A novel O-methyltransferase involved in the first methylation step of yatein biosynthesis from matairesinol in Anthriscus sylvestris

Abstract

Yatein is an important biosynthetic precursor of the lignan podophyllotoxin. In Anthriscus sylvestris, yatein biosynthesis is preceded by two regioselective methylations involving the intermediates: thujaplicatin and 5-Omethylthujaplicatin. The two methylation steps are most likely catalyzed by plant O-methyltransferases (OMTs). In this paper, we report the isolation and characterization of a cDNA encoding an OMT involved in the first methylation step. The OMT cDNA was isolated from an A. sylvestris cDNA library prepared from roots and young shoots. The OMT was expressed as a recombinant protein using the pET expression system. Of the substrates that were tested, the recombinant OMT exclusively catalyzed regioselective methylation of thujaplicatin to produce 5-O-methylthujaplicatin, and thus was designated as A. sylvestris thujaplicatin OMT (AsTJOMT). Kinetic analysis with its substrate (thujaplicatin) showed that AsTJOMT had a Km value of 3.8 μM and kcat value of 0.29 min-1. Quantitative real-time polymerase chain reaction showed that AsTJOMT had the highest expression level in roots compared with other organs. This was in accordance with plant protein assays in which specific activity for thujaplicatin was significantly higher in roots compared with other organs. To the best of our knowledge, this is the first report on the isolation and characterization of a thujaplicatin-specific plant OMT. © 2013 The Japanese Society for Plant Cell and Molecular Biology.