Xie, Lina (2018-12). Enzyme-Catalyzed Acylium Ion Formation and Reagents for the Detection of Thiocarboxylates Involved in the Biosynthesis of the NAD Derived Pincer Cofactor. Doctoral Dissertation. Thesis uri icon

abstract

  • In this dissertation, I describe: 1) the first case of experimental trapping of the acylium ion intermediates formed in an enzyme (1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase, QDO) catalyzed reaction using interdisciplinary approaches; 2) the computational supports on the QDO acylium ion involved mechanism; 3) the selectively trapping of the protein thiocarboxylates generated from a NAD derived thiocarboxylate cofactor (Nickel pyridinium-3,5 bisthiocarboxylic acid mononucleotide, NiP2TMN) biosynthesis and its partner enzyme (lactate racemase, LarA) activation pathway in the bacterial proteomes using the aromatic sulfonyl azide "click" probes; 4) the selectively detection of the small thiocarboxylate metabolites involved in the NiP2TMN pathway in the bacterial metabolomes using the similar "click" strategies mentioned in 3). Two acylium ion intermediates in the QDO catalyzed reaction were successfully trapped by the solvent water. The trapping was monitored by ??O isotope labeling assays. The design and synthesis of the substrate analog, 3,7-dihydroxyquinolin-4(1H)-one, and the QDO W153G protein mutant were crucial for the solvent trapping. The former stabilized the first acylium ion intermediate by the enhanced electron donating effect, while the latter opened up the QDO binding pocket for the solvent water better approaching the acylium ion intermediates. DFT energy profile and the MM optimized QDO W153G structure with the substrate docking into the binding pocket provided the computational support for the acylium ion mechanism. The active form of lactate racemase (LarA) in Lactobacillus plantarum had a cofactor NiP2TMN covalently bound to lys184, which was obtained by the coexpression of the complete operon larABCDE. Lissamine Rhodamine B Sulfonyl Azide (LRSA) and biotin-4 carboxybenzene sulfonyl azide (BiotinSA) were used for selectively trapping the two protein thiocarboxylates, LarA-COSH, LarE-COSH, generated in this NiP2TMN biosynthesis and LarA activation pathway in the bacterial proteomes. BiontinSA labeled LarA-COSH from the protein mixtures was enriched using the streptavidin resin and characterized by LC-MS/MS peptide analysis. The LRSA and dansyl azide (DanA) were further used to specifically trap the two small thiocarboxylate metabolites, pyridinium-3-carboxy-5-thiocarboxylic acid mononucleotide (PCTMN) and pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN) in the bacterial metabolomes. High-resolution LC-MS and LC-MS/MS techniques were used to characterize the "click" labeling products.
  • In this dissertation, I describe: 1) the first case of experimental trapping of the acylium
    ion intermediates formed in an enzyme (1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase, QDO)
    catalyzed reaction using interdisciplinary approaches; 2) the computational supports on the QDO
    acylium ion involved mechanism; 3) the selectively trapping of the protein thiocarboxylates
    generated from a NAD derived thiocarboxylate cofactor (Nickel pyridinium-3,5
    bisthiocarboxylic acid mononucleotide, NiP2TMN) biosynthesis and its partner enzyme (lactate
    racemase, LarA) activation pathway in the bacterial proteomes using the aromatic sulfonyl azide
    "click" probes; 4) the selectively detection of the small thiocarboxylate metabolites involved in
    the NiP2TMN pathway in the bacterial metabolomes using the similar "click" strategies
    mentioned in 3).

    Two acylium ion intermediates in the QDO catalyzed reaction were successfully
    trapped by the solvent water. The trapping was monitored by ??O isotope labeling assays. The
    design and synthesis of the substrate analog, 3,7-dihydroxyquinolin-4(1H)-one, and the QDO
    W153G protein mutant were crucial for the solvent trapping. The former stabilized the first
    acylium ion intermediate by the enhanced electron donating effect, while the latter opened up the
    QDO binding pocket for the solvent water better approaching the acylium ion intermediates.
    DFT energy profile and the MM optimized QDO W153G structure with the substrate docking
    into the binding pocket provided the computational support for the acylium ion mechanism.

    The active form of lactate racemase (LarA) in Lactobacillus plantarum had a cofactor
    NiP2TMN covalently bound to lys184, which was obtained by the coexpression of the complete
    operon larABCDE. Lissamine Rhodamine B Sulfonyl Azide (LRSA) and biotin-4
    carboxybenzene sulfonyl azide (BiotinSA) were used for selectively trapping the two protein
    thiocarboxylates, LarA-COSH, LarE-COSH, generated in this NiP2TMN biosynthesis and LarA
    activation pathway in the bacterial proteomes. BiontinSA labeled LarA-COSH from the protein
    mixtures was enriched using the streptavidin resin and characterized by LC-MS/MS peptide
    analysis. The LRSA and dansyl azide (DanA) were further used to specifically trap the two small
    thiocarboxylate metabolites, pyridinium-3-carboxy-5-thiocarboxylic acid mononucleotide
    (PCTMN) and pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN) in the bacterial
    metabolomes. High-resolution LC-MS and LC-MS/MS techniques were used to characterize the
    "click" labeling products.

publication date

  • December 2018