SNAPsHOTs

Fatty acid photodecarboxylase (FAP) is a photoenzyme recently discovered and characterized by the coordinator and a partner of this proposal (Sorigué et al. 2017 Science 357:903). FAP catalyses the light-driven decarboxylation of fatty acids into hydrocarbons and CO2. It is only the third photoenzyme to be identified and thus represents a unique opportunity to deepen the understanding of light-driven catalysis. In spite of our initial characterization of FAP, 3D structures of intermediate-states and a detailed mechanistic understanding of FAP catalytic events have remained elusive. Our goal is to gain insight into the mechanistic events along the FAP photocycle, occurring from the ultra-fast time scale (femto- to picoseconds) right after photon absorption to product formation on slower time scales (nano- to milliseconds). Our central hypothesis is that photon absorption by FAD leads to product formation in FAP via a sequence of intermediate states within a photocycle that involves distinct spectroscopic and structural changes. Our objectives are the structural and spectroscopic characterization of FAD excited states on the ultra-fast time scale, the structure determination of catalytic intermediate states, the spectroscopic elucidation of electron and proton transfer steps, the structural and spectroscopic observation of the cleavage of the C-C bond leading to substrate decarboxylation, and the identification of a proton (or hydrogen atom) donor (amino acid or water molecule) whose existence has been postulated in the photocycle. The chosen methodology consists of a combination of experimental biophysical and biochemical techniques, including time-resolved crystallography at synchrotrons and X-ray free electron lasers (XFEL), FTIR and time-resolved infrared, absorption and fluorescence spectroscopy on FAP in solution and in crystals and complementary QM/MM computational methods. In particular, the project SNAPsHOTs will fully exploit the unique capabilities of the European facility for X-ray free electron laser (XFEL) that has been recently inaugurated and to which France contributed financially. Funding will be critical to maintain French leadership in a highly competitive field with potential industrial applications. Ultimately, our project will provide a molecular movie of the FAP photocycle that features the structural changes occurring during light-driven enzyme catalysis at atomic resolution. Structural and mechanistic information should also be useful to improve stability, turnover or specificity of FAP in view of biotechnological applications.