Probing the chemistry of the prenylated flavin cofactor: Synthesis, reactive intermediates, and biocatalysis

The portfolio of chemical reactions mediated by enzymes is greatly enhanced by cofactors: e.g. small organic molecules that are bound by the protein to constitute the active holo-enzyme. The Leys team has recently discovered a new prenylated-flavin cofactor that is an integral part of the widespread ubiD/ubiX enzyme system and exhibits striking chemical properties. This project will address many outstanding questions regarding the chemistry of prenylated-flavin and use the improved understanding of mechanism to develop better biocatalysts. The prenylated-flavin cofactor has the potential to play a major role in synthesis as the diverse UbiD (de)carboxylase enzyme family has a pivotal role in bacterial ubiquinone biosynthesis and microbial biodegradation of aryl and alkenyl compounds. The new cofactor significantly extends flavin chemistry by allowing access to new flavin-derived radical species by oxidation. Crucially, it is believed that a particular reactive intermediate, called an 'azomethine ylide', is ultimately formed by oxidation. It is this key reactive intermediate that appears to instigate the important decarboxylation processes mediated by these enzymes by undergoing ring-forming or 'cycloaddition' reactions. This is believed to be the first example of a cycloaddition reaction of this kind being employed by an enzyme.

If enzyme systems using prenylated-flavin are to achieve their full potential for the sustainable production of key commodity chemicals, an improved mechanistic understanding of the chemical role played by the cofactor is needed. Using synthetic analogues of prenylated-flavin we will explore the electron transfer oxidation chemistry of the new cofactor and investigate the formation and reactivity of key intermediates (using EPR spectroscopy, for example). Armed with improved mechanistic understanding, we will engineer artificial prenylated-flavin-dependent enzymes for the transformation of important chemical feedstocks.