Fellowship: Theresa Downey: Enzyme Engineering of MtmOIV from the Mithramycin Biosynthetic Pathway for the Production of Novel Natural Product Analogues

Mithramycin (MTM) is an antineoplastic antibiotic whose clinical use has been hindered by its high toxicity. Recently there has been a revived interest in this compound as new potential clinical uses have been discovered. The trisacchride side chain at the 2-position of MTM is known to interact with the base pairs located in the minor groove of CG rich sequences of DNA. By altering this trisaccharide chain it may be possible to create MTM analogues with improved or new activity and/or decreased toxicity. Due to MTM’s complex chemical structure combinatorial biochemistry provides a promising tool in creating saccharide chain analogues where synthetic methods have failed. MtmOIV is a highly specific homodimeric FAD- and NADPH-dependent Baeyer-Villiger monooxygenase (BVMO) that catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway in a step following the glycosylation reactions that yield premithramycin B (PreB), the substrate of MtmOIV. Thus, if combinatorial biochemistry is to be used to create new MTM analogues from PreB-analogues with altered saccharide patterns, it is essential that MtmOIV can act on those derivatives, necessitating that MtmOIV’s catalytic mechanism must be elucidated in detail. The structure of MtmOIV has previously been solved with and without substrate PreB bound. Using the structure as a starting point our research goal is to understand MtmOIV’s catalytic mechanism and engineer an enzyme capable of utilizing new unnatural substrates.