Time-resolved serial femtosecond crystallography on fatty-acid photodecarboxylase: lessons learned
Time-resolved serial femtosecond crystallography on fatty-acid photodecarboxylase: lessons learned
Upon absorption of a blue-light photon, fatty-acid photodecarboxylase catalyzes the decarboxylation of free fatty acids to form hydrocarbons (for example alkanes or alkenes). The major components of the catalytic mechanism have recently been elucidated by combining static and time-resolved serial femtosecond crystallography (TR-SFX), time-resolved vibrational and electronic spectroscopies, quantum-chemical calculations and site-directed mutagenesis [Sorigué et al. (2021), Science, 372, eabd5687]. The TR-SFX experiments, which were carried out at four different picosecond to microsecond pump–probe delays, yielded input for the calculation of Fourier difference maps that demonstrated light-induced decarboxylation. Here, some of the difficulties encountered during the experiment as well as during data processing are highlighted, in particular regarding space-group assignment, a pump-laser power titration is described and data analysis is extended by structure-factor extrapolation of the TR-SFX data. Structure refinement against extrapolated structure factors reveals a reorientation of the generated hydrocarbon and the formation of a photoproduct close to Cys432 and Arg451. Identification of its chemical nature, CO2 or bicarbonate, was not possible because of the limited data quality, which was assigned to specificities of the crystalline system. Further TR-SFX experiments on a different crystal form are required to identify the photoproducts and their movements during the catalytic cycle.
- Commissariat à l’Energie Atomique et aux Energies Alternatives France
- French National Centre for Scientific Research France
- Grenoble Alpes University France
- Institut de Biologie Structurale France
- Stanford University United States
serial femtosecond crystallography, Light, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], structure-factor extrapolation, MESH: Spectrum Analysis, Crystallography, X-Ray, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, fatty-acid photodecarboxylase, photoenzymes, Crystallography, Molecular Biology/Structural Biology [q-bio.BM], MESH: Crystallography, Lasers, Spectrum Analysis, Fatty Acids, 500, 540, Research Papers, MESH: Light, MESH: Fatty Acids, time-resolved crystallography, X-Ray, MESH: Lasers
serial femtosecond crystallography, Light, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], structure-factor extrapolation, MESH: Spectrum Analysis, Crystallography, X-Ray, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, fatty-acid photodecarboxylase, photoenzymes, Crystallography, Molecular Biology/Structural Biology [q-bio.BM], MESH: Crystallography, Lasers, Spectrum Analysis, Fatty Acids, 500, 540, Research Papers, MESH: Light, MESH: Fatty Acids, time-resolved crystallography, X-Ray, MESH: Lasers
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