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Title:Diverse enzyme reactions in phosphonate natural product biosynthesis
Author(s):Ulrich, Emily C.
Director of Research:van der Donk, Wilfred A.
Doctoral Committee Chair(s):van der Donk, Wilfred A.
Doctoral Committee Member(s):Martinis, Susan A.; Nair, Satish K.; Silverman, Scott K.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Natural product
Abstract:A number of new biosynthetic gene clusters encoding phosphonate natural products have been characterized by mining of microbial genomes. Phosphonates, which are characterized by their hydrolytically stable carbon-phosphorus bond, are prevalent in everyday life, such as in commercially available herbicides and an antibiotic deemed critically important in the clinic. Their biosynthetic pathways are rich in novel chemistry, ranging from iron-dependent radical rearrangements to multiple methods of forming amide bonds. This thesis will include my contributions to understanding phosphonate enzymatic chemistry in more detail. Methylphosphonate (MPn) was recently isolated from marine surface waters and is proposed to be a precursor in the production of methane, which is supersaturated in these environments. The oxygenase MPn synthase (MPnS) produces MPn from 2-hydroxyethylphosphonate (2-HEP), a substrate also acted on by 2-HEP dioxygenase (HEPD) to make hydroxymethylphosphonate (HMP). Both enzymes are proposed to share a common mechanism up until the final intermediate consisting of an MPn radical and formate. Chapter 2 will discuss efforts to elucidate how each enzyme dictates individual product formation at this key branching step between the two mechanisms. An unprecedented 2-His-1-Gln facial triad, along with the identity of a second sphere residue, were determined to contribute to MPn versus HMP formation. This sequence motif was used to predict the prevalence of MPnS in other microbial genomes, leading to the functional characterization of an MPnS from the abundant marine bacterium Pelagibacter ubique. This discovery further supports MPn as a possible source of the supersaturated methane levels in the aerobic ocean. The tripeptide antibiotic dehydrophos consists of a phosphonate warhead attached to two proteinogenic amino acids which function to allow entry into target cells. During biosynthesis, these amino acids are transferred to the phosphonate through the action of aminoacyl-tRNA (aa-tRNA)-dependent ligases. Chapter 3 will discuss efforts to characterize the interaction of the enzyme DhpH-C with Leu-tRNALeu. Mutagenesis studies of both the enzyme and tRNA led to the conclusion that DhpH-C shares features of aa-tRNA recognition common to other aa-tRNA-dependent enzymes. In addition, DhpH-C was found to accept other amino acids for the production of multiple dipeptide phosphonates, although Leu-tRNALeu was the most efficient substrate with the fastest rate of product formation over time. These features of DhpH-C may aid in future studies to produce phosphonate analogs chemoenzymatically, and its modes of aa-tRNA substrate recognition appear to be applicable to the study of aa-tRNA-dependent enzymes in the production of different natural product classes. Biosynthesis of the clinically used antibiotic fosfomycin can occur by two convergent routes in Streptomyces and Pseudomonas species. The pathways only share the first and last steps, which are catalyzed by phosphoenolpyruvate mutase and 2-hydroxypropylphosphonate epoxidase (HppE), respectively. Chapter 4 will discuss efforts to elucidate the more recently discovered pathway in Pseudomonas syringae PB-5123. Analysis of the gene cluster led to the characterization of reductase Psf3 as the penultimate step converting 2-oxopropylphosphonate (2-OPP) to (S)-2-hydroxypropylphosphonate ((S)-2-HPP). Active site mutants were analyzed for activity to probe how Psf3 interacts with 2-OPP and performs its stereospecific reduction. Combining our investigation of Psf3 with the previous characterization of Psf4 (HppE) led to the formulation of a one-pot assay to produce fosfomycin from racemic 2-HPP. Further elucidation of the fosfomycin pathway in P. syringae should uncover new biosynthetic mechanisms in phosphonate metabolism and provide insight into how two almost completely different sets of enzymes evolved to accomplish the same task.
Issue Date:2018-02-02
Rights Information:Copyright 2018 Emily C. Ulrich
Date Available in IDEALS:2018-09-04
Date Deposited:2018-05

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