Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase.
Isoflavonoids are distributed predominantly in leguminous plants, and play pivotal roles in the interaction of host plants with biological environments. Isoflavones in the diet also have beneficial effects on human health as phytoestrogens. The isoflavonoid skeleton is constructed by the CYP93C subfamily of cytochrome P450s in plant cells. The reaction consists of hydroxylation of the flavanone molecule at C-2 and an intramolecular 1,2-aryl migration from C-2 to C-3 to yield 2-hydroxyisoflavanone. In this study, with the aid of alignment of amino acid sequences of CYP93 family P450s and a computer-generated putative stereo structure of the protein, candidates for key amino acid residues in CYP93C2 responsible for the unique aryl migration in 2-hydroxyisoflavanone synthase reaction were identified. Microsomes of recombinant yeast cells expressing mutant proteins of CYP93C2 were prepared, and their catalytic activities tested. The reaction with the mutant in which Ser 310 in the centre of the I-helix was converted to Thr yielded increased formation of 3-hydroxyflavanone, a by-product of the 2-hydroxyisoflavanone synthase reaction, in addition to the major isoflavonoid product. More dramatically, the mutant in which Lys 375 in the end of beta-sheet 1-4 was replaced with Thr produced only 3-hydroxyflavanone and did not yield the isoflavonoid any longer. The roles of these amino acid residues in the catalysis and evolution of isoflavonoid biosynthesis are discussed.
Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan.
Yuji
Y
Sawada
Kengo
K
Kinoshita
Tomoyoshi
T
Akashi
Toshio
T
Aoki
Shin-Ichi
S
Ayabe
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044950:Flavanones; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007529:Isoflavones / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
C420227:2,7,4'-trihydroxyisoflavanone; C468910:3-hydroxyflavanone; D000596:Amino Acids; D044950:Flavanones; D005419:Flavonoids; D007529:Isoflavones; C402170:cytochrome P-450 CYP93C2 (Glycyrrhiza echinata); D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C114032:cytochrome P-450 CYP93B1 (Glycyrrhiza echinata); C083152:liquiritigenin