Exploring biosynthetic routes to novel histidine containing cyclodipeptides

Abstract

Histidine containing cyclodipeptides have gathered attention for their important biological potential as anti-cancer drugs and neuroprotective agents. Thus, the creation of facile synthetic routes to these compounds are essential to exploit their potential as therapeutic targets. Biosynthetic pathways of natural products recently revealed a family of enzymes called cyclodipeptide synthases (CDPSs) which use aminoacylated tRNAs as substrates to yield an array of cyclodipeptides. From the current set of characterised CDPSs however, only two CDPSs accept histidine as a substrate: ParaCDPS and ParcuCDPS. Extensive research has led to thorough characterisation of the catalytic mechanism of CDPSs, however their exact substrate recognition and specificity determinants remain poorly understood.

Initial investigation of CDPSs demonstrated the inherent promiscuity of this class by generating a library of cyclodipeptides from canonical and non-canonical amino acids. By solving the structure of ParcuCDPS, key residues for substrate recognition were unveiled in the first binding pocket of the CDPS. Rationally engineered mutants subsequently displayed altered substrate scope, rejecting histidine, and accepting phenylalanine and leucine. This research is the first instance of successfully engineering a CDPS to yield multiple products and paves the way for directed modifications to enhance the promiscuity of these enzymes to produce molecules of our choosing. Furthermore, tailoring enzymes are counterparts to CDPSs in nature and create an additional layer of diversity to cyclodipeptides. Preliminary investigations sought to characterise a cyclodipeptide oxidase to identify the substrate specificity of these rather mysterious enzymes.

As a final observation, the work presented here complements the current understanding of biosynthetic pathways to novel cyclodipeptides. Cyclodipeptide synthases pose as a facile tailored route with the potential to generate a large range of cyclic products from just one enzyme. Additionally, the introduction of tailoring enzymes enhances the diversity possible from such a small family of enzymes.