Kinetic Analysis of the Catabolism of Tabersonine in Catharanthus roseus Leaf Protein Extracts

Abstract

Vinblastine and vincristine are valuable chemotherapy compounds produced by Catharanthus roseus. However, they are produced in small quantities in the plant because of their cytotoxicity and spatial separation of the metabolic pathways of the two monomeric molecules, vindoline and catharanthine, that condense to form them. There have been extensive attempts to metabolically engineer greater production of these molecules, but the specialized cell types needed for the terminal steps of vindoline biosynthesis from tabersonine and the high regulation of the pathway in the plant have made use of a homologous host difficult. Recently, all metabolic steps of the pathway have been elucidated, but there remains difficulty in culturing a heterologous host to express all metabolic steps. There is almost no knowledge of the catabolism of these alkaloids in the plant, and this knowledge could be used in future metabolic engineering attempts. Leaf protein extracts were incubated with tabersonine to attempt to find possible catabolic products, and initial rate experiments were used to calculate kinetic parameters of a possible catabolic enzyme. Results showed no formation of previously known or unknown metabolites. After performing kinetic studies, evidence was found of an allosteric enzyme acting on tabersonine with a vm of 5.4 ± 1.9 μM min-1, a Km " of 16,000 ± 14,000 μM, and a Hill coefficient of 2.0 ± 0.2.