In silico enzymology: A mechanistic study of prenylated flavin-dependent enzymes

Many enzymes make use of cofactors to aid in catalysis, as they offer alternative reactivities to the canonical amino acids found in the enzyme active site. One such cofactor is the recently discovered prenylated flavin (prFMN), which is used in the UbiD family of enzymes to catalyse reversible decarboxylation of a range of substrates; many of which are of interest to the bioenergy and biotechnology industries. This project will use a range of computational chemistry approaches, including density functional theory (DFT) modelling and molecular dynamics (MD) simulations, to study the mechanism of prFMN-dependent enzymes. Of particular interest is the reversible 1,3-dipolar cycloaddition thought to be crucial to catalysis, and the reversible (de)carboxylation that occurs readily under atmospheric CO2. In order to benchmark these calculations, the project will also include an experimental component, comprising enzyme kinetic measurements made using steady state and rapid-mixing methods and kinetic isotope effect (KIE) measurements made using NMR. An element of both computational and experimental method development will be required and it is envisaged an iterative compute-test cycle will be used to validate proposed mechanism. Once the mechanism of wild-type enzyme(s) is established, the approach will be expanded to identify related enzymes with new substrate profiles and/or to improve the catalytic performance of established enzymes and to expand their substrate scope. The synthesis of prFMN by UbiX enzymes will also be investigated to explore the potential of the biosynthesis of new prFMN-like cofactors, which may expand the reactivity of prFMN-dependent enzymes.

The project is a computationally-driven study of a family of biotechnologically-important enzymes. It falls within the remit of both 'technologies and methodological development' and 'molecules, cells and industrial biotechnology' and is firmly embedded at the interface of chemistry, biology and physics, a key driver for BBSRC in the 'Exploiting new ways of working' agenda. It draws on the core bioscience skills of bioinformatics and mathematics, and provides advanced research training in (biologically-relevant) computational chemistry, recombinant protein production and biophysical characterisation and NMR spectroscopy. Overall, the work will provide a highly interdisciplinary approach to (bio)chemistry/ biophysics-based research, offering highly diverse training opportunities to a PhD student, who will have the additional benefit of being able to access the supervisors' laboratories on a daily basis, as they are all co-located within the same building, the Manchester Institute of Biotechnology (MIB).