Exploiting alkaloid biosynthesis in Madagascar periwinkle to obtain natural product derivatives and new biocatalysts

Plant alkaloid biosynthesis produces many natural products with medicinal value. For example, vinblastine and vincristine from Catharanthus roseus monoterpene indole alkaloid biosynthesis, and camptothecin derivatives from Ophiorrhiza pumila quinoline alkaloid biosynthesis, are anticancer agents currently used in the clinic. Strictosidine synthase is a key enzyme in the biosynthesis of these medicinal natural products, but its narrow substrate scope limits precursor-directed biosynthesis of alkaloid analogs in plant cell cultures. I describe two new assays to monitor strictosidine synthase activity, which enable the rapid screening of enzyme mutant libraries to identify two strictosidine synthase variants that accept new substrates. A transgenic plant cell culture that contains one of these mutants generated "unnatural" monoterpene indole alkaloids in C. roseus. I also describe the characterization of 0. pumila strictosidine synthase, which has considerably broader substrate specificity than the homologous enzyme from C. roseus. This alternative catalyst is a candidate enzyme for construction of transgenic cell cultures, and potentially useful as a biocatalyst, since it catalyzes the asymmetric Pictet- Spengler reaction to form tetrahydro-p-carboline pharmacophores. I used computer modeling to propose a model for how strictosidine synthase achieves its high stereoselectivity; this model may be used to engineer a Pictet-Spenglerase that forms the alternative stereoisomer.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2010

Author dc:creator

• Bernhardt, Peter, Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Sarah E. O'Connor.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/58384

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/58384