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

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-occurrencebased 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 >1000 unique gene clusters, evidence of an unexplored biosynthetic landscape. Herein, we assess an MNIO-RiPP cluster from this network that is encoded in Proteobacteria and Actinobacteria. The cluster, which we have termed mov (for methanobactinlike 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 Cterminally amidated peptide. This transformation expands the reaction scope of the enzyme family, marks the first example of an MNIO-catalyzed modification that does not involve Cys, and sets the stage for future exploration of other MNIO-RiPPs.

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

“…S1). In light of recent discoveries of MNIOs involved in RiPP biosynthesis (7)(8)(9)(10)(11)(12)(13), we hypothesized that BufB1 and BufC1 would install modifications on Cys residues of BufA1. Finally, CCNA_03366 encodes a putative DoxX-type oxido-reductase, BufD1 (22,23).…”

“…The conversion of Cys to 5-thiooxazole represents a new reaction catalyzed by MNIOs, further highlighting their chemical versatility. Intense efforts have been made recently to explore the chemical space of RiPPs undergoing MNIO-catalyzed transformations (7, 8, 11, 13, 38). In line with previous work on Cys-modifying MNIO enzymes, BufB would similarly make use of the mixed-valent iron center and proceed by an intermediate step of proton abstraction from the Cβ of cysteine (9, 10, 12, 13) (SI Fig.…”

“…The vast structural diversity of RiPPs is conferred by virtually unlimited possibilities of chemical transformations. An emerging family of PTM enzymes is the DUF692 family of multinuclear non-heme iron dependent oxidases (MNIOs) that catalyze various reactions in the biosynthesis of diverse RiPPs, mostly but not exclusively on Cys residues (7)(8)(9)(10)(11)(12)(13). These reactions include oxazolone coupled to thioamide formation, excision of cysteine β-methylene, Cys-involved macrocyclization, and C-C and C-N cleavages (7,8,(11)(12)(13).…”

“…An emerging family of PTM enzymes is the DUF692 family of multinuclear non-heme iron dependent oxidases (MNIOs) that catalyze various reactions in the biosynthesis of diverse RiPPs, mostly but not exclusively on Cys residues (7–13). These reactions include oxazolone coupled to thioamide formation, excision of cysteine β-methylene, Cys-involved macrocyclization, and C-C and C-N cleavages (7, 8, 11–13). However, the full spectrum of MNIO activities remains far from being fully appreciated.…”

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

“…Perhaps the most interesting modification observed was catalyzed by ApyHI (a member of the multinuclear non-heme iron-dependent oxidative enzyme superfamily, MNIO), which are universally conserved among apy BGCs and are thus considered class-defining. Previously characterized MNIOs catalyze distinct chemical reactions exclusively on Cys, although a very recent report describes an MNIO catalyzed N–Cα bond cleavage of an Asn to generate a C-terminal amide in a methanobactin-like pathway . ApyHI instead performs an unprecedented enzymatic modification on a C-terminal Asp.…”