Graduate Thesis Or Dissertation
 

Advanced studies on cyclic amino acids in neurological signaling and peptide antibiotics

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  • L-pipecolic acid (L-PA) is the higher homolog of proline. It occurs naturally in many organisms, including primates, as an intermediate in lysine degradation. The pathway by which lysine is converted into L-pipecolic acid employs the enzyme Lpipecolate oxidase (L-PO), and appears to be tissue specific to the central nervous system (CNS). The oxidation facilitated by L-PO is the rate limiting step of lysine degradation in the CNS. For this reason, the mechanism of action for L-PO may be useful in the development of neuromodulation. This thesis describes efforts to probe the mechanism of action of L-PO through the synthesis of substrate analogs as alternate substrates and inhibitors of L-PO. Analogs that contain heteroatoms and other functionalities at key positions have been synthesized and analyzed as both alternate substrates and inhibitors of L-PO. The 4,5-methanopipecolic acid has been shown to be an excellent substrate for L-PO. The 5- keto analog was not a substrate or inhibitor of the enzyme. The 5-hydroxy and 5,5- 2 difluoro analogs have been shown to be weak inhibitors of L-PO. 6S-methyl-L-pipecolic acid (35) was shown to be a weak substrate and strong inhibitor while the enantiomeric 6R methyl-D-pipecolic acid (36) was neither a substrate nor inhibitor. These results suggest flexibility within the binding pocket of L-pipecolate oxidase toward analogs containing substituents at the 5-position. Additionally, these studies demonstrate the potential to develop mechanism-based inhibitors that could be used to control the rate of L-pipecolic acid consumption as well as the production of downstream products. A hallmark of peptide antibiotics are the varied and unique amino acids they employ. Capreomycidine and enduracididine are two such examples found in the peptide antibiotics muraymycin and enduracidin, respectively. Both are cyclic amino acids derived from arginine. Muraymycin is produced by Streptomyces sp. 30471 and has been shown to be active against a number of Gram-positive bacteria including Methicillin-resistant Staphylococcus aureus (MRSA). In an effort to further our understanding of this antibiotic, efforts to isolate the muraymycin gene cluster have begun. A genomic library has been constructed in the pCCFos1 Copy Control vector. Efforts to identify the genes encoding for the enzyme involved in the conversion of L-arginine to capreomycidine are described. Enduracidin is another peptide antibiotic which contains a cyclized form of arginine, enduracididine. Enduracidin has potent activity against Gram-positive bacteria including Methicillin-resistant Staphylococcus aureus. The enduracidin gene cluster has been cloned and sequenced by Yin and Zabriskie . Efforts to express and characterized the genes involve in the biosynthesis of enduracididine are described. Labeled feeding studies were also employed to determine the precursor to enduracididine. ¹³C-γ-hydroxyarginine was synthesized and β- and γ-hydroxyarginine were used in feeding experiments to determine which, if either, would be incorporated into enduracidin. Labeled enduracidin was isolated and characterized by LC-MS from feeding studies using ¹³C-γ-hydroxyarginine, identifying γ-hydroxyarginine as a precursor to enduracididine. Additional studies were performed using 3-fluoro-L-tyrosine. The fluoro-tyrosine was converted to 3-fluoro-4-hydroxyphenylglycine through a pathway utilized for hydroxyphenylglycine biosynthesis. The 3-fluoro-4-hydroxyphenylglycine was found to be incorporated into multiple positions within enduracidin by LC-MS.
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