title: Multiple mutagenesis of P450 isoflavonoid synthase reveals a key active-site residue.
abstract: The leguminous isoflavonoid skeleton is constructed by P450 2-hydroxyisoflavanone synthase (CYP93C). Two active-site residues of CYP93C2, Ser 310 and Lys 375, are critical for unusual aryl migration of the flavanone substrate. Leu 371 is located near the substrate in a homology model, and mutant proteins regarding this residue were expressed in recombinant yeast microsomes. The single mutant, L371V, yielded only inactive P420, but multiple mutants incorporating K375T restored the P450 fold: the S310T-L371V-K375T triple mutant showed four times higher P450 level than the wild type. L371V-K375T and S310T-L371V-K375T produced a mixture of major 3beta-hydroxyflavanone and minor flavone, and 100% flavone, respectively, from a flavanone. Thus, Leu 371 appeared to control the substrate accommodation in favor of hydrogen abstraction from C-3 of the flavanone molecule and contribute to the P450 fold under the presence of Lys 375, the residue responsible for aryl migration. The molecular evolution of CYP93 enzymes is discussed.
authors.affiliation: Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sawada
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Ayabe
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D014158:Transcription, Genetic
chemical_list: C402170:cytochrome P-450 CYP93C2 (Glycyrrhiza echinata); D003577:Cytochrome P-450 Enzyme System