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Vitamin C Inhibits the Enzymatic Activity ofStreptococcus pneumoniae Hyaluronate Lyase*

Open AccessPublished:January 01, 2001DOI:https://doi.org/10.1074/jbc.M011102200
      Enzyme activity measurement showed thatl-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.1f9g
      Vc
      l-ascorbic acid known as vitamin C
      HA
      hyaluronan
      SpnHL
      S. pneumoniae 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 (
      • Khurana S.
      • Powers D.B.
      • Anderson S.
      • Blaber M.
      ). 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 (
      • Levine K.
      ). Vc is a multifunctional molecule in tissues. It usually acts as an antioxidant (
      • Hwang J.
      • Peterson H.
      • Hodis H.N.
      • Choi B.
      • Sevanian A.
      ), free radical scavenger (
      • Kalka K.
      • Mukhtar H.
      • Turowski-Warke A.
      • Merk H.
      ), neuroprotectant, and neuromodulator (
      • Rice M.E.
      ). It also plays an important physiological function in activating peptide hormones (
      • Levine K.
      ) and regulating cell division and growth (
      • Smirnoff N.
      ). Vc is the single synthetic chemical that is manufactured and consumed in the greatest quantity in the world (
      • Khurana S.
      • Powers D.B.
      • Anderson S.
      • Blaber M.
      ). 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 ofd-xylose isomerase (Protein Data Bank accession number1xid) in which Vc was present between two tryptophan residues (
      • Carrell H.L.
      • Glusker J.P.
      • Burger V.
      • Manfre F.
      • Tritsch D.
      ). It clearly emphasizes the importance of hydrophobic interactions in the protein-Vc interface. Here we present the crystal structure ofStreptococcus 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” (
      • Li S.
      • Kelly S.J.
      • Lamani E.
      • Ferraroni M.
      • Jedrzejas M.J.
      ,
      • Jedrzejas M.J.
      ). 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.

      Acknowledgments

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

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