# Structural Biology of Complex Enzymes

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2020 · $418,036

## Abstract

Project Summary
Polyketide natural products are the basis for a substantial number of pharmaceuticals, including some with
newly discovered indications for inflammatory diseases. Development of polyketide lead compounds is
challenging due to their chemical complexity and the inability to obtain most of them from natural sources.
Thus the biosynthetic pathways are attractive targets for expanding chemical space through chemo-enzymatic
synthesis and for engineering pathways to new function. The type I polyketide synthases (PKS) are assembly-
line megasynthases where several distinct modules sequentially extend and modify a polyketide intermediate.
This project examines fundamental aspects of PKS module function and of PKS enzyme domains that catalyze
unusual reactions. In Aim 1, a robust assay system using authentic, late-stage polyketide intermediates as
substrates will be employed to determine the selectivity of PKS catalytic domains for their natural acyl carrier
protein (ACP) partners and the limits of enzyme tolerance for foreign ACPs. Details of ACP-enzyme
interaction will be visualized in crystal structures of crosslinked ACP-enzyme complexes and will aid future
efforts to design of a broadly tolerated generic ACP. In Aim 2, dehydratase domains will be investigated to
determine the structural basis for two unusual activities: a rare dual dehydratase-isomerase activity and the
unprecedented dehydration of δ-hydroxyacyl substrates. In Aim 3, the structural basis for C-methyltransferase
activity by domains embedded in PKS modules will be investigated and the substrate range will be explored.
Macrolide antibiotics are among the most interesting polyketides, and a final aim will address the determinants
of macrocycle formation by the thioesterases (TE) that offload polyketide products. Using four macrocycle-
forming TEs of known structure and a panel of natural and non-natural substrates, Aim 4 will test the
hypothesis that the active site of each TE is adapted to bind its natural substrate in conformations that are
most conducive to cyclization and not hydrolysis to a linear product. Details of trapped acyl-enzyme
intermediates will be visualized in crystal structures. These fundamental studies will have direct relevance to
efforts to tap the amazing diversity of polyketide natural products by exploiting the biosynthetic pathways in
development of new compounds. The enzymes that catalyze reactions of dehydration, isomerization, methyl
transfer and macrocycle formation can be deployed as tools to expand the chemical potential of polyketide bio-
activity.

## Key facts

- **NIH application ID:** 9895732
- **Project number:** 5R01DK042303-30
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** JANET L. SMITH
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $418,036
- **Award type:** 5
- **Project period:** 1990-02-01 → 2021-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9895732

## Citation

> US National Institutes of Health, RePORTER application 9895732, Structural Biology of Complex Enzymes (5R01DK042303-30). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9895732. Licensed CC0.

---

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