Radical-based photoinactivation of fatty acid photodecarboxylases
| Autor: | Tobias M. Hedison, Robin Hoeven, Balaji Lakavath, Muralidharan Shanmugam, Nigel S. Scrutton, Derren J. Heyes, Michiyo Sakuma, Viranga Tilakaratna |
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| Rok vydání: | 2020 |
| Předmět: |
Free Radicals
Carboxy-Lyases photoinactivation Radical Biophysics 01 natural sciences Biochemistry Catalysis Palmitic acid 03 medical and health sciences chemistry.chemical_compound flavoprotein Manchester Institute of Biotechnology Pentadecane Escherichia coli Enzyme kinetics decarboxylation Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences Chemistry Fatty Acids 010401 analytical chemistry Fatty acid Substrate (chemistry) Cell Biology Photochemical Processes ResearchInstitutes_Networks_Beacons/manchester_institute_of_biotechnology Combinatorial chemistry fatty acid photodecarboxylase 0104 chemical sciences Kinetics Enzyme Biocatalysis |
| Zdroj: | Lakavath, B, Hedison, T, Heyes, D, Shanmugam, M, Sakuma, M, Hoeven, R, Tilakaratna, V & Scrutton, N 2020, ' Radical-based photoinactivation of fatty acid photodecarboxylases ', Analytical Biochemistry|Anal Biochem, vol. 600, 113749 . https://doi.org/10.1016/j.ab.2020.113749 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2020.113749 |
| Popis: | Fatty acid photodecarboxylases (FAP) are a recently discovered family of FAD-containing, light-activated enzymes, which convert fatty acids to n-alkanes/alkenes with potential applications in the manufacture of fine and speciality chemicals and fuels. Poor catalytic stability of FAPs is however a major limitation. Here, we describe a methodology to purify catalytically stable and homogeneous samples of recombinant Chlorella variabilis NC64A FAP (CvFAP) from Escherichia coli. We demonstrate however that blue light-exposure, which is required for photodecarboxylase activity, also leads to irreversible inactivation of the enzyme, especially in the absence of palmitate substrate. Photoinactivation is attributed to formation of protein based organic radicals, which were observed by EPR spectroscopy. To suppress photoinactivation, we prepared stable and catalytically active FAP in the dark. The steady-state kinetic parameters of CvFAP (kcat: 0.31 ± 0.06 s−1 and KM: 98.8 ± 53.3 μM) for conversion of palmitic acid to pentadecane were determined using gas chromatography. Methods described here should now enable studies of the catalytic mechanism and exploitation of FAPs in biotechnology. |
| Databáze: | OpenAIRE |
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