Gut Bacterial β-Glucuronidases: Structural Basis of Substrate Specificity, Inhibitor Potency and Selectivity to Provide a Solution for Xenobiotic-Induced Toxicity

Autor: Punsaldulam Dashnyam, 朋莎朗
Rok vydání: 2019
Druh dokumentu: 學位論文 ; thesis
Popis: 107
Gut bacterial β-D-glucuronidases (GUSs) catalyze the removal of glucuronic acid from liver-produced β-D-glucuronides. These reactions can have deleterious consequences when they reverse xenobiotic metabolism. The human gut contains hundreds of GUSs of variable sequences and structures. To understand how any particular bacterial GUS(s) contributes to global GUS activity and affects human health, the substrate preference(s) of individual enzymes must be known. Herein, we report that representative GUSs vary in their ability to produce various xenobiotics from their respective glucuronides. In an attempt to explain the distinct substrate preference, we solved the structure of a bacterial GUS complexed with coumarin-3-D-glucuronide. Comparisons of this structure with other GUS structures identified differences in TIM barrel loop 3 (or the 2-helix loop) and loop 5 at the aglycone-binding site, where differences in their conformations, hydrophobicities and flexibilities appear to underlie the distinct substrate preference(s) of the GUSs. Additional sequence, structural and functional analysis indicated that several groups of functionally related gut bacterial GUSs exist. Our results pinpoint opportunistic gut bacterial GUSs as those that cause xenobiotic-induced toxicity. We propose a structure-activity relationship that should allow both the prediction of the functional roles of GUSs and the design of selective inhibitors. Furthermore, we demonstrated how charge, conformation, and substituents of several uronic iminocyclitols and analogues contribute to the inhibitory potency and selectivity for gut bacterial GUSs by using crystallographic and biochemical methods. Uronic isofagomine was a potent inhibitor (Ki up to 4 nM) unlike uronic deoxynojirimycin that showed a less potent inhibition (Ki up to 930 nM), indicating that the positive charge at the anomeric position, but not endocyclic oxygen position, was favorable to form electrostatic interactions with the two catalytic glutamates of GUSs. Moreover, D-glucaro-δ-lactam displayed good inhibitions (up to 280 nM) for bacterial GUSs owing to the half-chair conformation that favored several polar contacts and a charge-dipole interaction. Additionally, C6-alkylated uronic isofagomine displayed selective inhibition for opportunistic bacterial GUSs, which was attributed to the hydrophobic interactions between the propyl group and the loop 5 residues of the GUSs.In contrast, N1-alkylated analogue was a non-selective and moderate inhibitor for GUSs, which was explained by a steric hindrance between the alkyl group of the inhibitor and the catalytic acid/base glutamate of GUSs. In summary, we identified the responsible group of GUSs for the xenobiotic-induced toxicity, and developed a selective inhibitor targeting these GUSs.
Databáze: Networked Digital Library of Theses & Dissertations
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