Stuffed Epimerase Domains of Pyochelin Biosynthesis are Defunct Methyltransferases

Bacteria and fungi use nonribosomal peptide synthetases (NRPSs) to generate secondary metabolites called nonribosomal peptides (NRPs). A common feature of NRPs is the incorporation of D-stereocenter amino acids, which enhance chemical functionality and confer resistance to proteolytic degradation. Typically, these stereochemical inversions arise from dedicated epimerase domains, which are well-characterized. However, 2-hydroxyphenylthiazoline-containing natural products lack these conventional tailoring domains and are instead proposed to utilize noncanonical epimerase domains embedded, or "stuffed", within the mobile loop of NRPS adenylation domains. The stereochemistry of the final natural product does not align with the absence or presence of the stuffed epimerase domain, so we examined the adenylation-epimerase didomains of 2-hydroxyphenylthiazoline siderophores from <i>Pseudomonas aeruginosa</i> (pyochelin, <i>R</i> stereochemistry at 4') and <i>Streptomyces venezuelae</i> (watasemycin, <i>S</i> at 4'). For comparison, we also examined the biosynthesis of enantiopyochelin (<i>S</i> at 4') by the homologous enzyme from <i>Pseudomonas protegens</i>, which lacks the stuffed epimerase domain. Substrate and product analogs were synthesized to probe the epimerase chemistry. While the variants performed adenylation chemistry, epimerase activity was not enzymatically catalyzed. Indeed, racemization was spontaneous for the 2-hydroxyphenylthiazoline ethyl ester analogues and for intermediates isolated from the enzymes. These data suggest that noncanonical stuffed epimerase domains of 2-hydroxyphenylthiazoline natural products are catalytically defunct methyltransferases and possibly an evolutionary remnant.