Studies on the biosynthesis of indole alkaloids in Catharanthus roseus

Abstract

Catharanathus roseus (Madagascar periwinkle) is one of the few plants which produces all the four major classes of indole alkaloids. Out of the 90 alkaloids which have been isolated and characterized, vinblastine and vincristine (bis-indoles) are of great clinical importance due to their anti-tumor activity. A better understanding ofthe various enzyme catalyzed steps in the biosynthetic pathways is important because of the extremely low yields in chemical syntheses and very low availability of these alkaloids from the plant. This dissertation has focussed on the first two steps of the indole alkaloid pathway--(a) the stereospecific condensation of tryptamine and secologanin which leads to the formation of 3($alpha$)-S strictosidine, which is catalyzed by strictosidine synthase, and (b) the hydrolysis of strictosidine by a $beta$-glucosidase to form cathenamine. An oligonucleotide probe derived from the sequence of the Rauvolfia serpentina strictosidine synthase gene was used to probe a Catharanthus roseus cDNA library in $lambda$gt11. The nucleotide sequence of the C. roseus strictosidine synthase gene was determined and analysis of the predicted amino acid sequences revealed an 80% identity between the two species. The strictosidine synthase gene was cloned into an E. coli inducible vector, pHN1+ and induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 28 mg of active enzyme from a single liter of cell culture (specific activity = 30 nkat/mg). It was demonstrated that hydrolysis of strictosidine in C. roseus takes place by a strictosidine-specific glucosidase and a second enzyme known as cathenamine synthase is required for the stereospecific formation of cathenamine. Absence of the second enzyme leads to a mixture of cathenamine and 19-epicathenamine formed by chemical cyclization. Various 1D and 2D NMR techniques were used for the complete $sp1$H and $sp{13}$C assignments and structure elucidation of new addition products of cathenamine. The complete $sp1$H and $sp{13}$c assignment and conformational analysis of strictosidine and strictosidine lactam has been carried out and the chemical shift values for strictosidine lactam tetera-acetate reassigned.