Radical-based photoinactivation of fatty acid photodecarboxylases

Balaji Lakavath, Tobias Hedison, Derren Heyes, Muralidharan Shanmugam, Michiyo Sakuma, Robin Hoeven, Viranga Tilakaratna, Nigel Scrutton

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Article number113749
JournalAnalytical Biochemistry|Anal Biochem
Volume600
DOIs
Publication statusPublished - 26 Apr 2020

Keywords

  • fatty acid photodecarboxylase
  • flavoprotein
  • decarboxylation
  • photoinactivation

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology

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  • Biochemical and Biophysical Sciences Technology Platform

    Heyes, D. (Senior Technical Specialist), Boothman, C. (Senior Technical Specialist), Cliffe, L. (Technical Specialist), Dunstan, M. (Senior Technical Specialist), Golovanova, M. (Senior Technician), Hoeven, R. (Technical Specialist), Lopez Perez, R. (Senior Technician), Sakuma, M. (Senior Technician), Tait, S. (Senior Technician) & Tilakaratna, V. (Senior Technician)

    FSE Research

    Facility/equipment: Platform

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