions with 7 residues of

the enzyme (Table 1). These residues were also shown to interact with the substrate (8).

The relative positions of these residues to the bound Vc were shown in Figure 3b. Five

out of the seven interface residues, Arg243, Asn290, Trp292, Tyr408, and Asn580, were

extensively studied in our previous biochemical and structural studies and were shown to

play important roles in the normal function of this enzyme (8, 20, 21).

Trp292 forms 8 interactions with Vc which accounts for the most interactions


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among all these 7 residues in the protein-Vc interface. The indole group of Trp292 is in

parallel to the five member ring of Vc (Figure 3a). This structural arrangement provides

the main hydrophobic interaction that stabilizes the Vc molecule inside the cleft. Trp292

is one of the aromatic patch residues and is responsible for the selection of the cleavage

sites on the substrate chain. The binding of Vc, therefore, likely competes with the

binding of hyaluronan substrate at the HA2 position which is located at the middle of the

cleft.

Tyr408 is one of the three key catalytic residues in the hyaluronan degradation. It

donates one proton to the glycosidic oxygen to break the β-1,4 glycosidic linkage

between HA1 and HA2 disaccharide units (8). In the complex structure, Tyr408 forms

one salt bridge with the Vc O1 oxygen (refer to Figure 4 for Vc atom labelings).

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.

Residues Asn290 and Asn580 form the narrowest part across the cleft. Asn580 is

the only residue from the β-domain which 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, 21).

SpnHL-Vc complex structure showed that Vc is also in contact with Asn580 and

Asn290.

The cleft is highly positively charged by the accumulation of lysine and arginine

residues inside the cleft. There are 9 conserved arginine residues present in the cleft.

Three of them are involved in the interaction with Vc. Arg462 and Arg466 are


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extensively involved in Vc binding (Table 1). Arg243 forms one salt bridge with Vc

carboxyl group. When the substrate is bound into the cleft, Arg243 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. Mutation R243V

surprisingly decreased the enzyme activity by 33% (8). In our previous studies, we

proposed that Arg243 plays an important role in the substrate translocation after the

initial glycosidic bond is degraded (9, 19). The glycosidic linkage in contact with Arg243

is the next linkage to be degraded. The interaction with Arg243 suggest 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 likely one of the

characteristic features in the protein-Vc interface.

The 25 interactions in the SpnHL-Vc interface (Table 1) can be classified into

two groups, hydrophobic and ionic interactions. Trp292 contributes mostly to the

hydrophobic interactions with Vc. Three arginine residues, Arg243, Arg462, and Arg466

form several salt bridges, while Tyr408, Asn290, and Asn580 form hydrogen bonds with

the ligand. In comparison with the protein-Vc interface observed in the D-xylose

isomerase where 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 are very small. On the contrary, Vc itself has relatively significant


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structural changes compared to its native crystal structure (23) used for the modeling and

refinement. The carboxyl group is forced to bend towards the plane of the Vc ring (Figure

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 Trp292 indole group which is not in favor of the Vc binding.

Discussion

Inhibitory effect of Vc on SpnHL enzyme activity Vitamin C, salicylate, and

flavonoids had 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 inhibits 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 β-1,4-glucuronic-β-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


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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 (Figure 5). At our experimental condition, the

IC50 of this inhibition is about 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 fit to both the competitive model and to the noncompetitive model by nonlinear

regression. The fitting attempts with the noncompetitive model repeatedly resulted in an

unreasonably high value (>50,000 mM) for the parameter Ki, which is not present in the

competitive model. Since this parameter indicates the concentration of Vc 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 here. 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 virulant than those strains

producing less (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 likely important in the control of

the pneumococcal invasion. And since animals usually use hydrolases to degrade


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hyaluronan, pneumococcal hyaluronate lyase becomes a potential target for developing

novel antibacterial agent. Vc is the first chemical shown to have inhibition effect on the

activity of this pneumococcal enzyme.

The adult minimum daily requirement for Vc is approximately 10 mg. Vc exists

in human tissues at the level of 0.2 mM to over 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 about 1.33 mg Vc per gram of wet

weight tissue (27), which corresponds to about 7.5 mM Vc. In the activated human

neutrophils, internal Vc concentrations as high as 14 mM were detected when external

vitamin is 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 deficiency of Vc may cause decreased resistance to some bacterial infections.

