Mode-of-action and evolution of methylenedioxy bridge forming P450s in plant specialized metabolism

Abstract

(+)-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 MDBforming CYP719As functionally conserved in Ranunculales.

Oxidative rearrangement of (+)-sesamin by CYP92B14 co-generates twin dietary lignans in sesame

“…(+)-Sesamolinol, although previously proposed to be a putative precursor for (+)-sesamolin biosynthesis 38 , is unlikely to be directly related to the biosynthesis of (+)-sesamolin, since (+)-sesamolinol and (+)-piperitol were not accepted as substrates by CYP81Q1 and CYP92B14, respectively (Fig. 4 ) 10 , 39 . However, there might be additional enzymes, other than CYP92B14, that are responsible for (+)-sesaminol biosynthesis in S .…”

“…For the synthesis of (+)-samin, (+)-sesamolin was treated with 3N aqueous hydrogen chloride in dioxane at 45 °C for 1 h 37 . For the synthesis of (+)-piperitol, (+)-sesamin was treated with diisobutyl aluminum hydride in toluene at 140 °C for 1 h 39 . Sesamol and other chemical reagents, including solvents, were purchased from Tokyo Chemical Industry, Sigma-Aldrich or Nacalai Tesque.…”

Gene isolation, heterologous expression, purification and functional confirmation of sesamin synthase from Sesamum indicum L.

“…However, isolation and characterization of enzymes involved in sesamin biosynthesis for biotechnological application has been very few. CYP81Q1 was earlier expressed in heterologous yeast system or in Baculovirus / Sf9 cell system with the enzyme activity assayed using microsomal fractions from the recombinant cells [ 23 , 37 ]. Recently Hemati and Nayeri [ 26 ] reported amplification and cloning of CYP81Q1 gene from the genomic DNA isolated from an Iranian S. indicum variety Karaj-1.…”

“…Recently by homology modelling and site directed mutagenesis it was shown that residue A308 is important for catalysis leading to formation of characteristic methylenedioxy bridge and a mutation in this residue resulted in inhibition of PSS [ 37 ]. Precise catalytic role of the amino acid residues is yet to be known.…”

(+)‐Sesamin‐oxidising CYP92B14 shapes specialised lignan metabolism in sesame

“…Individual genes cloned in each vector were verified by DNA sequencing of both strands. Expression vectors for SiCYP92B14 and SrCYP92B14 mutants were generated from the wild‐type by PCR‐based mutagenesis, as described previously (Noguchi et al ., 2014; Ono et al ., 2018). All primer sets used for PCR are described in Table S3.…”