Natural products are key contributors to the drug discovery pipeline. Numerous drug candidates have come from the family of compounds called diketopiperazines (DKPs), which are cyclic dipeptides constructed from two amino acids and often heavily tailored. One DKP important to natural product drug discovery is cyclo-L-Trp-L-Trp (LL-cWW), which is the enantiomer to cyclo-D-Trp-D-Trp (DD-cWW). DD-cWW forms the backbone of the nocardioazines, DKPs with unique biological activity produced by a marine Nocardiopsis sp. actinomycete. The goal of this research was to identify the biosynthetic origin of the unique DD-cWW configuration of the nocardioazines. We identified NozR as a novel isomerase that catalyzes the transformation of LL-cWW to DD-cWW as a nocardioazine precursor. To our knowledge, this marks the first reported enzyme to catalyze the inversion of configuration of any DKP. To evaluate the substrate range of NozR, ten DKPs with varying amino acid residues and/or configurations were tested for NozR-catalyzed stereoisomerization in vivo. NozR was found to catalyze isomerization reactions for any cWW stereoisomer. NozR also catalyzed isomerization reactions for all evaluated DKPs containing at least one aromatic group, while isomerization was not observed for DKPs without aromatic group(s). Thus, NozR catalyzes isomerization of a broad spectrum of DKPs that contain at least one aromatic amino acid residue. The discovery of this enzyme’s promiscuity is anticipated to open doors for the biological synthesis of novel drugs built from the DKP scaffold.

Probing the substrate specificity of a diketopiperazine racemase to expand natural product diversity

ACS Spring 2022

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