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Title:Mechanistic investigations on polyprenyl transferases and multi-target antibiotic discovery
Author(s):Chen, Lu
Director of Research:Oldfield, Eric
Doctoral Committee Chair(s):Gennis, Robert
Doctoral Committee Member(s):Metcalf, William; Procko, Erik
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
natural products
protein structures
X-ray diffraction
structure elucidation
antibiotic resistance
C. elegans
multi-drug resistance
product chain length
site-directed mutagenesis
enzyme catalysis
multi-target antibiotics
Abstract:Terpenoid biosynthesis plays essential structural and functional roles in almost all life forms. Prenyl transferases, enzymes involved in the isoprenoid metabolism related to prenyl chains, transfer allylic prenyl groups to acceptor molecules. Head-to-tail condensations, usually of allylic diphosphates (isoprene units), are catalyzed by head-to-tail prenyl transferases (commonly referred to prenyl diphosphate synthase), and produce regular terpenes which are by far the most common isoprenoid compounds. Head-to-middle prenyl transferases have been reported recently and they catalyze the formation of branched products which belong to irregular terpenes. Based on the stereochemical outcome of the linear products, head-to-tail prenyl transferases can be categorized to two major classes, trans- and cis- prenyl tranferases. Trans- prenyl transferases are critical players in bacterial respiratory systems while cis-prenyl transferases are involved in the biosynthesis of peptidoglycan, essential in bacterial cell wall synthesis. Thus, both classes of head-to-tail prenyl transferases are druggable targets in antibiotic development. In Chapter 2, I discuss the new series of lipophilic bisphosphonates that inhibit the growth of various bacteria and target multiple prenyl transferases. To determine and verify the enzyme targets of antibiotic leads, enzyme inhibition assays and bacterial rescue assays were performed. In Chapter 3, I discuss the antimicrobial agents phenylthiazoles which target bacterial UPPS, YubB (undecaprenyl diphosphate phosphatase (UPPP)) deducted from transposon mutagenesis. Putative inner membrane family proteins YubA and YubD are annotated as transporters and may also be targets because the compounds collapsed the proton motive force in membrane vesicles. Cis-prenyl transferase UPPS serves as a lipid carrier in cell wall peptidoglycan synthesis and catalyzes consecutive condensations of isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP) to produce C55-PP. The UPPS product has the specific chain length essential for the biological function and an intriguing question is how UPPS regulates the product chain length. Chapter 4 is focused on chain length regulation mechanism of UPPS and its interaction with bacterial membrane. Head-to-middle prenyl transferases catalyze the formation of branched terpenoid products, which are reported more recently. It was found that they are highly structurally similar to the cis class of head-to-tail prenyl transferases. In Chapter 5, I discuss the first X-ray crystal structure of head-to-middle monoterpene synthase, lavandulyl pyrophosphate synthase (LPPS). Irregular prenyl transferase LPPS catalyzes two DMAPP molecules and form LPP, a precursor of the fragrances (R)-lavandulol and (R)-lavandulyl acetate. The active sites of LPPS were examined and compared with cis- head-to-tail prenyl transferase UPPS, and site-directed mutagenesis study was conducted. We proposed the first structure-based mechanism of action of this unusual prenyl synthase. In Chapter 6, a homolog of LPP, isosesquilavanduly diphosphate (ILPP), and its specific enzyme ILPPS were studied, and they are involved in the biosynthesis of the merochlorin class of antibiotics found in Streptomyces sp. strain CNH-189. We determined the crystal structure of ILPPS, substrate binding, substrate specificity, product length regulation, structure-based mechanism and the results were surprising and gave an unexpected perspective into catalysis by prenyl transferases. Based on ligand-bound crystal structure, the large, hydrophobic side chain of one substrate does not occupy a central hydrophobic channel. Instead, it occupies a surface pocket approximately 90° to the chain axis (hydrophobic tunnel) in other enzymes with cis- prenyl transferase fold. Interestingly, the proton abstraction is achieved with a diphosphate-Asn- Ser relay, which is shared in head-to-middle and cis- head-to-tail prenyl transferases.
Issue Date:2018-12-02
Rights Information:Copyright 2018 Lu Chen
Date Available in IDEALS:2019-02-08
Date Deposited:2018-12

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