Present address: Division of Structural Bioinformatics, Graduate School of Integrated Science, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan.
Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase
Article first published online: 30 AUG 2002
The Plant Journal
Volume 31, Issue 5, pages 555–564, September 2002
How to Cite
Sawada, Y., Kinoshita, K., Akashi, T., Aoki, T. and Ayabe, S.-i. (2002), Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase. The Plant Journal, 31: 555–564. doi: 10.1046/j.1365-313X.2002.01378.x
- Issue published online: 30 AUG 2002
- Article first published online: 30 AUG 2002
- Received 21 November 2001; revised 16 April 2002; accepted 14 May 2002.
- CYP93C subfamily;
- homology modelling;
- intramolecular aryl migration;
- site-directed mutagenesis
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 β-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.