Structural and Biochemical Characterization of the Early Steps in Fungal Indole Alkaloid Biosynthesis

Abstract

Prenylated indole alkaloids are a class of natural products with great structural diversity and pharmaceutical potential. These alkaloids, which are isolated from various fungi, mostly Aspergillus and Penicillium, are often produced by homologous dimodular non-ribosomal peptide synthetase (NRPS) pathways that combine two amino acids, typically tryptophan, proline, histidine or phenylalanine, to form the alkaloid skeleton. A unique bicyclo[2.2.2]diazaoctane group is a distinctive feature of the NRPS pathway of malbrancheamide, a calmodulin inhibitor produced by Malbranchea aurantiaca. The bicyclo[2.2.2]diazaoctane is proposed to form via an intramolecular Diels-Alder reaction, but the protein that ensures stereospecificity of the reaction is unknown. This thesis describes research focused on the structural and biochemical characterization of the early steps in the malbrancheamide biosynthetic pathway, which precedes the proposed Diels-Alder reaction. In collaborative studies, I solved the first crystal structure of a fungal NRPS terminal reductase domain, PhqB R in the homologous paraherquamide pathway, which indicates that it functions as a 2-electron or 4-electron reductase. I also solved a 1.6 Å crystal structure of MalC, a candidate for re-oxidation of a potential 4-electron reduction product. However, the MalC structure strongly indicates that it cannot catalyze a redox reaction and its function remains to be characterized. Furthermore, MalB and MalE, two prenyltransferases in the pathway, were characterized in detail. In summary, the dissertation research provides the first structural and biochemical insights into the early steps of malbrancheamide biosynthesis and will guide protein engineering and chemoenzymatic synthesis of related compounds in the future.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/137149/1/qingydan_1.pd