J. Am. Chem. Soc. 134, 2844–2847 (2012)

The tirandamycins are biosynthesized by hybrid PKS-NRPS machinery, with several tailoring steps occurring after their release from the terminal carrier protein. TrdE is part of the tirandamycin cluster and shows high identity to glycoside hydrolase family 16, including the catalytic EXDXXE motif. However, tirandamycin does not contain any sugars, raising questions as to the true role of this enzyme. Mo et al. now demonstrate that TrdE provides a new biosynthetic mechanism for double-bond formation. Deletion of trdE generated an intermediate with a pendant hydroxyl group on the bicyclic ring, suggesting TrdE might remove this group prior to further tailoring. In vitro testing of TrdE confirmed this function, showing conversion of the hydroxylated compound—which the authors noted does structurally resemble a glycoside hydrolase substrate—into a dehydrated analog. To determine whether the glycoside hydrolase motif is relevant for catalysis, the authors mutated the conserved residues as well as four histidines on the basis of the known requirement for a histidine in other dehydratases. Only two of these mutants retained activity, in support of a typical glycoside hydrolase mechanism. Interestingly, mutations to three of the four histidines, close to each other in the primary sequence, caused substantial changes to the protein conformation, perhaps suggesting that a metal may be involved in stabilizing TrdE's structure. This functional reassignment may have relevance to other biosynthetic pathways with uncharacterized enzymes.