This project consisted of three main strands of research: 1) understanding the mechanism of UbiD and UbiX, 2) exploring the natural diversity of the UbiD/UbiX system and 3) creation of novel prFMN-dependent enzymes. The majority of results have already been published in open access scientific publications as well as a range of review and method/protocol papers. Where appropriate these have been accompanied by press-release statements. In addition, several talks were given at international meetings highlighting introducing this work to the scientific audience. We have made significant progress in understanding the mechanism of UbiD and UbiX by detailed biochemical studies of model systems. We have been able to conclusively demonstrate the mechanism of the UbiX flavin prenyltransferase reaction highlighting how a multistep process requires various molecular components to reach the ultimate product. This knowledge was used to create variants able at by-passing the prenyltransferase step and producing prenyl-derived hydrocarbons instead. For fungal UbiDs, we have been able to demonstrate that the usual reaction, 1,3-cycloaddition, underpins the reversible (de)carboxylation. Furthermore, we demonstrated the enzyme plays a key role in ensuring that prFMN-substrate adducts formed remain on the catalytic trajectory. These insights have been used to develop novel fungal UbiD variants using laboratory evolution approaches to yield novel routes to isobutene production as well as aromatic C-H activation using carboxylation.
Given the diversity of UbiD enzymes, we carried out biochemical and structural studies of a distinct representatives, including those that are believed to work in the carboxylative direction. We solved the crystal structures of 9 distinct classes of UbiDs (6 have been published already), the comparison of which has allowed us to understand the common features as well as determine which elements govern substrate specificity. Our work has revealed enzyme dynamics are a key part of the UbiD reaction, as well as reveal considerably heterogeneity in terms of oxygen sensitivity, oxidative maturation, substrate specificity and stability. This has been used to guide direct application in the production of the polymer precursor FDCA, but also laboratory evolution studies of the more robust fugal UbiD yielding demonstration of challenging transformations such as naphthalene carboxylation at ambient conditions are within scope of this enzyme family. We also show how Nature uses a combination of UbiD enzymes to catalyse the carboxylation of phenol through coupling with a phosphorylation-dephosphorylation step (publication in preparation). Our most recent studies have revealed UbiX can be evolved to make prenylated FAD while additional prFMN binding folds were discovered as part of a chaperone system aiding the prFMN incorporation and maturation process for some UbiD enzymes. Both suggest the natural prenylated flavin-repertoire might well extend beyond the UbiX-UbiD system.
