A novel 41 kDa methyltransferase displaying high regiospecificity towards the allylic hydroxyl moiety of coniferyl alcohol was cloned from suspension cultures of Linum nodiflorum L. and expressed in E. coli. The apparent K m for coniferyl alcohol is 7.23 μM with a V max of 707.5 pkat mg−1 protein at 30°C, whereas the K m for the co-substrate S-adenosyl-l-methionine is 18.5 μM. Structure-function relationship studies revealed stringent structure requirements. Even minor substructure deviations as the side-chain saturation or changes in the phenyl ring substitution result in activities decreased by 75–90%. Crotyl and allyl alcohols are not substrates, confirming that the aromatic ring itself is indispensable, and solely the derivatives with a C3 side-chain are accepted. The enzyme shares only similarities under 46% on amino acid level with other known methyltransferases. The designated reaction product, coniferyl alcohol 9-methyl ether, could be detected in suspension cells. The highest content of up to 0.02% of the dry mass is concurrent with an increase of the specific enzyme activity that reaches its maximum of 3.94 pkat mg−1 on day 6 of the culture period. Transcript levels estimated by semi-quantitative RT-PCR remain constant until day 6 and recede thereafter. The corresponding methyltransferase from Linum flavum L. differs mainly by one short variable fragment. Biochemical characterization revealed a higher catalytic efficiency and a slightly broader substrate plasticity together with a lower sensitivity to the presence of Zn2+, Cu2+ and Co2+. This is to our knowledge the first report of a regiospecific allylic O-methylation of phenylpropanoids in plants.
Plant Molecular Biology – Springer Journals
Published: Mar 2, 2007
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