Architecture of a PKS-NRPS hybrid megaenzyme involved in the biosynthesis of the genotoxin colibactin Original paper

What was studied?

This study used structural biology to define how ClbK, a PKS–NRPS hybrid “megaenzyme” encoded by the pks biosynthetic gene cluster in colibactin-producing Enterobacteriaceae, is built and how its architecture could support a key elongation step in colibactin assembly. The authors focused on the complete trans-AT PKS module of ClbK and the full-length hybrid protein because ClbK helps incorporate an unusual aminomalonyl extender unit and contributes to building a colibactin precursor that ultimately links to host DNA injury and colorectal cancer risk.

Who was studied?

The investigators studied purified bacterial proteins, not patients or clinical cohorts. They expressed and purified multiple E. coli–derived ClbK constructs, including a crystallizable PKS module fragment (residues 1–787) and full-length ClbK, then analyzed these macromolecules in vitro using X-ray crystallography, SEC-MALLS, and SEC-SAXS to determine oligomeric state, flexibility, and overall shape. In practical terms, the “subjects” were the ClbK enzyme domains that physically execute colibactin precursor transfer inside pks-positive bacteria.

What were the most important findings?

The study solved a 2.98 Å crystal structure of the complete ClbK PKS module and showed it forms a dimeric KS-centered core with features that look adapted for NRPS/PKS hybrids. The ketosynthase (KS) domain displayed a remodeled “cap” and loop architecture that opens an additional bottom entrance to the active-site tunnel, a change the authors argue could help accommodate bulky, amino acid–containing intermediates that arise when NRPS and PKS chemistry merges. The acyltransferase region proved to be a degenerate, truncated AT* lacking essential catalytic motifs, supporting a model in which the standalone trans-AT ClbG loads aminomalonyl directly onto the ClbK ACP. The ACP domain appeared near the KS bottom entrance but too far for catalysis in the captured conformation, consistent with a pre- or post-condensation snapshot and emphasizing the enzyme’s dynamic carrier-protein choreography.

What are the greatest implications of this study/ review?

By providing a concrete structural framework for a central colibactin biosynthetic enzyme, this work strengthens functional interpretation of the microbiome signature “pks-positive Enterobacteriaceae” by explaining how a specific enzymatic step can plausibly control flux toward genotoxic metabolites. The identification of an inactive AT* and the proposed direct ACP docking by the trans-AT partner offer rational targets for pathway disruption that would reduce colibactin production without requiring broad bacterial eradication. The SAXS-supported view of full-length ClbK as a flexible dimer with multiple catalytic chambers also supports future efforts to design small-molecule inhibitors or engineer hybrid megaenzymes, which could translate into strategies that dampen colibactin-associated DNA damage risk in susceptible clinical contexts