Article ID: 14.0828a
(+)-Sesamin is a major furofran-class lignan in sesame seeds and harbors characteristic two methylenedioxy bridges (MDB) that are sequentially formed from (+)-pinoresinol via (+)-piperitol by a Sesamum indicum P450, CYP81Q1. However, the molecular basis for this unique catalytic activity of CYP81Q1 has been poorly understood. To elucidate MDB formation, we tested various natural and non-natural metabolites as substrates for CYP81Q1. A synthetic (+)-SC1mr and a naturally occurring (+)-kobusin showed inhibitory effect on the production of (+)-sesamin by CYP81Q1 unlike (+)-epipinoresinol and (−)-pinoresinol, indicating the strict diastereomer and enantiomer selectivity. Homology modeling followed by site-directed mutagenesis of CYP81Q1 showed that an amino acid residue crucial for MDB formation is a unique Ala residue (A308), located in I-helix proximal to the substrate pocket, responsible to the conserved distal-Thr residue. MDB by CYP81Q1 is produced possibly through the formation of a substrate-participated hydrogen-bonding network, since single replacement of the Ala by Thr severely and specifically lowered the MDB forming activity. This hypothesis is supported by a newly identified MDB-forming enzyme CYP81Q38 from Phryma leptostachya harboring an Ala responsible to Ala308 in CYP81Q1. An evolutional perspective of CYP81Q1 is discussed in relation to another MDB-forming CYP719As functionally conserved in Ranunculales.