The Fdc1 and Pad1 decarboxylase system from Saccharomyces cerevisiae has been identified as a potential candidate to feature in novel biofuel production pathways based on its ability to catalyze the transformation of sorbic acid, an antimicrobial compound, to 1, 3-pentadiene, a volatile hydrocarbon. Although information on the system is currently insufficient to permit a full assessment of its potential for future commercialization, it is hoped that (rational) engineering approaches can be used to evolve the enzymes to produce more desirable hydrocarbons. This requires biochemical characterization of the proteins. Genetic manipulation experiments have indicated that both enzymes are required for activity. However, no in vitro studies were conducted to verify the function, determine the relationship or establish the cofactor requirements of Fdc1 and Pad1. Results reported here establish that Fdc1 is the enzyme responsible for catalyzing decarboxylation, requiring a novel cofactor synthesized by Pad1 (or the bacterial homologue UbiX) for activity. High resolution crystal structures and mass spectrometry data from Fdc1 co-expressed with UbiX have indicated that the cofactor corresponds to a modified flavin mononucleotide (FMN) that has been extended with a C5-unit through linkages at the N5 and C6 atoms, creating a fourth, non-aromatic ring on the isoalloxazine group. Subsequent solution studies have established that this modification is achieved through isoprene chemistry, with UbiX facilitating prenyl transfer from dimethylallyl monophosphate (DMAP) to FMN. Analysis of wild type and mutant UbiX constructs by kinetic X-ray crystallography has allowed several distinct stages of the prenyl transfer reaction to be trapped, establishing that the protein uses a number of chemical strategies similar to terpene synthases to generate its product. The active site is dominated by pi systems, which aid heterolytic cleavage of the isoprene precursors phosphate-C1' bond following FMN reduction, leading to the formation of an N5-C1' intermediate. UbiX then acts as a chaperone for adduct reorientation, potentially via a transient tertiary carbocation, ultimately resulting in ring closure between the C6 and C3'. This work has established the biochemical principles underpinning the Fdc1 and Pad/UbiX decarboxylase system, providing a platform from which rational evolution approaches can be applied to the enzymes, specifically Fdc1, to improve their validity in the biofuels industry. It has also identified a novel cofactor that extends the previously well-documented flavin and isoprenoid repertoire.
|Date of Award||31 Dec 2015|
- The University of Manchester
|Supervisor||David Leys (Supervisor) & Nigel Scrutton (Supervisor)|