Characterization of ent-kaurene oxidase activity from Gibberella fujikuroi

Abstract

Cell extracts of wild-type and mutant strains of Gibberella fujikuroi were assayed for kaurene oxidase activity, using ent-[3H]kaurene as the substrate. Extracts from strain SG78 exhibited the highest specific activity, and were used in subsequent experiments. The microsomal enzyme activity was solubilized with buffers or salt solutions at a concentration of 400 mM. Both the soluble and microsomal preparations showed characteristic cytochrome P-450 spectra, ligand binding spectra with the substrate and with the plant growth regulator, paclobutrazol, and inhibition of enzymic activity by carbon monoxide. The addition of 20% glycerol to the extraction buffer stabilized the activity to some extent. Loss of enzymic activity on storage was accompanied by conversion of P-450 to P-420. Michaelis-Menten kinetic parameters for the membrane-bound and soluble enzyme have been estimated, as have constants for the binding of ent-kaurene and paclobutrazol to the P-450 and P-420 forms of the protein.