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-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 >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 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 example of an MNIO-catalyzed modification that does not involve Cys, and sets the stage for future exploration of other MNIO-RiPPs.

Figure 1.

Sequence similarity network (SSN) of MNIO-RiPPs and the mov BGC. (A) SSN of RiPP BGCs that encode an MNIO and an adjacent transporter. Each node marks an individual MNIO enzyme, and lines connecting them indicate sequence similarity. Nodes are colored based on phyla: Proteobacteria (red), Actinobacteria (green), Firmicutes (yellow), Bacteroidetes (purple), and other phyla (blue). MNIO-RiPP families investigated in this work (MovX, MovB), related ones in Actinobacteria (MoaX, MoaB), and previously characterized ones (MbnB, TglH, and ChrH) are highlighted; reactions are shown for the latter. (B) The mov BGC from Vibrio fluvialis encodes a precursor peptide (MovA), two MNIOs (MovX and MovB), a discrete RRE (MovC), and a transporter (MovT). Genes are color-coded; the sequence of the precursor peptide is shown.

Figure 2.

HR-MS and HR-MS/MS analyses of the heterologous expression constructs. Shown are data for trypsin-cleaved MovA after coexpression with no enzymes as control (A), MovBC (B), and MovX (C). The construct is shown above each HR-MS profile, which is zoomed in on the [M+H]⁺ ion of the trypsin-cleaved product, consisting of 9mer (A,B) or 7mer (C) peptides. Coexpression of MovA with MovBC and MovX gives products that are 4.0313 and 243.1219 Da lighter than the linear 9mer, respectively. The HR-MS/MS fragmentation pattern of the MovBC product is consistent with an oxazolone-thioamide motif. HR-MS/MS analysis of the MovX product reveals that the peptide is devoid of the C-terminal Asn-Lys dipeptide; while b-ions are unaffected, all observed y-ions exhibit a −0.9840 Da shift relative to the unmodified 7mer peptide.

Figure 3.

HR-MS and HR-MS/MS analyses of MovBC and MovX products. (A) Enzymatic activity assays of MovBC using MovA as a substrate. Shown are extracted ion chromatograms for the −4.0313 Da product of MovBC relative to the linear 23mer MovA. Product is not observed when MovB, MovBC, or substrate is omitted from the reaction, while the addition of ascorbate increases turnover. (B) Enzymatic activity assays of MovX using MovBC-modified MovA, generated through coexpression of 6HMBP-MovA and MovBC in E. coli, as a substrate. Shown are extracted ion chromatograms for the −247.1532 Da product of MovXBC, which corresponds to the loss of the Asn-Lys dyad and the introduction of a C-terminal amide. Product is not observed when MovX or the substrate is omitted from the reaction, while the addition of ascorbate increases turnover. In panels (A) and (B), traces are offset in both axes for clarity and color-coded as indicated. (C) Substrate preference of MovBC (red) and MovX (blue), as assessed by monitoring conversion of MovA substrate variants by HPLC-coupled HR-MS. MovBC converts both unmodified and MovX-modified MovA to the same extent, while MovX displays a clear preference for MovBC-modified MovA, followed by hydrolyzed MovBC-modified MovA, and unmodified MovA. (D,E) HR-MS/MS analyses of MovBC (D) and MovXBC (E) products. Mass shifts relative to the linear MovA are shown. (F) Ultraviolet–visible spectra of unmodified, MovBC-, and MovXBC-modified MovA. Unmodified MovA solely absorbs at 278 nm, while MovBC- and MovXBC-modified MovA exhibit additional absorption features at 264 and 302 nm.

Figure 4.

