# Structure and Function of Biosynthetic Enzymes

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2022 · $426,836

## Abstract

The proposed research explores the structure and mechanism of terpene cyclases, which are unique
among enzymes in that they catalyze the most complex carbon-carbon bond forming reactions in nature: on
average, more than half of the substrate carbon atoms undergo changes in bonding and/or hybridization during
the course of a typical enzyme-catalyzed reaction. Notably, many terpenoids exhibit useful pharmacological
properties, such as the blockbuster cancer chemotherapy drug Taxol (paclitaxel) and the antimalarial drug
artemisinin. Thus, a better understanding of terpene cyclase structure and mechanism will enable drug
discovery and manufacturing at the interface of natural products chemistry, enzymology, structural biology, and
synthetic biology. To advance our understanding of structure-function relationships in terpene cyclases, we will
pursue the following lines of investigation:
 (1) We will determine the structural basis of substrate binding, transit, and catalysis in a class I assembly-
line terpene synthase, fusicoccadiene synthase from Phomopsis amygdali (PaFS). We will determine cryo-EM
structures of PaFS complexes with an inhibitor and with a substrate analogue, and we will determine the
influence of oligomeric structure as well as the interdomain linker on substrate channeling between the
prenyltransferase and cyclase domains. These studies will broaden our understanding of substrate channeling
– perhaps better designated as "directed substrate transit" – between covalently-linked enzymes catalyzing
consecutive reactions in a biosynthetic pathway.
 (2) We will determine the structural basis of substrate binding, transit, and catalysis in class II assembly-
line terpene synthases, the copalyl diphosphate synthases from Penicillium verruculosum (PvCPS) and
Penicillium fellutanum (PfCPS). We will complete the cryo-EM structure determination of PfCPS, and we will
determine the cryo-EM structures of its complexes with a substrate analogue and product. We will also
determine whether directed substrate transit occurs between the prenyltransferase and cyclase domains in
both PfCPS and PvCPS, and we will ascertain the importance of oligomeric structure for catalytic function.
 (3) We will explore and exploit the structural basis of chemodiversity in terpene biosynthesis, focusing on
sesquiterpene synthases that quench reactive carbocation intermediates with hydroxyl or amino nucleophiles.
We will convert our paradigm for protein engineering, epi-isozizaene synthase, into a sesquiterpene alcohol
synthase. We will also determine structure-function relationships for the sesquiterpene synthase FlvF from
Aspergillus flavus to understand how it catalyzes the condensation of a cyclic sesquiterpene with
dimethylcadaverine. Importantly, FlvF represents the first example of a synthase that catalyzes C–N bond
formation with a cyclic terpene.

## Key facts

- **NIH application ID:** 10516850
- **Project number:** 2R01GM056838-25
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** DAVID W CHRISTIANSON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $426,836
- **Award type:** 2
- **Project period:** 1998-08-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10516850

## Citation

> US National Institutes of Health, RePORTER application 10516850, Structure and Function of Biosynthetic Enzymes (2R01GM056838-25). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10516850. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*