Structure and Activity of the Paramecium Bursaria Chlorella Virus Arginine Decarboxylase



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The substrate specificity of enzymes has been studied with keen interest for many years. An understanding of the structural basis of specificity may help to explain how enzymes have evolved such enormous rates of catalysis above uncatalyzed reactions. By understanding how enzymes coordinate residues within and outside of the active site, enzyme engineering efforts may be aided. Finally, in the case of enzymes as drug targets, the structural basis of enzyme-substrate interactions may facilitate medicinal chemistry efforts to modulate the activity of a targeted enzyme. The investigations presented here focus on a homolog of ornithine decarboxylase, a proven drug target in the treatment of parasitic infections. The Paramecium bursaria Chlorella virus arginine decarboxylase is a member of the Group IV pyridoxal-5'-phosphate-dependent decarboxylase family. The enzyme is a close homolog of eukaryotic ornithine decarboxylases and is only distantly related to bacterial arginine decarboxylases. The goals of my dissertation project were: 1) to determine the substrate preference of the Paramecium bursaria Chlorella virus arginine decarboxylase, 2) to determine the structural basis of specificity of this enzyme, 3) to attempt to define the amino acid determinants of specificity for both ornithine and arginine decarboxylases, 4) to provide a more thorough understanding of the catalytic cycle of theses decarboxylases. To accomplish these goals of my thesis I employed biochemical and biophysical techniques ranging from HPLC-based analysis of reaction products to x-ray crystallography to fluorescence spectroscopy. The results of these efforts have produced a number of unique and intriguing observations. First, I have demonstrated that the Chlorella virus arginine decarboxylases prefers arginine by over 600-fold compared ornithine or lysine, and, therefore, represents a new activity within the ODC clade of Group IV decarboxylases. Second, I have shown that the structural basis of specificity of Group IV decarboxylases is a short helix which functions as a molecular ruler that selects substrates based on chain-length. Mutants of both arginine and ornithine decarboxylase that I created demonstrate the importance of the sequence identity and precise positioning of this helical ruler in determining substrate specificity.