# Determining the Architectures and Activities of Polyketide Synthase Modules

> **NIH NIH R01** · UNIVERSITY OF TEXAS AT AUSTIN · 2020 · $306,252

## Abstract

ABSTRACT
Our research on polyketide assembly lines is helping bring about a paradigm shift for how sets of component
enzymes cooperate to biosynthesize polyketide natural products. The updated definition of a module, with the
ketosynthase domain positioned at its downstream end, affects every level of modular polyketide synthase
enzymology. Each of these levels must be further explored to achieve our long-term goal of reprogramming
polyketide assembly lines to synthesize designer molecules and accelerate the drug discovery process. Our
highest-resolution proposal is to study how ketosynthases gatekeep such that only one type of polyketide
intermediate is selected by a module to be further elongated by the downstream assembly line (Specific Aim
1). This will be accomplished through methodology we have developed to crystallographically observe
polyketides bound in ketosynthase active sites and measure the activity of ketosynthases mutated at
suspected gatekeeping residues; engineered triketide synthases will also aid in this effort. From structures
determined by our lab and others, we hypothesize that several uncharacterized domain interfaces are present
within modules. We seek to structurally elucidate these interfaces within the context of the newly-defined
module (Specific Aim 2). Thus, through crystallography and cryo-electron microscopy the structures of
multidomain fragments possessing upstream processing enzymes and a downstream ketosynthase will be
determined. We will also continue our efforts to characterize transient interfaces that form during the reaction
cycle as acyl carrier protein domains present polyketide intermediates to cognate enzymes for catalysis. Our
lab has collected several pieces of structural evidence for higher-order architecture. In the bacillaene
polyketide synthase, a three-helix element adjacent to the ketosynthase domain seems to zipper homodimeric
assembly lines into ~100 MDa assembly sheets observable within Bacillus subtilis cells. We propose to
understand the structures of such biosynthetic megacomplexes by reconstituting them in vitro and observing
them through electron microscopy (Specific Aim 3). Our lab is already visualizing Pks12 from Mycobacterium
tuberculosis both in its “bimodular” and its polymeric assembly line states. We seek to determine how modules
stack to construct higher-order architecture in such systems. Through investigations at each of these levels, an
overall picture of the architectures and activities of polyketide assembly lines will emerge that will be
particularly significant to the future engineering of these medicinally-relevant molecular machines.

## Key facts

- **NIH application ID:** 9918938
- **Project number:** 5R01GM106112-08
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** Adrian Tristan Keatinge-Clay
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $306,252
- **Award type:** 5
- **Project period:** 2013-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9918938

## Citation

> US National Institutes of Health, RePORTER application 9918938, Determining the Architectures and Activities of Polyketide Synthase Modules (5R01GM106112-08). Retrieved via AI Analytics 2026-06-07 from https://api.ai-analytics.org/grant/nih/9918938. Licensed CC0.

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