# Malonyl-thioester Isosteres to Determine Enzyme Structure-Function Relationships

> **NIH NIH R01** · PURDUE UNIVERSITY · 2021 · $303,787

## Abstract

Project Summary/Abstract
Malonyl-thioesters are one of the major reactive intermediates in the biosynthesis of fatty acids and polyketides.
Because fatty acids are essential to cellular life, the inhibition of fatty acid synthases is a viable mechanism for
the generation of antimicrobials, anticancer agents and control of metabolic disease. Polyketides on the other
hand are widely used as antibiotics and anticancer agents, making polyketide synthases targets for enzyme
engineering. While most intermediates in fatty acid and polyketide biosynthesis are used in reversible reactions,
malonyl-thioesters are created in and used in essentially irreversible reactions. This makes studying the
enzyme:malonyl-thioester interactions virtually impossible because the malonyl-thioesters are destroyed in the
process. To overcome this problem analogs of malonyl-thioesters were generated by other researchers. These
analogs replace the thioester ketone with a thioether or oxetane, both of which are stable to enzymatic activity.
However, neither of these analogs bind in enzyme active sites in catalytically relevant orientations. Thus, there
is a critical need to develop stable malonyl-thioester isosteres capable of binding in enzyme active sites to
elucidate molecular interactions and conformational changes leading to efficient catalysis. The objective of this
proposal is to overcome problems associated with the natural malonyl-thioesters and previously synthesized
isosteres. We have a panel of malonyl-thioesters that preserve a key ketone lost in the previous isosteres. Our
first aim is to solve crystal or cryo-EM structures of acyl-CoA carboxylase enzymes in complex with our best
isosteres to elucidate the enzyme:substrate interactions and conformational changes. Our second aim is to solve
crystal, cryo-EM or NMR structures of ȕ-ketoacyl synthase enzymes in complex with our best isosteres to
elucidate enzyme:substrate interactions and conformational changes. Together these studies will validate the
use of malonyl-thioester analogs with carboxylate isosteres to capture enzyme:substrate interactions. Our
structures will reveal conformational changes during catalysis that can be targeted for drug design and that need
to be accounted for during enzyme engineering.

## Key facts

- **NIH application ID:** 10263242
- **Project number:** 5R01GM140290-02
- **Recipient organization:** PURDUE UNIVERSITY
- **Principal Investigator:** Jeremy Ray Lohman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $303,787
- **Award type:** 5
- **Project period:** 2020-09-20 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10263242

## Citation

> US National Institutes of Health, RePORTER application 10263242, Malonyl-thioester Isosteres to Determine Enzyme Structure-Function Relationships (5R01GM140290-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10263242. Licensed CC0.

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