The terpene indole alkaloids are a diverse group of molecules with a range of chemical structures and medicinal uses. Understanding the enzymes that catalyze natural product biosynthesis may enable production in more tractable host organisms, and may also allow reprogramming of biosynthetic pathways to produce """"""""unnatural"""""""" natural products with improved pharmacological activities. Our laboratory seeks to understand, and ultimately harness, the metabolic pathways that direct the biosynthesis of plant-derived terpene indole alkaloids. This proposal describes the use of the terpene indole alkaloid biosynthetic pathway to make novel alkaloid structures.
Specific Aim 1. The First Committed Step of Terpene Indole Alkaloid Biosynthesis This aim focuses on strictosidine synthase, the enzyme that catalyzes the first committed step of the terpene indole alkaloid biosynthetic pathway. The major focus of this aim is to understand and modify the substrate specificity of strictosidine synthase using rational and random mutagenesis. Synthesis of substrates, development of assays and design of mutants are described.
Aim 1 A Tryptamine Substrate Specificity Aim 1B Secologanin Substrate Specificity Aim 1C Altering Strictosidine Synthase Substrate Specificity Specific Aim 2. Later Steps in the Terpene Indole Alkaloid Pathway The substrate specificity of the second committed step of terpene indole biosynthesis, catalyzed by strictosidine glucosidase, will be examined. Precursor directed biosynthesis in C. roseus cell culture and plants are used to probe the substrate specificity of the later steps of the pathway. Alkaloid analogues are isolated and structurally characterized. Use of a substrate analogue to purify a later enzyme in the pathway is described.
Aim 2 A Substrate Specificity of Strictosidine Glucosidase Aim 2B Probing the Biosynthetic Pathway Specificity in vivo ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM074820-01A2
Application #
7263536
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Jones, Warren
Project Start
2007-04-01
Project End
2012-02-29
Budget Start
2007-04-01
Budget End
2008-02-29
Support Year
1
Fiscal Year
2007
Total Cost
$294,787
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Runguphan, Weerawat; O'Connor, Sarah E (2013) Diversification of monoterpene indole alkaloid analogs through cross-coupling. Org Lett 15:2850-3
Glenn, Weslee S; Runguphan, Weerawat; O'Connor, Sarah E (2013) Recent progress in the metabolic engineering of alkaloids in plant systems. Curr Opin Biotechnol 24:354-65
Ruff, Bettina M; Bräse, S; O'Connor, Sarah E (2012) Biocatalytic production of tetrahydroisoquinolines. Tetrahedron Lett 53:1071-1074
Geu-Flores, Fernando; Sherden, Nathaniel H; Courdavault, Vincent et al. (2012) An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 492:138-42
Hicks, Michael A; Barber 2nd, Alan E; Giddings, Lesley-Ann et al. (2011) The evolution of function in strictosidine synthase-like proteins. Proteins 79:3082-98
Liscombe, David K; O'Connor, Sarah E (2011) A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus. Phytochemistry 72:1969-77
Glenn, Weslee S; Nims, Ezekiel; O'Connor, Sarah E (2011) Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor. J Am Chem Soc 133:19346-9
Giddings, Lesley-Ann; Liscombe, David K; Hamilton, John P et al. (2011) A stereoselective hydroxylation step of alkaloid biosynthesis by a unique cytochrome P450 in Catharanthus roseus. J Biol Chem 286:16751-7
Runguphan, Weerawat; Qu, Xudong; O'Connor, Sarah E (2010) Integrating carbon-halogen bond formation into medicinal plant metabolism. Nature 468:461-4
Liscombe, David K; Usera, Aimee R; O'Connor, Sarah E (2010) Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis. Proc Natl Acad Sci U S A 107:18793-8

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