Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - pre-FAB (Prenylated-flavins: Application and Biochemistry)

Teaser

Enzymes catalyse a wealth of reactions underpinning biochemistry, and thus life itself. For many of these protein catalysts, a small molecule bound to the protein is essential for activity. These molecules, so-called cofactors that are often derived from vitamins, provide the...

Summary

Enzymes catalyse a wealth of reactions underpinning biochemistry, and thus life itself. For many of these protein catalysts, a small molecule bound to the protein is essential for activity. These molecules, so-called cofactors that are often derived from vitamins, provide the necessary chemical reactive groups that are controlled and manipulated by the protein scaffold. Examples include PLP (Vit B6), NAD(P)H (derived from Vit B3), TPP (Vit B1) and FMN/FAD (Vit B2). The flavins FMN/FAD catalyse a very diverse repertoire of reactions, including redox reactions, light-dependent reactions and isomerisations. Many of these enzymes, and flavin-dependent enzymes in particular, have found application in industry. The catalysis of industrially relevant reactions by enzymes offers an alternative green process, but also has the potential to occur with higher efficiency.

Our discovery of a new cofactor, derived from FMN (Vit B2) in 2015 opened up a new area of research. 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. While still resembling FMN, prFMN cannot catalyse any FMN-type reactions, instead, it is proposed to be able to undergo a reaction previously only associated with organic chemistry. This particular reaction, believed to be a so-called 1,3 dipolar cycloaddition, is key to how prFMN helps proteins catalyse particular conversions. At present, the only enzymes found to use prFMN belong to the UbiD/UbiX system. This is a wide spread microbial system, containing an enzyme UbiD and the helper protein UbiX. The latter catalyses the conversion of FMN to prFMN. UbiD in turn uses the prFMN produced by UbiX to interconvert unsaturated acids with corresponding alkenes. The latter reaction 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, before such applications can be considered and developed, we need to know more about the UbiD/UbiX system, and about the prFMN cofactor properties itself. This project seeks to explore in detail how prFMN is synthesised and what its properties are. It is focussed initially on exploring the natural variability of the UbiD/UbiX system to uncovering the mechanism of these enzymes, and to highlight what capabilities already exist in Nature. It furthermore seeks to develop new prFMN-dependent enzymes, making use of the existing diversity of flavin binding enzymes, and combining these with the new capabilities of the prFMN cofactor. The knowledge gathered will underpin future application of the UbiD/UbiX system, but also prFMN in general. We hope these will contribute to both the development of sustainable biofuel production methods, as well as offering new catalytic capabilities in the CO2 sequestration field.

Work performed

Our project has 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. We have made significant progress in understanding the mechanism of UbiD and UbiX by biochemical studies of our 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 is now being used to inform studies aimed at by-passing the prenyltransferase step and producing prenyl-derivid hydrocarbons in stead. For UbiD, we have been able to demonstrate that cycloaddition indeed underpins the reversible (de)carboxylation and that the enzyme plays an key rol in ensuring that prFMN-substrate adducts formed remain on the catalytic trajectory. These insights are now used to develop novel UbiD variants using rational evolution approaches aimed at developing novel (de)carboxylases with substrate specificities of biotechnological interest.

We have started biochemical and structural studies of a wide range of UbiD enzymes, including representatives that are believed to work in the carboxylative direction. At present we have solved the crystal structures of 10 distinct UbiDs (4 have been published), the comparison of which allows 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, and we are actively studying the exact role of protein motion in this reaction. We are using these insights to further inform the development of novel UbiD variants, and to generate new systems capable of CO2 sequestration at ambient conditions.

As we have gather more information on the UbiX/UbiD system, we have been able to produce sufficient amounts of the prFMN cofactor to study its properties in isolation. We have also started combining this cofactor with flavin enzymes, and are set to explore whether the properties of hybrid prFMN-flavin enzymes resemble UbiX/UbiD. We ultimately seek to understand what the inherent prFMN chemical repertoire is, and what aspects can be used to support enzyme catalysis.

Final results

At present, we have moved far beyond our initial understanding of the UbiX/UbiD system in terms of mechanism. The UbiD reaction in particular is unprecedented in Nature (ie dependent on a 1,3 dipolar cycloaddition) and we have been able to provide a full account of how the enzyme is able to guide the reaction through a range of distinct intermediates. We have preliminary evidence to show that enzyme dynamics is coupled to catalysis, especially of the more difficult aromatic substrates. We are seeking to demonstrate that protein motion is able to compress the substrate-cofactor complex, guiding the cycloaddition step.

While we keep expanding our fundamental knowledge of UbiD, both through detailed focussed research on model systems, as well as exploring natural diversity within the UbiD family, we have started laboratory evolution of UbiD enzymes. We have been able to create variants that can take (hetero)aromatic substrates, and are ultimately seeking to demonstrate that very difficult reactions such as benzene carboxylation are within the UbiD scope. We fully expect to demonstrate benzoic acid decarboxylation and the reverse reaction, benzene carboxylation using UbiD variants by the end of the project.

The creations of entirely new prFMN dependent enzymes has been started, and we are poised to explore the biophyscial and biocatalytic properties of these novel entities. We hope to demonstrate that prFMN can support novel mono oxygenation reactions.