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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.

Protein modification is an essential tool in Chemical Biology, allowing a functional biomolecule to be equipped with a small molecule tag or label. However, as proteins are constructed from a limited palette of around 20 canonical amino acids, achieving selective modification can be problematic. Previously reported methods for protein modification will be discussed in Chapter 1; these often rely on alteration of the protein sequence to introduce a uniquely reactive (often non-canonical) amino acid which may then be covalently modified in a bioorthogonal manner. An alternative approach is to identify a uniquely reactive site within the native protein sequence, such as the protein N-terminus or the reactive side chain of an amino acid with low frequency, and modify this using selective chemistry. In this project, modification of a native sequence protein was achieved by targeting a low abundance residue, tyrosine (Tyr), in a selective manner. Tyr was identified as the ideal candidate as it displays only ~3% frequency in the proteome and, due to its electron-rich aryl ring, it can be selectively modified by electrophilic aromatic substitution. Using a diazonium salt as the tuned electrophile, modification results in formation of an azobenzene motif which may be orthogonally cleaved under mild reducing conditions. The resulting cleavage product bears an o-aminophenol modification on the Tyr side chain, which can then be conjugated to a fluorescent label using established chemistry. This system has been developed on a solid-phase platform to give further control over the extent of modification achieved. In Chapter 2, the component parts of this method are developed through reactions performed in-solution on small molecule substrates. In Chapter 3, this work is then moved onto a solid-phase resin in order to 'catch-and-release' small molecule and peptide substrates. Finally in Chapter 4, the resin-based catch-and-release system is optimised for use in protein modification, and analysis of the modification site is explored.

This research investigated the mechanism of action in bacteria of 3,4-methylenedioxy-β-nitropropene (BDM-I), a very broad spectrum antimicrobial lead compound in development as an anti-infective drug. The thesis proposes that BDM-I inhibits bacterial protein tyrosine phosphatases, a novel mechanism of action for an antimicrobial agent and a new target in microorganisms. This very open investigation was directed by considerable biological information on the effects of BDM-I in microorganisms and animals which provided insights into possible and improbable cellular targets. The biological effects of BDM-I were investigated using biochemical and cell-based assays, transmission electron microscopy and whole genome DNA microarray analysis. The specific experiments and order of execution were largely dependent on information gained as the project progressed. BDM-I was shown not to target the metabolic pathways of the major classes of antibacterial drugs, which supports a novel mechanism of action. Investigation of several species-specific effects suggested that cell signalling pathways were a possible target. Based on the structure of BDM-I and review of the scientific literature on cell signalling in bacteria, the hypothesis that BDM-I acted by inhibition of protein tyrosine phosphatases (PTP) was supported by demonstrating inhibition of human and bacterial PTP's in an enzyme assay. This mechanism was consistent with other demonstrated effects: inhibition of the intracellular pathogen, Chlamydia trachomatis; inhibition of swarming in Proteus spp. and inhibition of pigment production in Serratia marcescens; and with kill kinetics in bacteria and yeast. A pilot global genome analysis of BDM-I treated Bacillus subtilis did not detect differential expression of PTP genes but has provided many avenues for further investigation. This research further supports the development of BDM-I as a broad spectrum anti-infective drug.