Molecular cloning and functional expression of O-methyltransferases commonto isoquinoline alkaloid and phenylpropanoid biosynthesis

In cell suspension cultures of the meadow rue Thalictrum tuberosum, biosynt hesis of the anti-microbial alkaloid berberine can be induced by addition o f methyl jasmonate to the culture medium. The activities of the four methyl transferases involved in the formation of berberine from L-tyrosine are inc reased in response to elicitor addition. Partial clones generated by RT-PCR with methyltransferase-specific primers were used as hybridization probes to isolate four cDNAs encoding O-methyltransferases from a cDNA library pre pared from poly(A)+ RNA isolated from methyl jasmonate-induced cell suspens ion cultures of T. tuberosum. RNA gel blot hybridization indicated that the transcripts for the methyltransferases accumulated in response to addition of methyl jasmonate to the cell culture medium. The cDNAs were functionall y expressed in Spodoptera frugiperda Sf9 cells and were shown to have varyi ng and broad substrate specificities. A difference of a single amino acid r esidue between two of the enzymes was sufficient to alter the substrate spe cificity. The four cDNAs were expressed either as four homodimers or as six heterodimers by co-infection with all possible combinations of the four re combinant baculoviruses. These 10 isoforms thus produced displayed distinct substrate specificities and in some cases co-infection with two different recombinant baculoviruses led to the O-methylation of new substrates. The s ubstrates that were O-methylated varied in structural complexity from simpl e catechols to phenylpropanoids, tetrahydrobenzylisoquinoline, protoberberi ne and tetrahydrophenethylisoquinoline alkaloids, suggesting that some bios ynthetic enzymes may be common to both phenylpropanoid and alkaloid anaboli sm.