Enzymatic halogenation during natural product biosynthesis

Organohalogens feature prominently in the inventory of medicinal natural products. Halogenation often confers chemical properties that contribute to biological activity. Thus, enzymes that catalyze formation of carbon-halogen bonds are of interest both for their intriguing chemistry and their role in natural product biosynthesis. In this dissertation, investigation of flavin-dependent halogenases and a nonheme Fe(lI)/alphaKG-dependent halogenase involved in biosynthesis of several natural products demonstrate the elegant chemical and biosynthetic logic of these enzymes and the pathways in which they participate.;A series of studies on the halogenase RebH from rebeccamycin biosynthesis aimed at elucidating the chemical mechanism of the class of flavin-dependent halogenases. Flavin-dependent halogenases utilize FADH2, O 2, and Cl- to catalyze chlorination of aromatic substrates, however the identity of the chlorinating species formed in the active site was not known. Rapid reaction kinetics using stopped-flow spectroscopy and quench-flow kinetics support the formation of reactive HOCl in the halogenase active site. Furthermore, we provide biochemical and biophysical evidence suggesting formation of a Lys-NH-Cl chloramine intermediate in the enzyme active site. A mechanism for flavin-dependent halogenases is proposed based on these findings.;Several flavin-dependent and nonhemeFe(II)/alphaKG-dependent halogenases act as tailoring enzymes during assembly-line nonribosomal peptide synthetase and polyketide synthase pathways, in which biosynthetic intermediates are covalently attached to carrier proteins. The timing and substrate specificity of halogenases in the assembly lines is a major challenge for investigation of these biosynthetic pathways. In the formation of the 4,5-dichloropyrrole moiety of pyoluteorin, biochemical assays coupled with ESI-FTMS demonstrate that the flavin-dependent halogenase PltA carries out dichlorination of a pyrrolyl ring presented on a carrier protein domain.;Finally, a nonheme Fe(II)/alphaKG-dependent halogenase CmaB is found in the biosynthetic gene cluster for construction of coronamic acid, which contains a cyclopropane ring but no halogen modification. We show that CmaB catalyzes a cryptic chlorination step which is required to functionalize an inert carbon center before closure of the cyclopropane ring by metalloenzyme CmaC. Elucidation of the coronamic acid pathway clarifies the role of these enzymes and the strategy employed for executing this chemical transformation.