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). The extensive diversification of diterpenoids indicates that the manifold hydrocarbon skeletons that can be formed from this C20 backbone provide privileged scaffolds for derivation of biological activity. Indeed, a number of diterpenoids are used as pharmaceuticals (e.g., the anti-cancer paclitaxel/TaxolTM and antibiotic mutilins) or research tools (e.g., the protein kinase C activating and tumor promoting phorbol esters, and adenylate cyclase activator forskolin). In addition, we have contributed to the discovery that the human pathogen Mycobacterium tuberculosis utilizes diterpenoid metabolism in construction of an immune-modulatory factor. The enzymes that produce the underlying hydrocarbon scaffolds catalyze complex electrophilic reactions that form new carbon-carbon bonds, which are of significant mechanistic interest. Our long-term goal is to engineer enzymes and metabolic pathways for the production of targeted libraries and specific individual terpenoid ?natural? products for pharmaceutical investigation and use. This revised renewal proposal focuses on the critically important scaffold assembling diterpene synthases/cyclases. Here we propose to build on our findings from the previous grant period, which includes discovery of means by which these enzymes may be re-engineered for novel product outcome. In particular, the objective of this proposal is to build on our on-going detailed studies of enzymatic structure-function relationships, and to also demonstrate our understanding of their catalytic mechanism by re-engineering activity, specifically to incorporate the addition of water to generate hydroxylated products. Such re-engineering will be enabled by the innovative approach taken here of combining quantum chemical calculations on simplified theozymes to identify key functional groups and their optimal positioning relative to high-energy intermediates, which will guide Rosetta based enzyme (re)design, followed by biochemical characterization of the (re)designed enzymes, with the results then informing subsequent (re)design efforts. We further will investigate the potential pharmaceutical activity of the resulting hydroxylated diterpenes, leveraging our modular metabolic engineering system used to characterize biochemical function. In particular, the resulting recombinant bacteria will be fed to a variety of Caenorhabditis elegans disease models to determine the ability of the the produced hydroxylated diterpenes to alleviate the relevant symptoms.

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., triptolide) pharmaceutical use, as well as an immuno- modulatory factor produced by the human pathogen Mycobacterium tuberculosis. Here we propose to continue our fruitful investigations of the enzymes that form the hydrocarbon scaffolds that underlie the structural diversity of these natural products. This will not only increase our understanding of the relevant enzymatic mechanisms, but also enable engineering of such biosynthesis to increase access to these often scarce natural products.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Bond, Michelle Rueffer
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Iowa State University
Schools of Arts and Sciences
United States
Zip Code
Jia, Meirong; O'Brien, Terrence E; Zhang, Yue et al. (2018) Changing Face: A Key Residue for the Addition of Water by Sclareol Synthase. ACS Catal 8:3133-3137
Liang, Jin; Liu, Jiang; Brown, Reid et al. (2018) Direct production of dihydroxylated sesquiterpenoids by a maize terpene synthase. Plant J 94:847-856
O'Brien, Terrence E; Bertolani, Steven J; Zhang, Yue et al. (2018) Predicting Productive Binding Modes for Substrates and Carbocation Intermediates in Terpene Synthases-Bornyl Diphosphate Synthase as a Representative Case. ACS Catal 8:3322-3330
Schulte, Samuel; Potter, Kevin C; Lemke, Cody et al. (2018) Catalytic Bases and Stereocontrol in Lamiaceae Class II Diterpene Cyclases. Biochemistry 57:3473-3479
Nagel, Raimund; Thomas, Jill A; Adekunle, Faith A et al. (2018) Arginine in the FARM and SARM: A Role in Chain-Length Determination for Arginine in the Aspartate-Rich Motifs of Isoprenyl Diphosphate Synthases from Mycobacterium tuberculosis. Molecules 23:
Xu, Meimei; Jia, Meirong; Hong, Young J et al. (2018) Premutilin Synthase: Ring Rearrangement by a Class II Diterpene Cyclase. Org Lett 20:1200-1202
Inabuy, Fainmarinat S; Fischedick, Justin T; Lange, Iris et al. (2017) Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures. Plant Physiol 175:92-103
Jia, Meirong; Zhou, Ke; Tufts, Samuel et al. (2017) A Pair of Residues That Interactively Affect Diterpene Synthase Product Outcome. ACS Chem Biol 12:862-867
Xu, Meimei; Hillwig, Matthew L; Tiernan, Mollie S et al. (2017) Probing Labdane-Related Diterpenoid Biosynthesis in the Fungal Genus Aspergillus. J Nat Prod 80:328-333
Jia, Meirong; Peters, Reuben J (2017) cis or trans with class II diterpene cyclases. Org Biomol Chem 15:3158-3160

Showing the most recent 10 out of 59 publications