N–Cα Bond Cleavage Catalyzed by a Multinuclear Iron Oxygenase from a Divergent Methanobactin-like RiPP Gene Cluster

18 December 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

DUF692 multinuclear iron oxygenases (MNIOs) are an emerging family of tailoring enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Three members, MbnB, TglH, and ChrH, have been characterized to date and shown to catalyze unusual and complex transformations. Using a co-occurrence-based bioinformatic search strategy, we recently generated a sequence similarity network of MNIO-RiPP operons that encode one or more MNIOs adjacent to a transporter. The network revealed >1,000 unique gene clusters, evidence of an unexplored biosynthetic landscape. In this work, we assess an MNIO-RiPP cluster from the network that is encoded in proteobacteria and actinobacteria. The cluster, which we have termed mov (for methanobactin-like operon in Vibrio), encodes a 23-residue precursor peptide, two MNIOs, a RiPP recognition element, and a transporter. Using both in vivo and in vitro methods, we show that one MNIO, homologous to MbnB, installs an oxazolone-thioamide at a Thr-Cys dyad in the precursor. Subsequently, the second MNIO catalyzes N–Cα bond cleavage of the penultimate Asn to generate a C-terminally amidated peptide. This transformation expands the reaction scope of the enzyme family, marks the first ex-ample of an MNIO-catalyzed modification that does not involve Cys, and sets the stage for future exploration of other MNIO-RiPPs

Keywords

biosynthesis
metalloenzyme
natural product
DUF692

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