One explanation to this is that animal cells (such as neutrophils) generate oxidants to kill

bacteria and 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, therefore,

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 likely 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


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about 10 times higher in lung, brain, kidney, lymph glands, and small intestinal mucosa

(29) 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 Trp292 indole group. At least one negative charge

provider, for example, a carboxyl group, 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.

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 this structural similarities of Vc, a sugar derivative, to polysaccharides.

This structural similarity confers 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.


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Conclusions and general implications Vc may compress or retard bacterial invasion

by directly inhibiting 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, S. agalactiae hyaluronate lyase (GbsHL) crystal structure was recently

determined (19, 30). Its active center construction and geometry is nearly the same as it is

in SpnHL. Therefore, the results shown may be applicable to GbsHL, 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 likely directly involved in the

inhibition of bacterial invasion, in addition to its antioxidant and free radical scavenger

properties.


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25. Berry A.M., and Paton J.C. (2000) Infect. Immun. 68, 133-140

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J. Biol. Chem. 265, 2584-2587

27. Brubaker, R.F., Bourne, W.M., Bachman, L.A., Mclaren, J.W. (2000) Invest.

Ophthalmol. Vis. Sci. 41, 1681-1683

28. Washko, P., Wang, Y., and Levine, M. (1993) J Biol. Chem. 268, 15531-15535

29. Lester, P., and Fuchs, J. (1997)Vitamin C in health and disease. Marcel Dekker, Inc.

30. Jedrzejas, M.J., and Chantalat, L. (2000) Acta Crystallogr. D 56, 460-463


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Footnotes:

Acknowledgments: Diffraction data were collected at the Brookhaven National

Laboratory, National Synchrotron Light Source at the beamline X25. This work was

supported by NIH grant AI 44079 (MJJ).

Figure legends:

Figure 1. 2Fo-1Fc electron density map (1 σ) for the refined Vc bound to the active site

of SpnHL. One water molecule hydrogen bonded to Vc O2 atom can be identified.

Figure 2. The Vc binding position in the SpnHL structure. The positions of the N- and

C- terminals, the α- and β-domains were shown. One Vc molecule is bound to the cleft

between two structural domains.

Figure 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.

Figure 4. Structural comparison of protein-bound Vc overlapped with the Vc crystal

structure. Hydrogen atoms were not shown. Vc atoms were labeled as conventional (23).

SpnHL bound Vc is in green color whereas the Vc crystal structure is in lavender color.

Large atomic displacement in the carboxyl group atoms can be seen, whereas the ring

atoms change less.


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Figure 5. Enzyme activity of SpnHL at the presence of ascorbic acid. The final enzyme

concentration was 0.33 mg/ml. The final Vc concentrations were 0.8 to 16 mM. Each

point represents the averaged value of seven parallel measurements. Vc inhibited SpnHL

activity shows in a dose-dependent manner with 50% inhibition (IC50) at around 5.8

mM.


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Table 1. Protein-Vc Interface

protein residues Vc atoms! No. of contacts* shortest dist. (Å)

Arg243 O6 1 3.99

Asn290 O2 1 3.20

Trp292 O2, O1, C1, O6 8 3.70

Tyr408 O1 1 3.44

Arg462 C1, C2, C3, O1, O2, O3 6 3.34

Arg466 C4, C6, O4 4 3.57

Asn580 C2, C3, O2, O3 4 3.28

! Refer to Figure 4 for Vc atom labels.

*Interactions within 4.1 Å were counted.


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C

Ascorbic acid

-domainβ

-domainα

N


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Songlin Li, Kenneth B. Taylor, Stephen J. Kelly and Mark J. Jedrzejaslyase

Vitamin C inhibits the enzymatic activity of streptococcus pneumoniae hyaluronate

published online January 12, 2001J. Biol. Chem.

10.1074/jbc.M011102200Access the most updated version of this article at doi:

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Vitamin C inhibits the enzymatic activity of Streptococcus pneumoniae hyaluronate lyase · 2001-01-12 · Summary Enzyme activity measurement showed that L-ascorbic acid (vitamin