Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin

Abstract

Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS-PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS-PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein-protein interactions supports a predicted molecular basis for enzyme-carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin.

Keywords: ClbL; amidases; colibactin; microbiome; natural product biosynthesis.

Figure 1

ClbL-mediated transacylation of colibactin NRPS–PKS intermediates. Phosphopantetheinyl thioesters of the ACP domains of ClbO and ClbI are substrates for ClbL transamidation. The bond-forming step (blue square) and product amide (blue circle) are highlighted.

Figure 2

Structure of ClbL along with comparison with a representative member of the AS superfamily. (a) Overall protein structure highlighting Ser179 in the active site (green) along with two disordered loop regions and the basic loop. (b) Structure of a bacterial aryl acylamidase (Lee et al., 2015 ▸) shown in the same orientation with the corresponding Ser174 highlighted.

Figure 3

Substrate–ClbL active-site interactions. (a) Schematic of the overall ClbL reaction and substrate interactions. The overall pathway is shown along with the interactions of a modeled PMSF adduct (boxed). R₁ and R₂ represent the extended end chains of precolibactin (Fig. 1 ▸). (b) Composite omit electron-density map, contoured at 1.0 Å, of the modeled phenylmethylsulfonyl–Ser179 adduct. Hydrogen-bonding distances are 2.7 Å (Ser155/sulfonyl) and 3.2 Å (Ser155 cis-amide/sulfonyl). (c) ClbL surface representation of a modeled bound substrate (Supplementary Fig. S5, yellow).