METHOD AND COMPOSITION FOR ENZYME STORAGE

摘要

We describe the detailed production, biochemical characterisation and storage of CYP74 enzymes, including CYP74C3, a recombinant plant cytochrome P450 enzyme with hydroperoxide lyase (HPL) activity from Medicago truncatula, and CYP74A1, an Allene Oxide Synthase (AOS), from Arabidopsis thaliana. Steady state kinetic parameters, substrate and product specificities, Rz, extinction coefficient, haem content, and new ligands are disclosed. We show, on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular weight) analyses that the CYP74 enzymes have low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by reactivation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably coordination of the haem-iron, are novel for 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 turnover number by an order of magnitude. The kcat/Km of up to 1.6 x 108 M-1. s-1 is amongst the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C-C bond. We carry out both kinetic and biophysical studies to demonstrate that these effects, and conclude that these result from 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 P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 and CYP74A1 proteins may be well suited for the purposes of crystallisation and structural resolution of the first plant cytochrome P450 enzyme. We further provide methods and compositions for stable storage of the CYP74 enzymes, including but not limited to HPL and AOS.