Spinosyn A is a particularly interesting natural product due to its structural complexity and potent insecticidal activity. The biosynthetic pathway of spinosyn A is interesting as it has two unusual features, the SpnF-catalyzed (4+2) cycloaddition and the SpnL-catalyzed cyclization to produce the perhydro-as-indacene core. The work described in this dissertation focuses on elucidating the mechanisms of the SpnF- and SpnL-catalyzed reactions. SpnF has attracted significant interest as a possible Diels-Alderase. To explain how SpnF catalyzes the formation of cyclohexene ring, three plausible mechanisms have been proposed, the Diels-Alder reaction mechanism, the ionic rearrangement mechanism, and the biradical rearrangement mechanism. Kinetic isotope effect studies were performed using four deuterium-labeled mechanistic probes, specially the C4-D, C7-D, C11-D, and C12-D analogs. Currently, the ionic rearrangement mechanism can be excluded, based on the results using the C4-D and C7-D analogs. In addition, how SpnF accelerates the reaction was studied to assess the contribution of an entropic x preorganization compared to enthalpic transition state stabilization. To measure the relative rate enhancements due to structural perturbations, three mechanistic probes were synthesized, the linear analog, the C13-14 Unc analog, and the C2-3 Unc analog. Unfortunately, the linear analog and C13-14 Unc analog didn’t show any turnover activity under either non-enzymatic or enzymatic conditions. Thus, no conclusion could be drawn from incubation with these substrate analogs. Mechanistic studies of SpnL-catalyzed cyclization were devoted to differentiating between the Rauhut-Currier type mechanism and the Michael addition mechanism. Biochemical studies using the C13-F analog as a mechanism-based inhibitor showed the formation of a covalent adduct with SpnL, which is consistent with the Rauhut-Currier type mechanism. Additional experimental data obtained from isotope trace experiments and kinetic isotope effect studies using C12-D analog supports the Rauhut-Currier type mechanism. Biochemical studies concerning the role of SAM in SpnF and SpnL showed that SAM is required for the activity of SpnL, and were inconclusive for SpnF. SpnL mutant studies showed that Cys60 and Glu96 may be important for the catalysis of SpnL. Chemoenzymatic total synthesis of spinosyn A was completed by chemical etherification of 17-pseudoaglycone and D-forosamine. / text

Natural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering. / text