Structural study on human microbiome-derived polyketide synthases that assemble genotoxic colibactin.

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Colibactin is a genotoxin from the human microbiome that drives colorectal cancer by damaging host gut epithelial DNA.
• Colibactin biosynthesis involves a hybrid non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway, but enzyme structures are poorly understood.

Purpose of the Study:

• To determine the cryo-electron microscopy (cryo-EM) structures of two critical colibactin-producing polyketide synthase (PKS) enzymes, ClbC and ClbI.
• To elucidate the structural basis of enzyme-substrate interactions and inter-enzyme complex formation within the colibactin assembly line.

Main Methods:

• Utilized cryo-electron microscopy (cryo-EM) to capture high-resolution structures of ClbC and ClbI.
• Employed a substrate-mimic crosslinker to trap enzymes in distinct functional states.
• Analyzed protein-protein interactions between NRPS and PKS enzymes.

Main Results:

• Determined the structures of ClbC and ClbI, revealing carrier protein (CP) domain binding sites on ketosynthase (KS) domains.
• Identified a novel docking interaction between ClbI and its upstream enzyme ClbH.
• Established a 1:2 stoichiometry for the ClbH-ClbI complex, mediated by ClbH's C-terminal peptide and ClbI's dimeric interface.

Conclusions:

• The structural insights advance the understanding of the colibactin biosynthetic machinery.
• Findings provide a foundation for future studies on NRPS-PKS pathway mechanisms and potential therapeutic targeting of colibactin production.