|Budget Amount *help
¥14,300,000 (Direct Cost: ¥14,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥11,300,000 (Direct Cost: ¥11,300,000)
1. The biosynthetic relationship between agatharesinol and trans-hinokiresinol was investigated by feeding experiments. Fresh sapwood sticks of Cryptomeria japonica was fed with p-coumaryl alcohol-[9,9-^2H], and then incubated under high-humidity, during which the two norlignans were produced simultaneously. Gas cluomatography-mass spectrometry (GC-MS) analyses of the extracts from the wood demonstrated that while trans-hinokiresinol was strongly deuterium-labeled, agatharesinol was only slightly labeled, suggesting that p-coumaryl alcohol, which is a precursor of hinolciresinol, is not involved in the biosynthesis of agatharesinol. Therefore, the norlignan carbon skeleton of agatharesinol must be formed from different types of phenylpropanoid monomer compared to those utilized by the trans-hinolciresinol pathway. The biosynthesis ofthese two norlignans seems to branch at an early stage, i. e., before the framing of the norlignan carbon skeleton.
2. The biosynthetic relationship between agatharesinol and sequirin C/metasequirin C was investigated by enzymatic experiments. Since it was found that sequirin C content is higher in the heartwood side of the intermediate wood, microsomal enzyme was prepared from this wood part. Enzyme assay was performed using the microsomal enzyme, agatharesinol as the substrate, in the presence of various cofactors, under various temperatures and pHs. GC-MS analyses of the reactants demonstrated that enzymatic formation of sequirin C and metasequirin C occurred with the highest yield after enzyme reaction in the presence of both NADH and FAD, at pH7, at 30oC. Therefore, an enzyme which hydroxylates agatharesinol to sequirin C/metasequirin C, namely agatharesinol hydroxylase was found, and enzymatic conversion between norlignans were demonstrated for the first time.
Norlignan biosynthesis is not well understood in comparison with other plant secondary metabolites, and this study contributes to the development in plant science.