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Title:Investigations of metalloenzymes involved in bacterial natural product biosynthesis
Author(s):McLaughlin, Martin Irving Harrison
Director of Research:van der Donk, Wilfred A
Doctoral Committee Chair(s):van der Donk, Wilfred A
Doctoral Committee Member(s):Lu, Yi; Metcalf, William W; Mitchell, Douglas A
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):3-thiaglutamate
B12
chiral acetate
chiral methyl
cobalamin
fosfomycin
Fom3
methyltransferase
nonheme iron
oxygenase
phosphonate
radical SAM
RiPP
s-adenosylmethionine
s-adenosyl-l-methionine
thiaglutamate
Abstract:Just as the structures of bacterial natural products have long been a source of inspiration for new developments in drug discovery and organic synthesis, so have investigations of their biosynthetic pathways led to the discovery of countless new enzyme-catalyzed reactions that open new frontiers of enzymology by defying conventional chemical intuition. Many of the most perplexing and challenging transformations are catalyzed by enzymes that use multiple redox-active metal ions to initiate and control radical chemistry, breaking inert C-H bonds and rearranging carbon skeletons with exquisite specificity. This work describes the characterization of two such reactions in the biosynthesis of two different natural products: fosfomycin, a clinically used phosphonate antibiotic discovered in 1969, and 3-thiaglutamate, a novel amino acid analog of unknown function discovered in 2019. In Chapter 2, newly available techniques were used to heterologously express and purify the fosfomycin biosynthetic enzyme Fom3 from Streptomyces wedmorensis, a methyltransferase in the radical S-adenosylmethionine (SAM) superfamily, with its required iron-sulfur cluster and cobalamin (B12) cofactors. The methyl transfer reaction catalyzed by Fom3 was then investigated in vitro using a combination of isotope labeling, enzymatic synthesis, liquid chromatography-mass spectrometry, and nuclear magnetic resonance methods. The results of these studies resolved a conflict in the literature about the stereochemistry of the product and supported a longstanding mechanistic hypothesis in which B12 serves as an intermediate methyl carrier, receiving a methyl group from SAM via a polar mechanism and transferring it to a substrate-derived species via a radical mechanism. In the collaborative study described in Chapter 3, the products of in vitro Fom3 reactions performed using SAM derived from methionine that was chirally labeled at the methyl position with deuterium and tritium were derivatized and analyzed, revealing overall retention of methyl stereochemistry during the reaction and therefore inversion during the radical methyl transfer step. The second reaction, oxidative excision of the β carbon from a peptide C-terminal cysteine residue by the complex of nonheme iron oxygenase TglH and putative substrate recognition protein TglI, was previously identified as a step in the biosynthetic pathway of the sulfur-substituted glutamate analog 3-thiaglutamate in a strain of Pseudomonas syringae. Chapter 4 describes initial studies of recognition of the 51-residue substrate peptide, TglACys, by the TglHI complex. In vitro activity assays of TglHI using synthetic TglACys variants with stretches of eight alanine mutations as well as C-terminal fragments of TglACys showed that no more than eighteen residues at the C-terminus of TglACys are required for modification by TglHI. Additionally, replacement of the C-terminal cysteine of TglACys by selenocysteine yielded a potent inhibitor of TglHI activity. Based on these results, several short, synthetically accessible peptide fragments were identified as candidate substrates and inhibitors for use in further mechanistic and structural studies.
Issue Date:2021-04-20
Type:Thesis
URI:http://hdl.handle.net/2142/110831
Rights Information:© 2021 Martin Irving Harrison McLaughlin
Date Available in IDEALS:2021-09-17
Date Deposited:2021-05


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