Thesis advisor: Steven D. Bruner / Bacteria utilize complex enzymatic machinery to create diverse secondary metabolites. The architectural complexities of these small molecules are enhanced by nature’s ability to synthesize non-proteinogenic amino acids for incorporation into these scaffolds. Many of these natural products are utilized as therapeutic agents, and it would be advantageous to understand how the bacteria create various non-natural amino acid building blocks. With a greater understanding of these systems, engineering could be used to create libraries of potentially useful natural product analogs. The tyrosine aminomutase SgTAM from the soil bacteria Streptomyces globisporus catalyzes the formation of tyrosine to generate (S)-B-tyrosine. The precise mechanistic role of MIO in this novel family of aminomutases has not been established. We report the first X-ray crystal--> structure of an MIO based aminomutase and confirm the structural homology of SgTAM to ammonia lyases. Further work with mechanistic inhibitors provide structural evidence of the mechanism by which MIO dependent enzymes operate. We have also investigated LnmQ, an adenylation domain in the biosynthetic pathway of leinamycin. Leinamycin is an antitumor antibiotic that was isolated from soil samples in 1989. LnmQ is responsible for the specific recognition of D-alanine and subsequent activation as an aminoacyl adenylate species. We have cloned the gene into a DNA vector and expressed it in E. coli. Upon purification of the protein, crystallization conditions have been tested. Synthesis of an inhibitor that mimics the aminoacyl adenylate product catalyzed by LnmQ has been completed. Crystallization with this--> inhibitor will provide better quality crystals and a catalytically informative co-complex. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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