Thesis advisor: Steven D. Bruner / Peptidic natural products are produced by diverse organisms ranging from bacteria to humans. These secondary metabolites can be assembled by the ribosome or by nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. The architectural complexity and biological activity of such compounds make them interesting targets for study. Frequently, nonribosomal peptides contain nonproteinogenic amino acid building blocks, and the biosynthetic routes to both ribosomal and nonribosomal peptides often utilize tailoring enzymes. These specialized enzymes catalyze mechanistically challenging reactions and provide peptidic natural products with structural motifs not normally found in proteins. Structural studies of these tailoring enzymes will further our understanding of biosynthetic pathways, and engineered tailoring enzymes could find use as promiscuous catalysts for the chemoenzymatic synthesis of natural product analogs. The L-tyrosine 2,3-aminomutase <italic>Sg</italic>TAM catalyzes the formation of &beta;-tyrosine from L-tyrosine, and is used in the biosynthetic pathway to the enediyne antitumor antibiotic C-1027. This enzyme contains the rare electrophilic prosthetic group 4-methylideneimidazole-5-one (MIO) and is homologous to the histidine ammonia lyase family of enzymes. While lyases form &alpha;,&beta;-unsaturated carboxylates as products, <italic>Sg</italic>TAM catalyzes additional chemical steps that result in an overall 2,3-amino shift. The precise mechanistic role of MIO in the ammonia lyase and aminomutase families of enzymes was actively debated for over 50 years. Here, we report the first x-ray crystal structure of an MIO-dependent aminomutase and detail the synthesis and characterization of mechanistic probes for this enzyme. Furthermore, we report several structures of <italic>Sg</italic>TAM bound to substrate analogs. These co-crystal structures reveal how <italic>Sg</italic>TAM achieves substrate recognition and suggest a specific role for MIO in catalysis. The results of our studies allow for the rational engineering of MIO-based aminomutases and ammonia lyases with altered physical properties and substrate specificities. Additionally, we are currently studying several enzymes involved in the biosynthesis of the tricyclic depsipeptide microviridin J. This ribosomal peptide natural product contains two lactones and one lactam, which are introduced by two enzymes belonging to the ATP-grasp ligase superfamily of proteins. Here, we detail the overexpression of these enzymes, MdnJ-B and MdnJ-C, in <italic>E. coli</italic> and report the optimization of conditions which lead to the crystallization of both enzymes. The structural characterization of MdnJ-B and MdnJ-C will lead to a greater understanding of macrocycle formation in ribosomal peptide biosynthesis, and engineered variants of these enzymes may find use as macrocylcization catalysts. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Glutathione syntethase (GS) is an enzyme that belongs to the ATP-grasp superfamily and catalyzes the second step in the biosynthesis of glutathione. GS has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. Four highly conserved residues were identified in the binding site of human GS. Additionally, the G-loop residues that close the active site during catalysis were found to be conserved. Since these residues are important for catalysis, their function was studied computationally by site-directed mutagenesis. Starting from the reported crystal structure of human GS, different conformations for the wild type and mutants were obtained using molecular dynamics technique. The key interactions between residues and ligands were detected and found to be essential for enzyme activity.