Amphipol trapping of a functional CYP system

Tomas Laursen, Peter Naur, Birger Lindberg Møller

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Abstract

In plants, some enzymes of the cytochrome P450 (CYP) superfamily are thought to organize into transient dynamic metabolons to optimize the biosynthesis of bioactive natural products. Metabolon formation may facilitate efficient turnover of labile and toxic intermediates and prevent undesired metabolic cross talk. Two CYPs, CYP79A1 and CYP71E1 involved in the synthesis of dhurrin, were used to assess the possibility to use amphipols (APols) to trap these membrane-bound enzymes in a soluble form in a detergent-free environment. APol surfactants are short polymers composed of a hydrophilic backbone randomly grafted with hydrophobic side chains. An optimal ratio of 1:2 w/w of protein to APol (A8-35) was required for trapping the single transmembrane helices of CYP79A1, CYP71E1, and the electron partner cytochrome P450 oxidoreductase (POR). CYP79A1 and POR retained their individual activity upon A8-35 trapping, whereas a direct interaction between CYP79A1 and POR was hampered, probably due to electrostatic repulsion caused by the negatively charged APol molecules. Upon substitution of POR with NADPH-ferredoxin oxidoreductase and ferredoxin as an electron donor system, the CYPs were shown to be catalytically active. The use of APol surfactants in functional and structural studies of membrane proteins is discussed. © 2013 International Union of Biochemistry and Molecular Biology, Inc.
Original languageEnglish
JournalBiotechnology and Applied Biochemistry
Volume60
Issue number1
Pages (from-to)119-127
ISSN0885-4513
DOIs
Publication statusPublished - 2013

Keywords

  • Biochemistry
  • Biological membranes
  • Enzymes
  • Surface active agents
  • Optimization

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