Browsing by Author "Mesecar, Andrew"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes
    (Public Library of Science, 2024-03-29) Visvanathan, Ramya; Utsuki, Tadanobu; Beck, Daniel E.; Clayton, W. Brent; Lendy, Emma; Sun, Kuai-lin; Liu, Yinghui; Hering, Kirk W.; Mesecar, Andrew; Zhang, Zhong-Yin; Putt, Karson S.; Pharmacology and Toxicology, School of Medicine
    The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes.
  • Thumbnail Image
    Item
    Probing the mechanism of Bacillus subtilis oxalate decarboxylase
    (2015-12-01) Zhu, Wen; Richards, Nigel G.; Li, Lei; Shah, Kavita; Mesecar, Andrew; Long, Eric C.
    Oxalate decarboxylase (EC 4. 1. 1. 2 OxDC) from Bacillus subtilis is a manganese-dependent enzyme that catalyzes the cleavage of the chemically inactive C-C bond in oxalate to yield formate and carbon dioxide. A mechanism involving Mn(III) has been proposed for OxDC, however no clear spectroscopic evidence to support this mechanism has yet been obtained. In addition, a recent study has shown that N-terminal metal binding site loop variants of OxDC were able to catalyze the oxidation of oxalate to yield hydrogen peroxide and carbon dioxide, which makes OxDc function as another oxalate degradation protein in the cupin superfamily, oxalate oxidase (EC 1.2.3.4 OxOx). In this work, wild-type (WT) Bacillus subtilis OxDC and a series of variants with mutations on conserved residues were characterized to investigate the catalytic mechanism of OxDC. The application of membrane inlet mass spectrometry (MIMS), electronic paramagnetic resonance (EPR) spectroscopy and kinetic isotope effects (KIEs) provided information about the mechanism. The Mn(III) was identified and characterized under acidic conditions in the presence of dioxygen and oxalate. Mutations on the second shell residues in the N-terminal metal binding site affected the enzyme activity properties of the metal. In the N-terminal domain, the functional importance of the residues in the active site loop region, especially Glu162, was confirmed, and evidence for the previously proposed mechanism in which OxDC and the OxDC/OxOx chimeric variant share the initial steps has been found. In addition, the mono-dentate coordination of oxalate in the N-terminal metal binding site was confirmed by X-ray crystallography. A proteinase cleavable OxDC was constructed and characterized, revealing the interaction between the N-terminal and C-terminal domains.
  • Thumbnail Image
    Item
    Structural and Kinetic Comparison of Acetolactate Synthase and Acetohydroxyacid Synthase from Klebsiella pneumoniae
    (2019-08) Latta, Alexander J.; McLeish, Michael; Li, Lei; Mesecar, Andrew; Laulhe, Sebastien
    Acetolactate synthase (ALS) and acetohydroxyacid synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that catalyze the formation of acetolactate from two molecules of pyruvate. In addition to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important precursors to the essential amino acids valine and isoleucine. Conversely, ALS forms acetolactate as a precursor to 2,3-butanediol, a product formed in an alternative pathway to mixed acid fermentation. While these enzymes catalyze the same reaction, they have been found to be quite different. Such differences include: biological function, pH optimum, cofactor requirements, reaction kinetics and quaternary structure. Importantly, AHAS has been identified as the target of the widely-used sulfonylurea and imidazolinone herbicides, which has led to many structural and kinetic studies on AHAS enzymes from plants, bacteria, and fungi. ALS, on the other hand, has only been identified in bacteria, and has largely not seen such extensive characterization. Finally, although some bacteria contain both enzymes, they have never been studied in detail from the same organism. Here, the ALS and AHAS enzymes from Klebsiella pneumoniae were studied using steady-state kinetic analyses, X-ray crystallography, site-directed and site-saturation mutagenesis, and cell growth complementation assays to i) compare the kinetic parameters of each enzyme, ii) compare the active sites to probe their differences in substrate profile and iii) test the ability of ALS to function in place of AHAS in vivo.