Investigations into the molecular basis of enzyme-catalyzed reactions in natural product biosynthesis

Bacteria synthesize a variety of compounds that can be grouped into two broad categories. Primary metabolites are involved in central metabolism and are essential for the survival of the organism. On the other hand, secondary metabolites are produced by organisms for an ‘added advantage’ effect. These molecules may be responsible for signaling and communication, antimicrobial defense, or metal import, for starters. Characterization of the enzymes responsible for the biosynthesis of these molecules will lead to a better understanding of specialized metabolism. In order to utilize these enzymes in a biotechnological setting, additional studies need to be performed on their substrate scopes and specificities. In this work, I describe my efforts to structurally and biochemically characterize enzymes capable of carrying out interesting chemical modifications. In the first chapter, I introduce a class of secondary metabolite termed the ribosomally synthesized and post-translationally modified peptides (RiPPs). The chemical modifications found in these peptide natural products further distinguish each them into specific types. The borosins constitute a class of RiPPs that are characterized by a cyclized N-C structure with methylations on the amide group of the peptide backbone. I describe the structural characterization of the tailoring enzyme capable of methylating the peptide backbone, dbOphMA, and attempts to delineate the substrate scope of this enzyme. In the second chapter, I introduce a class of RiPPs termed the lasso peptides. The maturation of a subset of lasso peptides requires the action of two proteins: a recognition element, and a protease. This subset is hence referred to as split-B lasso peptides. It has been previously demonstrated that split-B lasso peptides possess a conserved motif in their leader peptides that are key to recognition element binding. In this chapter, I investigate the prevalence of this binding motif across split-B lasso peptide biosynthetic gene clusters. I then elaborate on a series of experiments that investigate the portability of these systems using a ‘plug-and-play’ fashion. The focus of the third chapter is an enzyme involved in the biosynthesis of fosfomycin – a phosphonate antibacterial used to treat urinary tract infections. This enzyme, termed PsfC, possesses a metallohydrolase fold, but not share the canonical trinuclear metal center-binding motif associated with the class of enzymes. Structural characterization of PsfC reveals that it binds two equivalents of iron, which is necessary for the enzyme to decarboxylate its substrate. Our collaborative efforts demonstrate that PsfC is the enzyme that accepts the last known intermediate of fosfomycin biosynthesis in pseudomonads. The characterization of this enzyme thus completes the biosynthetic pathway. In the fourth chapter, I discuss the biochemical characterization of an Fe(II)- and alpha-ketoglutarate dependent dioxygenase that is capable of degrading phenoxy and aryloxyphenoxy propanoate based herbicides. Coupled with structural analyses performed by a previous lab member, these studies reveal the molecular basis behind herbicide degradation by this class of enzymes.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste