Thesis advisor: Steven D. Bruner / The enediyne antitumor antibiotics are produced by complex biosynthetic machinery in acetomycetes. This dissertation will focus on the study of three enzymes involved in key steps in the biosynthesis of two enediynes, neocarzinostatin and C-1027. Neocarzinostatin is biosynthesized by a number of enzymes that synthesize and decorate the enediyne core and the peripheral moieties. NcsB1 is one enzyme involved in functionalizing the naphthoic acid portion of neocarzinostatin, a key group involved in binding to target DNA duplexes. The enzyme has been shown to be a promiscuous (<italic>S</italic>)-adenosylmethionine-dependent <italic>O</italic>-methyltransferase responsible for methylating a variety of hydroxynaphthoic acids. Multiple crystal structures of NcsB1 cocomplexed to substrate and/or cofactor have been solved. These structures revealed a displacement of the C-terminal domain when not bound to substrate, a movement that likely opens up the active site for naphthoate binding. Additionally, the ternary complex structure of 1,4-dihydroxynaphthoic acid, (<italic>S</italic>)-adenosylhomocysteine, and NcsB1 was solved and showed a rotation of this alternate substrate in the binding pocket, allowing for methylation. These results led us to probe NcsB1 activity using active site mutants, demonstrating altered substrate specificity and revealing key residues in substrate binding. The final step of neocarzinostatin biosynthesis involves multiple enzymes that convergently assemble the multiple biosynthetic intermediates to form the chromophore. NcsB2, originally proposed to catalyze the attachment of the naphthoic acid moiety to the enediyne core, has been characterized <italic>in vitro</italic>. Studies into its substrate specificity as an adenylation domain led to a revised biosynthetic pathway of 2-hydroxy-7-methoxy-5-methyl naphthoic acid. Instead of catalyzing the attachment of an enzyme bound naphthoic acid to the enediyne core, NcsB2 was found to act as a CoA-ligase, activating a variety of naphthoic acids and forming a naphthoyl-CoA intermediate. The results of these studies present an outstanding opportunity to produce novel analogs of neocarzinostatin by manipulating its biosynthesis. C-1027 is an architecturally similar enediyne that is also biosynthesized in a convergent route. C-1027 is a member of a class of enediynes that contains a functionalized &beta;-tyrosine derived from L-tyrosine. The first catalytic step towards this beta-tyrosine moiety is achieved by <italic>Sg</italic>TAM, a tyrosine aminomutase that catalyzes a 2,3-amino shift on L-tyrosine to form (<italic>S</italic>)-&beta;-tyrosine. The first X-ray crystal structure of <italic>Sg</italic>TAM was recently solved by our group, revealing structural homology to ammonia lyases. Through site-directed mutagenesis, X-ray crystallography, and biochemical analysis, residues that influence the mechanism by which <italic>Sg</italic>TAM catalyzes this difficult transformation were explored. From these studies, the enzymatic base and other pertinent residues involved in catalysis have been identified. In addition, residues that close the tunnel leading to the active site, thought to play a key role in mutase activity, were probed. Further study of rational mutants of <italic>Sg</italic>TAM will allow us to engineer its activity to alter its substrate specificity and the type of product it produces. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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.