Structural elucidation of the MovBC and MovXBC products by 2D NMR and ¹⁵N labeling studies. (A–D) Comparative analysis of 2D NMR spectra of unmodified (green), MovBC-modified (red), and MovXBC-modified (blue) MovA. If present, impurity signals are shown in gray. All observed cross-peaks are labeled, and those that drop out post-modification are underlined. (A) HSQC spectra focusing on the Hα region. Compared to unmodified MovA, Cys-Hα is missing from the MovBC-modified product, and Thr-Hα is significantly downshifted. Moreover, MovXBC-modified MovA is devoid of Asn-Hα and Lys-Hα. (B) HSQC spectra focusing on the Hβ region. Compared to unmodified MovA, Cys-Hβ is missing from the MovBC-modified product. In addition, MovXBC-modified MovA lacks Asn-Hβ and Lys-Hβ. (C) HMBC spectra highlighting cross-peaks between Gly-Hα and Gly-C/Cys-Cβ. Compared to unmodified MovA, Cys-Cβ shifts upfield in both MovBC- and MovXBC-modified products, while Gly-C remains unchanged. (D) HMBC spectra highlighting cross-peaks between Tyr-Hα and Tyr-C and Ser-C. Compared to unmodified and MovBC-modified MovA, Tyr-C is downshifted in the product, while Ser-C remains unchanged. (E) Relevant NMR correlations used to solve the structure of MovXBC-modified MovA. (F) Reactions of MovX and MovXBC with unlabeled MovA and ¹⁵N₂-Asn-MovA. HR-MS focusing on the [M+3H]³⁺ ion of each peptide. Reactions with ¹⁵N₂-Asn-MovA yielded products 0.9970 Da heavier than the respective MovA products, indicating the incorporation of a single ¹⁵N label. (G,H) HR-MS/MS analyses of trypsin-cleaved ¹⁵N-labeled MovX (G) and MovXBC (H) products after reaction with ¹⁵N₂-Asn-MovA. The observed fragmentation patterns indicate ¹⁵N incorporation at the modified C-terminus.

Figure 5.

Proposed mechanisms for N–C bond cleavage catalyzed by MovX. (A) Heterolytic mechanism, in which succinimide formation is followed by Lewis acid-catalyzed hydrolysis to give the product. (B) Homolytic mechanism that involves O₂ activation, peroxide rebound, and hydrolysis of an imine intermediate. (C) Alternative homolytic mechanism involving O₂ activation, hydroxylation at Cβ followed by β-scission, and hydroxylation at Cα followed by cleavage of the N–C– bond. See text for further details.

Figure 6.

Mechanistic studies performed with MovX. (A) Reactions of MovX and MovXBC with¹⁵Nβ-Asn-MovA. HR-MS spectra are zoomed in on the [M+3H]³⁺ peptide ion. The ¹⁵N label is not incorporated in the observed products, indicating that the C-terminal amide originates from the α-amino group of Asn. (B) Enzymatic activity assays of MovX using MovBC-modified MovA as a substrate in the presence or absence of molecular oxygen. Shown are extracted ion chromatograms for MovA after reaction with MovXBC. The MovX reaction proceeds only under aerobic conditions. (C,D) Reaction of MovXBC with MovA (C) and N22G-MovA (D). HR-MS spectra are zoomed in onto the [M+3H]³⁺ peptide ions. The amidated product is observed for the Gly-substituted substrate, suggesting H-atom abstraction from Cβ is not required for the MovX reaction. (E,F) HR-MS and HR-MS/MS analyses of trypsin-cleaved MovXBC products using MovA (E) and N22G-MovA (F) as substrate. The products are identical, as demonstrated by analogous retention times and fragmentation patterns. The MovBC and MovX modifications, leading to losses of 4.0313 and 0.9840 Da, respectively, are marked.

Figure 7.

Proposed biosynthesis of RiPP produced by the mov cluster. Upon ribosomal synthesis of the MovA precursor peptide, MovBC catalyzes the formation of the oxazolone-thioamide. MovX then cleaves the N–Cα bond of the penultimate Asn to deliver a C-terminally amidated peptide. Following possible leader peptide processing, MovT likely exports the mature RiPP.