Comparative Investigation of Key Biosynthetic Transformations in Fungal Indole Alkaloid Natural Product Pathways.

Abstract

My dissertational research has focused on the elucidation of three key biosynthetic steps involved in the formation of fungal indole alkaloid natural products. Beginning with the comparative analysis of four assembled putative gene clusters, we performed a detailed annotation of the predicted gene functions and proposed a biosynthetic pathway for each compound: (+)-notoamide, (-)-notoamide, malbrancheamide, and paraherquamide. Although these molecules have demonstrated interest in biological activity and intriguing chemical synthesis, our understanding of their biosynthesis has been limited. Therefore, we sought a stronger biochemical understanding of the biosynthetic enzymes involved in the formation of these molecules. Firstly, NotI, NotI′, and PhqK were identified as flavin monooxygenases from the notoamide and paraherquamide gene clusters. These enzymes catalyze an epoxidation followed by semipinacol rearrangement to generate the spirooxindole moiety found in this class of compounds. Additionally, their utility as tools in synthetic biology was demonstrated by the wide range of flexibility of substrates accepted by these enzymes to broaden the pool of chemical diversity, yielding a new metabolite named notoamide T9. Second, we pursued a thorough biochemical investigation of the MalE prenyltransferase as the primary candidate for the intramolecular Diels-Alder reaction in the malbrancheamide biosynthetic pathway. Early experiments demonstrated the ability of MalE to reverse prenylate early intermediate dipeptides. However, reactions containing the putative native substrate led to the conclusion that MalE is a capable reverse prenyltransferase, but it does not seemingly perform the intramolecular Diels-Alder reaction. Finally, we investigated the halogenase MalA from the malbrancheamide pathway as an alternate candidate for the Diels-Alder reaction. The enzyme demonstrated catalytic activity and performed a halogenation event as predicted. However, contrary to expectations, the enzyme is only able to perform the second halogenation to form the final metabolite malbrancheamide. Interestingly, we were also able to generate new metabolites malbrancheamide D and isomalbrancheamide D. In summary, we elucidated three key steps in bicyclo[2.2.2]diazaoctane biosynthesis. These studies contribute directly to the understanding of complex fungal biosynthetic enzymes and the transformations they catalyze. Furthermore, these enzymes have demonstrated utility in diversification of chemical structures in this family of compounds, and thus may be optimized as biocatalysts.