Research Article

Mechanism and dynamics of fatty acid photodecarboxylase

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Science  09 Apr 2021:
Vol. 372, Issue 6538, eabd5687
DOI: 10.1126/science.abd5687

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Light makes light work of fatty acids

Photosynthetic organisms are notable for their ability to capture light energy and use it to power biosynthesis. Some algae have gone a step beyond photosynthesis and can use light to initiate enzymatic photodecarboxylation of fatty acids, producing long-chain hydrocarbons. To understand this transformation, Sorigué et al. brought to bear an array of structural, computational, and spectroscopic techniques and fully characterized the catalytic cycle of the enzyme. These experiments are consistent with a mechanism starting with electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. The wealth of experimental data explains how algae harness light energy to produce alka(e)nes and provides an appealing model system for understanding enzyme-catalyzed photochemistry more generally.

Science, this issue p. eabd5687

Structured Abstract


Photoenzymes are rare biocatalysts driven by absorption of a photon at each catalytic cycle; they inspire development of artificial photoenzymes with valuable activities. Fatty acid photodecarboxylase (FAP) is a natural photoenzyme that has potential applications in the bio-based production of hydrocarbons, yet its mechanism is far from fully understood.


To elucidate the mechanism of FAP, we studied the wild-type (WT) enzyme from Chlorella variabilis (CvFAP) and variants with altered active-site residues using a wealth of techniques, including static and time-resolved crystallography and spectroscopy, as well as biochemical and computational approaches.


A 1.8-Å-resolution CvFAP x-ray crystal structure revealed a dense hydrogen-bonding network positioning the fatty acid carboxyl group in the vicinity of the flavin adenine dinucleotide (FAD) cofactor. Structures solved from free electron laser and low-dose synchrotron x-ray crystal data further highlighted an unusual bent shape of the oxidized flavin chromophore, and showed that the bending angle (14°) did not change upon photon absorption (step 1) or throughout the photocycle. Calculations showed that bending substantially affected the energy levels of the flavin. Structural and spectroscopic analysis of WT and mutant proteins targeting two conserved active-site residues, R451 and C432, demonstrated that both residues were crucial for proper positioning of the substrate and water molecules and for oxidation of the fatty acid carboxylate by 1FAD* (~300 ps in WT FAP) to form FAD●– (step 2). Time-resolved infrared spectroscopy demonstrated that decarboxylation occured quasi-instantaneously upon this forward electron transfer, consistent with barrierless bond cleavage predicted by quantum chemistry calculations and with snapshots obtained by time-resolved crystallography. Transient absorption spectroscopy in H2O and D2O buffers indicated that back electron transfer from FAD●– was coupled to and limited by transfer of an exchangeable proton or hydrogen atom (step 3). Unexpectedly, concomitant with FAD●– reoxidation (to a red-shifted form FADRS) in 100 ns, most of the CO2 product was converted, most likely into bicarbonate (as inferred from FTIR spectra of the cryotrapped FADRS intermediate). Calculations indicated that this catalytic transformation involved an active-site water molecule. Cryo-Fourier transform infrared spectroscopy studies suggested that bicarbonate formation (step 4) was preceded by deprotonation of an arginine residue (step 3). At room temperature, the remaining CO2 left the protein in 1.5 μs (step 4ʹ). The observation of residual electron density close to C432 in electron density maps derived from time-resolved and cryocrystallography data suggests that this residue may play a role in stabilizing CO2 and/or bicarbonate. Three routes for alkane formation were identified by quantum chemistry calculations; the one shown in the figure is favored by the ensemble of experimental data.


We provide a detailed and comprehensive characterization of light-driven hydrocarbon formation by FAP, which uses a remarkably complex mechanism including unique catalytic steps. We anticipate that our results will help to expand the green chemistry toolkit.

Elucidation of the FAP photocycle by combining spectroscopic, biochemical, crystallographic, and computational studies.


Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

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