# Structure, Mechanism, and Engineering of Assembly Line Polyketide Synthases

> **NIH NIH R01** · STANFORD UNIVERSITY · 2020 · $466,003

## Abstract

Assembly line polyketide synthases (PKSs) are fascinating biological machines that catalyze vectorial
polyketide biosynthesis, namely the ability to channel a growing polyketide chain through a uniquely defined
sequence of acyl carrier protein (ACP) and ketosynthase (KS) domains via alternating chain translocation
and elongation reactions. The 6-deoxyerythronolide B synthase is a prototypical example, with 22 distinct
enzymes distributed over three large homodimeric proteins. We seek to understand the mechanisms enabling
vectorial biosynthesis along with those that diverge rapidly to spawn new biosynthetic pathways. We also
seek to exploit this knowledge for the design of chimeric PKSs with catalytic activity that compares well to
their naturally occurring counterparts. To these ends, the following Specific Aims are proposed:
1) Structural studies: We seek to solve the structures of a PKS module (or its catalytic core, comprised of
 its KS and acyltransferase (AT) domains along with flanking linkers) in states that can unequivocally be
 associated with either chain translocation or chain elongation, and to visualize how the KS-AT fragment
 interacts with its ACP partner in each of these states. Key tools already established for this purpose are:
 (i) Methods to crystallize the catalytic cores of two PKS modules; (ii) A method to crosslink the catalytic
 core of a module to either ACP partner; and (iii) High-affinity Fab antibodies that bind distinct domains of
 intact modules and stabilize their presumably dynamic conformations without inhibiting catalysis.
2) Engineering chimeric PKSs: Turnover of chimeric PKSs derived by linking intact modules from
 heterologous sources is invariably poor, principally due to suboptimal ACP-KS recognition at the chimeric
 junction. To solve this problem, we will develop streamlined methods to (i) identify heterologous module
 pairs that interface well with each other; and (ii) improve turnover of a given chimera by tuning ACP-KS
 interactions. Key tools already established for this purpose are (i) a panel of chimeric PKSs showing weak
 but measurable turnover; (ii) phage-display for selected ACPs; and (ii) identification of a specific helix in
 donor ACPs that promotes ACP  KS chain translocation.
3) Dissecting the turnstile mechanism: We have observed that vectorial polyketide biosynthesis is
 synchronized by a “turnstile” mechanism that energetically couples elongation of the growing polyketide
 chain to its intermodular translocation. Our working hypothesis is that the turnstile serves two important
 roles: (i) It prevents “stuttering” (back-transfer) of a newly elongated polyketide chain; and (ii) It prevents
 premature entry of reactive substrates into the KS active site. To test these hypotheses, we will better
 define the turnstile mechanism through a comparative study of a normal and a stuttering module. We will
 also construct and reconstitute PKSs harboring engineered modules that either stutter or on...

## Key facts

- **NIH application ID:** 9926263
- **Project number:** 5R01GM087934-26
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** CHAITAN KHOSLA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $466,003
- **Award type:** 5
- **Project period:** 1995-07-05 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9926263

## Citation

> US National Institutes of Health, RePORTER application 9926263, Structure, Mechanism, and Engineering of Assembly Line Polyketide Synthases (5R01GM087934-26). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9926263. Licensed CC0.

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