Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase

The terminal steps in the biosynthesis of the monoterpenoid indole alkaloid s vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-d ependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and wh ose deduced amino acid sequences were 78% identical. Active enzymes that we re expressed as recombinant His-tagged proteins in Escherichia coli were na med minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-ac etyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and horhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the cataly tic efficiency of recombinant MAT (rMAT) was very poor compared with that o f recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and de acetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical ce lls of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. Th e coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for th e biosynthesis of tabersonine and its substituted analogs occurs within the se cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvin doline with low efficiency suggests that this enzyme, rather than DAT, is i nvolved in vindoline biosynthesis within transformed cell and root cultures , which accumulate low levels of this alkaloid under certain circumstances.