Natural products have a proven record of providing a significant fraction, either directly or as lead compounds, of human medicines. Among natural products, the terpenoids (isoprenoids) stand out as being the largest class (>50,000 already known), with the 20-carbon diterpenoids targeted here forming a significant fraction of these (>12,000 known). Indeed, the extensive diversification of diterpenoids indicates that the manifold structures that can be formed from this C20 backbone provide a rich source of biological activity, and a number of these natural products are used as pharmaceuticals (e.g., the anti- cancer paclitaxel/TaxolTM and anti-biotic mutilins) or research tools (e.g., the protein kinase C activating and tumor promoting phorbol esters, and adenylate cyclase activator forskolin). Conversely, we have recently discovered that the human pathogen Mycobacterium tuberculosis utilizes a diterpenoid, isotuberculosinol, to modulate our initial immune response to infection. Accordingly, we propose here to continue our productive investigations of the enzymes, from both plants and microbes, that catalyze formation of the underlying hydrocarbon skeletal structures. Specifically, we will build on our findings from the previous grant period, which includes not only the discovery of isotuberculosinol, but elucidation of some of the first diterpene synthase crystal structures as well. This proposal then advances our long- term goal of engineering enzymes and metabolic pathways for the production of targeted libraries and specific individual terpenoid 'natural'products for pharmaceutical investigation and use. In particular, the objective of this proposal is a detailed structure-function investigation of the synthases/cyclases that catalyze formation of the hydrocarbon skeletons underlying the structural diversity of diterpenoid natural products. These enzymes catalyze complex electrophilic reactions that form new carbon-carbon bonds, and play important roles in initiating diterpenoid biosynthesis. Accordingly, our studies have implications not only for providing increased access to such natural products, but also insights into potential drug targets, specifically in the widespread pathogen M. tuberculosis.

Public Health Relevance

The diterpenoids form a very large class of natural products (>12,000 known), which includes many of realized (e.g., TaxolTM) or potential (e.g., prostratin) pharmaceutical use, as well as a compound (isotuberculosinol) that we have recently discovered is produced by the human pathogen Mycobacterium tuberculosis to suppress our immune response to infection. Here we propose to continue our fruitful investigations of the enzymes that form the hydrocarbon backbones that underlie the structural diversity of these natural products. This will not only increase our understanding of the relevant enzymatic mechanisms for engineering such biosynthesis to increase access to these often scarce natural products, but also provide insights into these enzymes as potential drugs targets against the pernicious M. tuberculosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM076324-06
Application #
8211972
Study Section
Special Emphasis Panel (ZRG1-BCMB-P (02))
Program Officer
Hagan, Ann A
Project Start
2005-12-01
Project End
2015-07-31
Budget Start
2011-08-15
Budget End
2012-07-31
Support Year
6
Fiscal Year
2011
Total Cost
$295,866
Indirect Cost
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
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