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Methyl allyl ether formation in plants: novel S-adenosyl l-methionine:coniferyl alcohol 9-O-methyltransferase from suspension cultures of three Linum species

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Abstract

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.

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Abbreviations

CA:

coniferyl alcohol

CA-Me:

coniferyl alcohol 9-methyl ether

DAD:

diode array detector

D.W.:

dry weight

IPTG:

isopropyl-1-thio-β-d-galactopyranoside

SAM:

S-adenosyl-l-methionine

(SM)OMT:

(small molecule) O-methyltransferase.

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Acknowledgments

We wish to thank Prof. Dr. Wibke Diederich (Institut für Pharmazeutische Chemie, Philipps-Universität Marburg) and her team for synthesizing for us 3,4-dimethoxycinnamyl alcohol. The financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

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Authors and Affiliations

  1. Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstr. 17A, Marburg, 35037, Germany

    Anna Berim & Maike Petersen

  2. Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Straße 8, Jena, 07745, Germany

    Bernd Schneider

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  1. Anna Berim
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Correspondence to Maike Petersen.

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Berim, A., Schneider, B. & Petersen, M. Methyl allyl ether formation in plants: novel S-adenosyl l-methionine:coniferyl alcohol 9-O-methyltransferase from suspension cultures of three Linum species. Plant Mol Biol 64, 279–291 (2007). https://doi.org/10.1007/s11103-007-9151-1

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