Micacocidin A

The thiazoline-containing siderophores pyochelin, yersiniabactin, and Micacocidin A all have D-thiazoline rings, participating in high-affinity chelation of ferric iron. However, studies with pyochelin (Pch) synthetase and yersiniabactin (Ybt) synthetase reconstituted from pure protein components have shown that only L-cysteine is activated and tethered as a covalent aminoacyl-S-enzyme intermediate. Nor are any of the canonical epimerase domains of nonribosomal peptide synthetase (NRPS) assembly lines found in the Ybt or Pch synthetase modules. Here, we report that the PchE subunit of the Pch synthetase exchanges solvent deuterium into the C(2) center of the thiazoline moieties during siderophore chain elongation. Both PchE and HMWP2, from Ybt synthetase, subunits have a 310-360-residue insert in their amino acid activation domains that look like defective methyltransferase (MT) domains. We suggest these inserts are noncanonical epimerase domains, reversibly deprotonating and reprotonating acyl-S-enzyme intermediates at the C(2) locus. The PchE subunit does not epimerize the Cys-S-enzyme intermediate, but once amide bond formation from a benzoyl-S-PchE donor is catalyzed by the cyclization (Cy) domain of PchE, the N-benzoyl-Cys-S-PchE intermediate is present as a D,L-mixture. The subsequent phenylthiazolinyl-S-PchE intermediate, arising from cyclodehydration of the N-benzoyl-Cys-S-PchE intermediate, is likewise a D,L-mixture on hydrolytic release and enantiomer analysis. These results suggest a default role for MT domains of NRPS assembly lines in generating alpha-carbanionic species from thioester intermediates during siderophore chain elongation.

Micacocidin is a thiazoline-containing natural product from the bacterium Ralstonia solanacearum that shows significant activity against Mycoplasma pneumoniae. The presence of a pentylphenol moiety distinguishes micacocidin from the structurally related siderophore yersiniabactin, and this residue also contributes to the potent antimycoplasma effects. The biosynthesis of the pentylphenol moiety, as deduced from bioinformatic analysis and stable isotope feeding experiments, involves an iterative type I polyketide synthase (iPKS), which generates a linear tetraketide intermediate from acyl carrier protein-tethered hexanoic acid by three consecutive, decarboxylative Claisen condensations with malonyl-coenzyme A. The final conversion into 6-pentylsalicylic acid depends on a ketoreductase domain within the iPKS, as demonstrated by heterologous expression in E. coli and subsequent site-directed mutagenesis experiments. Our results unveil the early steps in micacocidin biosynthesis and illuminate a bacterial enzyme that functionally resembles fungal polyketide synthases.

Synthetic chain terminators were used to capture the biosynthetic intermediates from a partially reducing iterative type I polyketide synthase, which is integrated into a multimodular biosynthesis enzyme. The off-loaded metabolites clarified the timing of ketoreduction and aromatization in the assembly of the antibiotic micacocidin.