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 K m value of 3.8 µM and k cat 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.

Recent advances in research on lignans and neolignans

“…13 In Linum, Anthriscus, and Podophyllum plants, matairesinol is also converted into hinokinin, yatein or podophyllotoxin via multiple biosynthetic pathways, even though all relevant enzymes have not yet been identied. 6 The isolation and characterization of a thujaplicatin-specic plant O-methyltransferase (TJOMT) from A. sylvestris that exclusively catalyzed regioselective methylation of thujaplicatin to produce 5-O-methylthujaplicatin, a biosynthetic precursor of the lignan podophyllotoxin, was recently reported by Ragamustari et al 14 Furthermore, next generation sequencing (NSG) coupled to bioinformatics and metabolome analyses of Podophyllum tissues led to the discovery of two putative unknown genes in the podophyllotoxin biosynthetic pathway. 15 The cytochrome P450s CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum were capable of specically converting (À)-matairesinol to (À)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates that were tested.…”

“…Strikingly, this class of lignans has seen remarkable growth in the number of new examples reported recently, with more than 130 in the period 2009 to 2015 compared to only 32 in the previous four years. The structures and absolute congurations of fourteen new dibenzocyclooctadiene lignans, ananolignans A-N (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) isolated from the seeds of Kadsura ananosma, were established using a combination of spectroscopic methods and CD techniques. Ananolignans F and ananolignan L showed the most promising in vitro cell survival data against oxidative stress-induced neurotoxicity.…”

Essences in Metabolic Engineering of Lignan Biosynthesis

“…In Linum , Anthriscus , and Podophyllum plants, matairesinol is also converted into hinokinin, yatein, or PTOX via multiple biosynthetic pathways, although all of the relevant enzymes have not yet been identified [ 1 , 2 , 55 ]. In A. sylvestris , AsTJOMT exclusively methylates the 5-hydroxyl group of thujaplicatin, an intermediate of PTOX [ 70 ]. The homologous enzymes, CYP719A23 (from P. hexandrum ) and CYP719A24 (from P. peltatum ) participate in the conversion of matairesinol into pluviatolide, a more downstream intermediate of PTOX ( Figure 2 ), via methylenedioxy bridge formation [ 71 ].…”

Towards Metabolic Engineering of Podophyllotoxin Production

“…Feeding experiments showed incorporation of matairesinol, thujaplicatin, 5-methylthujaplicatin, and 4,5-dimethylthujaplicatin into yatein [70]. This was followed by the discovery of the enzyme thujaplicatin O-methyltransferase (AsTJOMT), which methylates thujaplicatin to form 5-O-methylthujaplicatin [71]. Furthermore, they found incorporation of matairesinol and pluviatolide in bursehernin, but no further incorporation into yatein.…”