# Structure and Function of Biosynthetic Enzymes

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2020 · $357,032

## Abstract

The proposed research explores the structure and mechanism of terpenoid cyclases, which are unique
among enzymes in that they catalyze the most complex carbon-carbon bond forming reactions in biology: 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 terpenoid cyclase structure and mechanism will enable drug
discovery and manufacture at the interface of natural products chemistry, enzymology, structural biology, and
synthetic biology. To advance our understanding of structure-function relationships in terpenoid cyclases, we
will pursue the following lines of investigation:
 (1) We will determine the structural basis of reprogrammed cyclization cascades catalyzed by site-specific
mutants of the C15 sesquiterpene cyclase epi-isozizaene synthase from Streptomyces coelicolor (EIZS). We
will study the temperature dependence of cyclization fidelity in selected EIZS mutants and we will determine
crystal structures of complexes with analogues of substrate and carbocation intermediates, as well as the C15
hydrocarbon product. These "snapshots" of wild-type and mutant EIZS cyclization cascades will show us how
cyclization chemistry can be redirected to generate alternative products.
 (2) We will determine the structural basis of cyclization fidelity in the bifunctional C20 diterpene cyclase
fusicoccadiene synthase from Phomopsis amygdali (PaFS). We will study the temperature dependence of
PaFS cyclization fidelity and we will determine crystal structures of complexes with substrate analogues and
the hydrocarbon product to generate snapshots of the diterpene cyclization cascade. We will also study a
double mutant designed to introduce C25 sesterterpene cyclase activity by increasing active site volume. These
structures will be the first to map out the reaction coordinate of a diterpene cyclization reaction.
 (3) We will determine the structural basis of assembly-line biosynthesis in the bifunctional C25
sesterterpene synthases ophiobolin F synthase from Aspergillus clavatus (AcOS) and mangicdiene synthase
from Fusarium graminearum (FgMS). We will develop a radiolabeled substrate to measure the steady-state
kinetics of sesterterpene hydrocarbon formation, and we will determine whether channeling occurs between
the prenyltransferase and cyclase active sites of each bifunctional enzyme. We will also determine structures
of AcOS and FgMS using X-ray crystallography and/or electron microscopy to better understand assembly-line
terpenoid biosynthesis. These studies will provide the first structural views of sesterterpene synthases, a family
of terpenoid biosynthetic enzymes discovered only recently.

## Key facts

- **NIH application ID:** 9984396
- **Project number:** 5R01GM056838-23
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** DAVID W CHRISTIANSON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $357,032
- **Award type:** 5
- **Project period:** 1998-08-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9984396

## Citation

> US National Institutes of Health, RePORTER application 9984396, Structure and Function of Biosynthetic Enzymes (5R01GM056838-23). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9984396. Licensed CC0.

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