Uncovering the Biosynthesis of Natural Products Using Fourier-Transform Mass Spectrometry
Thomas, Paul Martin
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https://hdl.handle.net/2142/72253
Description
Title
Uncovering the Biosynthesis of Natural Products Using Fourier-Transform Mass Spectrometry
Author(s)
Thomas, Paul Martin
Issue Date
2009
Doctoral Committee Chair(s)
Kelleher, Neil L.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Chemistry, Biochemistry
Abstract
Within some of the simplest bacteria there exists the prospect of novel natural products with an exciting array of bioactivities. These natural products are assembled by a vast array of enzymes which can transform the simplest building blocks into large, complex molecules. Fourier-Transform mass spectrometry (FTMS) is a powerful technique for the accurate determination of mass in complex mixtures. A large portion of this research is focused on the multimodular assembly-line enzymes employed in the biosynthesis of polyketides (PK) and nonribosomal peptides (NRP). In this work, FTMS is used both in the discovery of novel natural products as well as in the characterization of novel enzymology.
An on-line extension of the phosphopantetheinyl (PPant) ejection assay has been developed and applied to the aflatoxin and andrimid biosynthetic gene clusters. In the biosynthesis of andrimid, the promiscuity of the transglutaminase homolog, AdmF, is assayed through Top Down mass spectrometry of AdmF and its two thiolation domain partners, AdmA and AdmI, with the observation of intact mass shifts and PPant ejection products. In the end, AdmF is a promiscuous condensation catalyst as long as the substrates are presented by the cognate thiolation domains. In aflatoxin biosynthesis, FTMS and the online PPant ejection assay provide the first glimpse into the cyclization process in a fungal iterative polyketide synthase. The PPant ejection assay is finally used in the detection of novel NRP/PK production by an organism with an unsequenced genome.
The accurate mass achievable with our 12 T LTQ-FT Ultra instrument has not only advanced the NRP/PK biosynthetic work, but also made possible the analysis of reduced phosphorus biosynthesis in phosphinothricin tripeptide, dehydrophos and FR900098 systems. Multiple unexpected intermediates have been discovered and the groundwork has been laid for the discovery of novel phosphonates with the development of a software package for automated data analysis. As the FTMS workflows advanced here mature, the speed at which hypotheses can be assessed will continue to accelerate a better understanding of the complex transformations occurring during natural product biosynthesis.
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