UC Berkeley

One of these methods, cryptic halogenation, involves the early enzymatic installation of a halogen to activate an otherwise unreactive C-H bond. A downstream enzymatic or non-enzymatic step can then use the halogen as a leaving group to facilitate the formation of a new chemical motif. Halogens also serve as reactive handles in synthetic chemistry and are essential in tuning the bioactive properties of small molecules. As such, we are interested in the discovery of new secondary metabolic pathways that utilize cryptic halogenation to build complex small molecules. By expanding the biochemical options available to modern chemical synthesis, we imagine a world in which the sustainable biomanufacturing of diverse chemical scaffolds becomes increasingly common and viable. In this dissertation, we describe how radical amino acid halogenases enabled us to identify and characterize biosynthetic gene clusters responsible for amino acid-derived natural product biosynthesis.We first describe how halogenation of the amino acid lysine enables a pyridoxal-5´-phosphate (PLP)-dependent enzyme, PazB, to catalyze intramolecular cyclization, giving rise to the cyclopropane-containing amino acid pazamine. We examine the substrate tolerance of PazB by creating non-natural biosynthetic pathways and demonstrate that it can also be used for cyclobutane biosynthesis. We further examine an ecological role for pazamine through plant-bacteria inoculation studies. We also examine the trv cluster, where halogenation enables substrate recognition by the diiron enzyme TrvC. Without the C4 chloride, L-ornithine is not a substrate for TrvC. The presumptive aldehyde product of these two enzymes is then likely phosphorylated by the semialdehyde dehydrogenase TrvE. Two electrophilic sites, the acyl phosphate and the C-Cl bond, may facilitate two independent intramolecular cyclizations in the biosynthesis of trv cluster product. We lastly demonstrate how choosing an enzymatic reaction, in this case halogenation, serves to inspire divergent research goals: biosynthesis, biocatalysis, and new enzyme discovery. As a whole, these collected works exemplify the power of enzymatic halogenation in the biosynthesis of small molecules and how it can be harnessed for the creation of new molecules.