Abstract:
Madagascar periwinkle (Catharanthus roseus) produces the well known and
remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are
derived by coupling vindoline and catharanthine monomers. This thesis describes the
novel application of carborundum abrasion (CA) technique as a tool for large scale
isolation of leaf epidermis enriched proteins. This technique was used to facilitate the
purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse
(l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine
into vindoline. This versatile tool was also used to harvest leaf epidermis enriched
mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and
biochemical characterization of recombinant 160MT enzyme showed that it had a very
narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other
closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition
to allowing the cloning of this gene, CA technique clearly showed that 160MT is
predominantly expressed in Catharanthus leaf epidermis, in contrast to several other
OMTs that appear to be expressed in other Catharanthus tissues. The results provide
compelling evidence that most of the pathway for vindoline biosynthesis including the 0-
methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with
subsequent steps occurring in other leaf cell types.
Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the
transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl
groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian
homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the
benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of
2 recombinant OMTs produced novel substrate specificities not found when each rOMT
was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can
fonn heterodimers that confer novel substrate specificities not possible with the
homodimer alone. The present study describes a 160MT model based strategy attempting
to modify the substrate specificity by site-specific mutagenesis. Our failure to generate
altered substrate acceptance profiles in our 160MT mutants has lead us to study the
biochemical properties ofhomodimers and heterodimers. Experimental evidence is
provided to show that active sites found on OMT dimers function independently and that
bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more
diverse range of natural products in plants.
