Molecular cloning and characterization of a novel S-adenosyl-L-methionine: coniferyl alcohol O-methyltransferase from suspension cultures of Linum nodiflorum L

Several methylation reactions occur in the course of aryl tetralin lignan biosynthesis in L. nodiflorum L., explaining our interest in this enzyme class. Using a homology-based RT-PCR strategy [1], we have cloned and functionally expressed in E. coli a novel 41 kDa methyltransferase displaying high regiospecificity towards the allylic OH-group of coniferyl alcohol (CA). The apparent K_m for CA was determined to be 6.77µM with V_max of 621.19µkat/kg protein at 30°C, whereas the K_m for the co-substrate S-adenosyl-L-methionine is 18.93µM Structure-activity relationship studies proved the double bond of the side-chain to be important, as the enzyme activity with dihydroconiferyl alcohol amounted to about 22.95% as compared to the best substrate (CA). The substitution pattern of the phenol ring is also essential, for both sinapyl and cinnamic alcohols were poorly accepted (7.86 and 15.69% activity of that with CA, resp.), whereas crotonyl and allyl alcohols are no substrates (<0.7% activity), confirming the aromatic ring itself is indispensable. The WU-BLAST2 (EMBL, Heidelberg) search revealed only low homology (<45%) to enzymes listed hitherto.

The transcription levels, determined by semi-quantitative RT-PCR, were highest between day 2 and 6 of the suspension culture period, whilst the corresponding enzyme activity declined for the first 4 days and rose from day 5 onwards, reaching its maximum of 612.31 nkat/kg on day 7.

By identifying a so far undescribed substrate preference of an enzyme with little homology to the already known, function attribution to newly discovered and/or yet unassigned genes might now be facilitated. The physiological role of this side-chain methylation of coniferyl alcohol, a precursor of both lignin and lignan biosyntheses, remains to be assessed yet.

Reference: 1. Ibrahim, R.K. et al. (1998), Plant Mol. Biol. 36: 1–10.