Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring

Abstract

Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity.

Figure 1. SAM is activated as a non-ribosomal peptide synthetase (NRPS) building block and loaded onto the phosphopantetheinyl arm of ClbH

(a) Pks-dependent, cyclopropane-containing metabolites isolated from pks+ E. coli mutant strains. (b) ATP-[³²P]-PPi exchange assay for the substrate specificity of full-length ClbH, ClbH-A1, ClbH-C-A2-PCP, and ClbH-C-A2-PCP-S1524A. C, condensation; A, adenylation; PCP, peptidyl carrier protein. Results of scintillation counting were averaged and scaled relative to the activation of L-Ser by full-length ClbH (data points are shown). Error bars represent the standard error of the mean (s.e.m.) of three replicates.

Figure 2. ClbH-mediated SAM elongation precedes ClbI-catalyzed cyclopropanation

(a) LC/MS analysis of pellet extracts of E. coli DH10B mutant strains expressing ClbA, ClbN, ClbB, ClbC, and either (i) ClbH, (ii) ClbH-C-A2-PCP, or (iii) ClbH and ClbI. (b) Biosynthetic hypothesis for the formation of SAM-containing assembly line intermediate 4 and cyclopropane-containing metabolites 2 and 1. (c) LC/MS analysis of in vitro assays of purified enzymes. These assays have been replicated three times in the laboratory. (a,c) Extracted ion chromatograms of m/z 567.3752.