Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2011.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2011
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| Online Access: | http://hdl.handle.net/1721.1/65266 |
| _version_ | 1826207100411314176 |
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| author | Giddings, Lesley-Ann |
| author2 | Sarah E. O'Connor. |
| author_facet | Sarah E. O'Connor. Giddings, Lesley-Ann |
| author_sort | Giddings, Lesley-Ann |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2011. |
| first_indexed | 2024-09-23T13:44:07Z |
| format | Thesis |
| id | mit-1721.1/65266 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T13:44:07Z |
| publishDate | 2011 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/652662019-04-12T13:31:29Z Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle Giddings, Lesley-Ann Sarah E. O'Connor. Massachusetts Institute of Technology. Dept. of Chemistry. Massachusetts Institute of Technology. Dept. of Chemistry. Chemistry. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2011. "February 2011." Vita. Cataloged from PDF version of thesis. Includes bibliographical references. The chemical diversity found in plants has served as a major source of inspiration to many synthetic and biological chemists. Nature has evolved enzyme active sites to catalyze the synthesis of structurally complex compounds that serve as pharmaceuticals, insecticides, dyes, perfumes, and biofuels. In Catharanthus roseus, approximately 130 structurally complex monoterpene indole alkaloids are produced, including the clinically used anti-mitotic drugs, vinblastine and vincristine. The common intermediate to all monoterpene indole alkaloids is strictosidine, the product of an asymmetric Pictet- Spengler condensation of tryptamine and the iridoid monoterpene secologanin. This reaction is catalyzed by the enzyme strictosidine synthase. This thesis describes the use of kinetic isotope effects, the rate dependence on pH, as well as structural and computational data to propose a mechanism by which strictosidine synthase catalyzes the Pictet-Spengler reaction. Notably, the data also shed light on the mechanism of the widely used nonenzymatic reaction. Interestingly, the Pictet-Spenglerase strictosidine synthase belongs to a superfamily of enzymes that have mainly been observed to catalyze ester hydrolysis. Using the [beta]-propeller fold conserved in both strictosidine synthase and the related hydrolase, paraoxonase, rational mutagenesis was used to convert strictosidine synthase into a hydrolase. Intriguingly, during the rational design process, the function of a closely related strictosidine synthase homolog was also functionally characterized as a hydrolase. In addition to reengineering proteins with new catalytic activity, the chemical diversity in plants can also modified using metabolic engineering. However, this approach requires knowledge of the genetic blueprints of the plant to be known. Using the recently released C. roseus transcriptome sequencing data along with co-expression analysis, this thesis describes the functional characterization of a new P450 gene involved in metabolizing a key intermediate in the biosynthesis of bioactive bisindole alkaloids. With the functional characterization of this new gene, a combination of gene silencing and synthetic biology techniques will provide a greater understanding on how to "tune" alkaloid biosynthesis in C. roseus in order to generate more functionally diverse molecules. by Lesley-Ann Giddings. Ph.D. 2011-08-18T19:10:54Z 2011-08-18T19:10:54Z 2011 Thesis http://hdl.handle.net/1721.1/65266 743736886 eng 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. http://dspace.mit.edu/handle/1721.1/7582 195 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemistry. Giddings, Lesley-Ann Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle |
| title | Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle |
| title_full | Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle |
| title_fullStr | Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle |
| title_full_unstemmed | Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle |
| title_short | Discovery, characterization, and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in Madagascar periwinkle |
| title_sort | discovery characterization and rational design of the enzymes involved in monoterpene indole alkaloid biosynthesis in madagascar periwinkle |
| topic | Chemistry. |
| url | http://hdl.handle.net/1721.1/65266 |
| work_keys_str_mv | AT giddingslesleyann discoverycharacterizationandrationaldesignoftheenzymesinvolvedinmonoterpeneindolealkaloidbiosynthesisinmadagascarperiwinkle |