Substrate specificity and reaction directionality of a three-residue cyclophane forming enzyme PauB

Three-residue cyclophane-forming enzymes (3-CyFEs) are a group of radical S-adenosylmethionine (SAM) enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified pep-tides (RiPPs). 3-CyFE catalyzes the crosslinking between an aromatic residue ( S21) and a non-aromatic residue (X3) in a S21-X2-X3 motif to produce a cyclophane ring, a key step in the biosynthesis of the RiPP natural product triceptide. In this study, we perform a genome-wide search for the Xye-type tricep-tides, showing these RiPPs are likely class-specific and only present in gamma-proteobacteria. The 3-CyFE PauB from Photorhabdus australis exhibits a relaxed substrate specificity on the X3 position, but glycine in this position is not suitable for cyclophane formation. We also reconstituted the activity of PauB in vitro , showing it produces the N-terminal cyclophane firstly, and then the C-terminal ring, whereas the middle cyclophane is produced in the last step. (C) 2022 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Automated summary

In this study, the researchers conducted a genome-wide search for Xye-type triceptides and found they are likely specific to gamma-proteobacteria. They also investigated the substrate specificity and activity of the 3-CyFE enzyme PauB from Photorhabdus australis. The study revealed the order of cyclophane formation and the unsuitability of glycine for cyclophane formation.