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.

The gateway pDEST17 expression vector encodes a −1 ribosomal frameshifting sequence

“…To verify directly that the ribosomal frameshifting had occurred at the AAA-AAA sequence in the RNA, the Mt HPLF-FS and -WT proteins were over-expressed in E. coli and homogeneous proteins were obtained by FPLC (22). MALDI-ToF MS analysis of tryptic peptides from purified Mt HPLF-FS and Mt HPLF-WT revealed that most of the peptides aligned over the entire length of proteins, but information was apparently lacking from the N-termini.…”

“…Homogenates were then transferred to 50-ml Oakridge tubes, vortexed for 1 min and mixed gently by inversion on a Spiramix 5 (Denley) for 20 min. His-tagged proteins were purified at 4°C as described earlier for Mt HPLF by immobilized metal affinity chromatography using cobalt as a ligand (22). For removal of detergent and histidine from the proteins the concentrated samples were then injected onto a HiLoad Superdex 26/60 gel filtration column (GE Healthcare) or a HiPrep 26/10 rapid desalting column (GE Healthcare) equilibrated with 100 mM sodium phosphate buffer, pH 6.5 and eluted with the same buffer at 2 ml/min (gel filtration) or 10 ml/min (desalting).…”

“…For removal of detergent and histidine from the proteins the concentrated samples were then injected onto a HiLoad Superdex 26/60 gel filtration column (GE Healthcare) or a HiPrep 26/10 rapid desalting column (GE Healthcare) equilibrated with 100 mM sodium phosphate buffer, pH 6.5 and eluted with the same buffer at 2 ml/min (gel filtration) or 10 ml/min (desalting). The concentration of Mt HPLF was determined using a molar extinction coefficient of 120 000 M −1  cm −1 at 391 nm (22). The Reinheitzahl (Rz) value of the purified Mt HPLF protein preparations was ∼1.3, indicating purification to homogeneity.…”

Design of a bivalent peptide with two independent elements of secondary structure able to fold autonomously

“…The design and study of oligomeric α-helical peptides [2][3][4] have contributed to a good understanding of the forces that control interhelical associations. However, the understanding of β-sheet structure lags behind that of α-helices due to the scarcity of simplified models [5][6][7][8]. The next degree of complexity is the design and study of mixed α/β structures.…”

Oxylipin-specific cytochrome P450s (CYP74s) inLotus japonicus: their implications in response to mechanical wounding and nodule formation

“…The symbiosis between legumes and rhizobium needs mutual exchange of signal molecules, such as flavonoids and Nodulation (Nod) factors (Ferguson et al 2010). Several CYP74 genes in leguminous plants have been isolated so far, and their biochemical nature has been studied (Noordermeer et al 2000;Stumpe et al 2005;Hughes et al 2006;De Domenico et al 2007;Kongrit et al 2007). However, their expression has been seldom reported.…”