Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Analysis of Natural Product Biosynthesis.

Natural products have provided some of our most clinically relevant drugs and continue to be a source of new leads. Indeed, our understanding of the fundamental mechanisms involved in their biosynthetic production is just beginning to develop. A FTICR-MS-centric analytical approach was applied to understand fundamental mechanistic details of natural product production, monitor chemoenzymatic generation of natural products, and characterize/identify novel natural product producing systems. Investigations in four different systems have highlighted the broad applicability of this analytical approach. Each of the chapters of this thesis seeks to examine these three themes in different contexts. In Chapter 2 the key step of coenzyme A extender unit selection in polyketide biosynthesis is explored by in vitro biochemistry of the PikAIV model system. Key findings developed allow a model for catalysis to be proposed. In Chapter 3, our investigations shift to the bryostatin biosynthetic system. The biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl-CoA synthase, implicated in β- of the core ring system from the bryostatin metabolic pathway, is reported. In Chapter 4, an unusual enzyme activity of the nonribosomal peptide synthase module CrpDm2 was explored in vitro by FTICR-MS—resulting in chemoenzymatic synthesis of three cryptophycin analogs. In Chapter 5, the biosynthetic pathway for the approved chemotherapeutic ET-743 is characterized from a tunicate/symbiont system using a novel workflow. Metabolite analysis, metagenomic sequencing, contig assembly, and metaproteomic analysis were used to probe the experimental system and a key enzyme activity was verified in vitro. Chapter 6 presents future directions based upon findings developed. We have focused on gaining a deeper understanding of key mechanisms in natural product biosynthesis by studying defined systems in vitro through the application of novel, FTICR-MS centric analytical technologies. These studies have illustrated the application of chemoenzymatic methodology to generate novel analogs of active natural products, with FTICR-MS as the key tool to investigate the final product and key enzyme bound intermediates. Together, the developed tools were applied to characterize a novel biosynthetic pathway that had previously been inaccessible with current analytical technologies. Fundamentally, these developments will inform our ability to access natural products for human health concerns.PhDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86408/1/crath_1.pd