Purification and substrate specificity of new C. roseus enzymes

Abstract

Terpene indole alkaloids (TIAs) are a class of natural products produced in plants. Many TIAs have medicinal uses; for example, vinblastine has anti-cancer activity and ajmaline has anti-arrhythmic activity. Many TIAs did not evolve to treat human disease, however, and thus most likely do not have optimal pharmacological properties. If TIAs could be modified, the novel TIAs produced could have improved bioactivities when compared with the unmodified natural TIAs. Unfortunately, the immense structural complexity of TIAs makes cost-effective industrial-scale synthesis of the majority of TIAs and TIA analogs unfeasible. Industrial-scale production of TIAs would be improved if TIAs could be produced via reconstitution of the enzymatic pathways in a heterologous organism such as yeast. However, many of the enzymes involved in TIA biosynthesis are unknown, thereby precluding these efforts. If more TIA biosynthetic enzymes were isolated, and the substrate specificity of the enzymes were known, both natural and novel TIA analogs could be more readily produced on an industrial scale. In this thesis I developed strategies to isolate new C. roseus enzymes and to make novel analogs of the anti-hypertensive agent ajmalicine and the anti-neoplastic agent isositsirikine. The NADPH-dependent reductases that produce ajmalicine and isositsirikine have not been isolated. To produce ajmalicine and isositsirikine analogs in vitro, two aims must be accomplished: first, the reductases forming ajmalicine and isositsirikine, ajmalicine synthase and isositsirikine synthase, must be partially purified, and second, the substrate specificity of those reductases must be determined. To satisfy the first of these aims, I developed a partial purification procedure for ajmalicine synthase and isositsirikine synthase from Catharanthus roseus tissue. My partial purification procedure involved acetone precipitation, ion exchange chromatography, and gel filtration chromatography. Analysis by 2D SDS-PAGE shows that the proteins have been significantly purified. I also performed crosslinking experiments with a substrate probe in attempts to isolate ajmalicine synthase and isositsirikine synthase. In the crosslinking studies four enzymes were isolated and cloned, and one has been found to have sinapyl alcohol dehydrogenase activity. I determined the substrate specificities of ajmalicine synthase and isositsirikine synthase' as well as the enzyme that precedes both enzymes in the biosynthetic pathway, strictosidine-pglucosidase (SGD). I found that SGD, ajmalicine synthase, and isositsirikine synthase all have broad substrate specificities, which is promising for the development of novel ajmalicine and isositsirikine analogs with potentially improved therapeutic activities.