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Date Submitted

Spring 4-18-2015

Research Mentor and Department

Timothy Wencewicz

Restricted/Unrestricted

Dissertation/Thesis

Abstract

In the golden age of drug discovery, scientists turned to nature to find and emulate molecules with pharmacological activity. The beta-lactam ring in penicillin has proven to be one of the most important classes of antibiotics of the last century. The molecule I am studying, obafluorine, was isolated from Pseudomonas fluorescens and has a beta-lactone ring in its structure—a close relative of the beta-lactam ring in Penicillin. Obafluorin was isolated as a broad-spectrum antibacterial and antifungal agent that could have therapeutic properties, but its cellular target is still unknown. The Wencewicz Lab in the WUSTL Department of Chemistry is studying the biosynthesis of obafluorine in order to understand how nature enzymatically constructs the rare beta-lactone ring. Moreover, the mechanism by which a P. fluorescens bacterial cell defends itself from the secreted toxic molecule is under investigation. The entire sequenced genome of P. fluorescens ATCC 39502 was searched for a putative cluster of genes responsible for obafluorine production. Then, a complete biosynthetic pathway was proposed, based on the predicted enzymatic reactions of the gene products. One of the enzymes I identified in the Obi cluster is a serine hydroxymethyltransferase (SHMT), which is critical in folate biosynthesis— a facet of bacterial growth. Interestingly, the gDNA of P. fluorescens codes for 2 unique SHMTs; one within (ObiH) and one outside of the Obi cluster, with significantly different amino acid sequences. These mutations alter conserved binding sites of the ObiH enzyme, hinting that ObiH could confer a new self-protection function in addition to the normal SHMT function. It is hypothesized that obafluorine will not inhibit the mutated SHMT enzyme (ObiH) as severely as the wild type SHMT, and the P. fluorescens cell is thereby protected during obafluorine production. If this turns out to be true, obafluorine represents the first non-folate inhibitor of SHMT which is a sought after anticancer, antimalarial, and antimicrobial biological target.