Periodic Reporting for period 5 - pre-FAB (Prenylated-flavins: Application and Biochemistry)
Reporting period: 2022-09-01 to 2023-08-31
Enzymes catalyse a wealth of reactions with many depending on a cofactor for activity. Many of these enzymes, and flavin-dependent enzymes in particular, have found application in industry.
Our discovery of a new cofactor, derived from the flavin FMN opened up a new area of research, and associated scope for development of novel green chemistry to underpin the burgeoning bioeconomy of the future. This new cofactor is obtained through prenylation of the FMN molecule, leading to prenylated FMN or prFMN. This chemical modification completely alters the properties of the FMN molecule, and affects a true metamorphosis. The prFMN form an integral part of the wide spread microbial UbiD/UbiX enzyme system. This consists of the prFMN-dependent (de)carboxylase enzyme UbiD and prFMN-synthesising UbiX. The reversible decarboxylation catalysed by UbiD is of considerable interest for two main reasons: one, it has the potential to contribute to CO2 sequestration/fixation (when used in the carboxylative direction), thereby converting alkene hydrocarbons to organic acids and two, when run in the decarboxylative direction (ie releasing CO2) it can be used to convert biomass derived organic acids to hydrocarbons (ie biofuels).
However, to develop these applications more fundamental understanding about the UbiD/UbiX system and the prFMN cofactor properties itself are required. This project focussed on exploring the natural variability of the UbiD/UbiX system with the aim to uncovering the mechanism of these enzymes, and to highlight what capabilities already exist in Nature. It has demonstrated UbiD enzymes and especially the robust fungal ferulic acid decarboxylases can be readily evolved and contribute to both the development of sustainable biofuel production methods, as well as offering new catalytic capabilities in the CO2 sequestration field.
Our discovery of a new cofactor, derived from the flavin FMN opened up a new area of research, and associated scope for development of novel green chemistry to underpin the burgeoning bioeconomy of the future. This new cofactor is obtained through prenylation of the FMN molecule, leading to prenylated FMN or prFMN. This chemical modification completely alters the properties of the FMN molecule, and affects a true metamorphosis. The prFMN form an integral part of the wide spread microbial UbiD/UbiX enzyme system. This consists of the prFMN-dependent (de)carboxylase enzyme UbiD and prFMN-synthesising UbiX. The reversible decarboxylation catalysed by UbiD is of considerable interest for two main reasons: one, it has the potential to contribute to CO2 sequestration/fixation (when used in the carboxylative direction), thereby converting alkene hydrocarbons to organic acids and two, when run in the decarboxylative direction (ie releasing CO2) it can be used to convert biomass derived organic acids to hydrocarbons (ie biofuels).
However, to develop these applications more fundamental understanding about the UbiD/UbiX system and the prFMN cofactor properties itself are required. This project focussed on exploring the natural variability of the UbiD/UbiX system with the aim to uncovering the mechanism of these enzymes, and to highlight what capabilities already exist in Nature. It has demonstrated UbiD enzymes and especially the robust fungal ferulic acid decarboxylases can be readily evolved and contribute to both the development of sustainable biofuel production methods, as well as offering new catalytic capabilities in the CO2 sequestration field.
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.
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.
The work carried out in this project has moved the prFMN field beyond our initial discovery of this cofactor. We have provided additional structures of key intermediates and variants of the model UbiD and UbiX originally reported, confirming hypothesis regarding mechanism and providing new insights into the use of strain to guide covalent catalysis. We have shown that enzyme dynamics is coupled to catalysis for UbiDs that act on aromatic substrates, revealing a domain motion is coupled to active site architecture. This suggests protein motion is able to compress the substrate-cofactor complex, guiding the covalent prFMN-substrate adduct formation step.
The diversity of UbiD has been demonstrated by biochemical and structural studies of a wide range of representatives, including those who act as unidirectional carboxylases. This has supported application of these enzymes, either directly of following laboratory evolution, with routes including both decarboxylation as well as carboxylative reactions. In case of the former, collaboration with an industry partner allowed us to demonstrate how laboratory evolution can yield robust routes to isobutene production. We show CO2 fixation under ambient conditions using UbiD enzymes can be achieved using coupled to other enzyme systems or the use of higher concentrations of CO2. The carboxylative synthesis of FDCA and the conversion of styrene to cinnamyl alcohol illustrate both approaches.
The most challenging transformations catalysed by the UbiD family consist of benzene and naphthalene carboxylation, postulated as part of the microbial process of anaerobic degradation of these compounds. We have been able to conclusively demonstrate naphthalene carboxylation in evolved fungal UbiD, although benzene carboxylation remains out of reach for the presently characterised enzymes.
The prFMN chemical repertoire is distinct from flavins, and we postulated Nature might have evolved additional prFMN-dependent enzymes that harness distinct aspects to catalyse other reactions. We have not found direct evidence for this, but have been able to evolve UbiX to make prFAD (as opposed to prFMN) through relatively modest changes, suggesting prFAD could exist in Nature. Furthermore, the unexpected discovery of a prFMN-binding chaperone with a fold distinct form UbiD or UbiD does suggest a wider range of protein-prFMN complexes exist in Nature, some of which might exhibit catalytic activity.
The diversity of UbiD has been demonstrated by biochemical and structural studies of a wide range of representatives, including those who act as unidirectional carboxylases. This has supported application of these enzymes, either directly of following laboratory evolution, with routes including both decarboxylation as well as carboxylative reactions. In case of the former, collaboration with an industry partner allowed us to demonstrate how laboratory evolution can yield robust routes to isobutene production. We show CO2 fixation under ambient conditions using UbiD enzymes can be achieved using coupled to other enzyme systems or the use of higher concentrations of CO2. The carboxylative synthesis of FDCA and the conversion of styrene to cinnamyl alcohol illustrate both approaches.
The most challenging transformations catalysed by the UbiD family consist of benzene and naphthalene carboxylation, postulated as part of the microbial process of anaerobic degradation of these compounds. We have been able to conclusively demonstrate naphthalene carboxylation in evolved fungal UbiD, although benzene carboxylation remains out of reach for the presently characterised enzymes.
The prFMN chemical repertoire is distinct from flavins, and we postulated Nature might have evolved additional prFMN-dependent enzymes that harness distinct aspects to catalyse other reactions. We have not found direct evidence for this, but have been able to evolve UbiX to make prFAD (as opposed to prFMN) through relatively modest changes, suggesting prFAD could exist in Nature. Furthermore, the unexpected discovery of a prFMN-binding chaperone with a fold distinct form UbiD or UbiD does suggest a wider range of protein-prFMN complexes exist in Nature, some of which might exhibit catalytic activity.