Trapping of a Polyketide Synthase Module after C-C Bond Formation Reveals Transient Acyl Carrier Domain Interactions

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Modular polyketide synthases (PKSs) are large enzymatic complexes responsible for synthesizing diverse bioactive compounds.
• The precise mechanism of substrate delivery from acyl carrier protein (ACP) to ketosynthase (KS) domains within PKSs is not fully understood.
• Understanding these dynamics is crucial for engineering PKSs for novel compound production.

Purpose of the Study:

• To elucidate the structural dynamics and inter-domain interactions in a chain-branching module of the rhizoxin PKS.
• To investigate the substrate delivery mechanism to the ketosynthase (KS) domain.
• To provide insights for the rational design of modular PKS assembly lines.

Main Methods:

• Mechanism-based crosslinking using a synthetic substrate surrogate.
• Cryo-electron microscopy (cryo-EM) to determine the structure of the crosslinked complex.
• AlphaFold2 predictions for identifying potential binding sites.
• Nuclear Magnetic Resonance (NMR) spectroscopy to study solution-state complex formation.
• Photochemical crosslinking/mass spectrometry of standalone domains.

Main Results:

• Identified an asymmetric state of the dimeric protein complex after C-C bond formation using cryo-EM.
• AlphaFold2 predictions suggested two distinct acyl carrier protein (ACP) binding sites, including a potential substrate loading site.
• NMR and crosslinking/mass spectrometry confirmed transient, linker-independent interdomain interactions in solution.

Conclusions:

• Structural insights into a branching PKS module captured after C-C bond formation enhance understanding of PKS domain dynamics.
• The findings reveal key inter-domain interactions critical for substrate processing.
• This work provides valuable information for the future engineering of PKS systems.