The study of the biosynthetic logic of natural products has established itself to be one of the more exciting areas of research and have become an important part of modern drug discovery and development efforts. Therefore, understanding the pathway and the chemical mechanism of the biosynthesis of natural products is important in that knowledge on these processes can be applied for combinatorial biosynthesis to generate new natural product derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating the chemical mechanism of a number of enzymes involved in natural product biosynthesis by utilizing the versatility of synthetic chemistry to prepare enzyme substrates and mechanistic probes. First, SpnF and SpnL responsible for constructing the tetracyclic architecture of spinosyn A have been investigated. In vitro assay revealed the importance of the highly conjugated system for the [4+2]cycloaddition catalyzed by SpnF. Biochemical studies strongly suggest that SpnL employs the Rauhut-Currier mechanism for the second cyclization step in the biosynthesis of spinosyn A. It was also demonstrated that SpnL requires SAM for its activity. Second, a radical SAM enzyme DesII involved in the desosamine pathway has been investigated. It has been demonstrated that DesII can catalyze the dehydrogenation of TDP-D-quinovose as well as the deamination of the natural substrate, which makes DesII unique among radical SAM enzymes. In vitro assays revealed that DesII requires stoichiometric amount of SAM, which. EPR study firmly established the intermediacy of a C-3 radical in the DesII-catalyzed dehydrogenation of TDP-D-quinovose. Finally, the chemical mechanism of AXS responsible for the biosynthesis of UDP-apiose has been investigated. In vitro activity assay using UDP-2F-glucuronic acid showed that the analog is a competitive inhibitor of AXS. A coupled assay strategy was also developed to investigate the chemical mechanism of AXS in the reverse direction. In addition, the stereospecificity of two separate hydride transfer steps of AXS reaction has been firmly established. / text

Diverse biological activities of polyketide natural products are often associated with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polykektide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating a number of post-PKS modifications involved in the biosynthesis of an insecticidal polyketide, spinosyn A, in Saccharopolyspora spinosa. First, three methyltransferases, SpnH, SpnI, and SpnK, responsible for the modification of the rhamnose moiety, have been investigated to verify their functions and to study how they are coordinated to achieve the desired level of methylation of rhamnose. In vitro assays using purified enzymes not only established that SpnH, SpnI, and SpnK are the respective rhamnose 4ʹ-, 2ʹ-, and 3ʹ-O-methyltransferase, but also validated their roles in the permethylation process of spinosyn A. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK, and SpnH in sequence is productive for the permethylation of the rhamnose moiety, which is likely achieved by the proper control of the expression levels of the methyltransferase genes involved in vivo. The key structural feature of spinosyn A is the presence of the unique tetracyclic architecture likely derived from the monocyclic PKS product. To elucidate this "cross-bridging" process, which had been hypothesized to involve four enzymes, SpnF, SpnJ, SpnL, and SpnM, the presumed polyketide substrate was chemically synthesized using Julia-Kocienski olefination, Stille cross-coupling, and Yamaguchi macrolactonization as key reactions. Incubation of the synthesized substrate with SpnJ produced a new product where the 15-OH group of the substrate is oxidized to the ketone. Next, it was demonstrated that incubation of this ketone intermediate with SpnM produces a tricyclic compound, via a transient monocyclic intermediate with high degree of unsaturation. Whereas it was initially thought that SpnM catalyzes both dehydration and [4+2] cycloaddition in sequence, detailed kinetic analysis revealed that SpnM is only responsible for the dehydration step, and the [4+2] cycloaddition step is indeed catalyzed by SpnF. Finally, successful conversion of the tricyclic intermediate to the tetracyclic core was demonstrated using SpnL. Proposed chemical mechanisms of SpnF and SpnL, Diels-Alder and Rauhut-Currier reactions, respectively, are interesting because enzymes capable of catalyzing these reactions have yet to be characterized in vitro. This work not only establishes the biosynthetic pathway for constructing the spinosyn tetracyclic core, but also epitomizes the significance of the post-PKS modification as a rich source of new enzyme catalysis. / text