title: Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer-micelle association.
abstract: We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/K(m) of up to 1.6x10(8) M(-1) x s(-1) is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C-C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.
authors.affiliation: John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. richard.hughes@bbsrc.ac.uk
authors.forename: Richard K
authors.initials: RK
authors.lastname: Hughes
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Belfield
authors.forename: Mylrajan
authors.initials: M
authors.lastname: Muthusamay
authors.forename: Anuja
authors.initials: A
authors.lastname: Khan
authors.forename: Arthur
authors.initials: A
authors.lastname: Rowe
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Harding
authors.forename: Shirley A
authors.initials: SA
authors.lastname: Fairhurst
authors.forename: Stephen
authors.initials: S
authors.lastname: Bornemann
authors.forename: Ruth
authors.initials: R
authors.lastname: Ashton
authors.forename: Roger N F
authors.initials: RN
authors.lastname: Thorneley
authors.forename: Rod
authors.initials: R
authors.lastname: Casey
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D046913:Medicago truncatula / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008823:Micelles; D011485:Protein Binding; D012995:Solubility; D013053:Spectrophotometry; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014867:Water
chemical_list: D002021:Buffers; D003902:Detergents; D008024:Ligands; D008823:Micelles; D014867:Water; D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase