Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3

Abstract

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.

Methanobactins (Mbns) are copper-chelating natural products produced by some species of methanotrophs,¹ methane-oxidizing bacteria that depend on copper as a cofactor for their primary metabolic enzyme, particulate methane monooxygenase (pMMO).² Under conditions of copper starvation, methanotrophs secrete Mbns to scavenge and import copper from the environment.^3–5 Due to their high copper binding affinity, Mbns are a promising therapy for Wilson disease, a genetic disorder of copper metabolism typically treated by copper chelation.^6–8 Mbns are ribosomally produced, post-translationally modified peptides (RiPPs) synthesized by modification of a precursor peptide, MbnA, by a series of biosynthetic enzymes encoded within the Mbn operon.^9–11 MbnA consists of a core peptide that is converted to mature Mbn and a leader peptide that is cleaved by an unknown mechanism. On the basis of phylogenetic analysis, Mbn operons have been divided into five groups, with methanotrophic Mbns classified in groups I and II.⁹ Genome mining studies have also identified Mbn operons in non-methanotrophic bacteria (groups III–V), which now constitute the majority of identified Mbn gene clusters, suggesting a larger role for Mbn-like natural products in microbial copper acquisition.^3,9,12

Several Mbns have been structurally characterized using X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy (Figure S1A–G).^11,13–16 In each Mbn, copper is coordinated by two bidentate ligands composed of nitrogen-containing heterocycles and neighboring thioamide groups. The group I Mbns from Methylosinus (Ms.) trichosporium OB3b¹³ and Ms. sp. LW4¹⁵ contain two oxazolone ring and thioamide pairs generated from two cysteine residues by the iron-dependent heterodimeric MbnBC enzyme complex (Figure S1A,B and S2).¹⁰ These MbnAs have two additional cysteine residues that form a disulfide bond, and the Mbns are further stabilized through conversion of the N-terminal amino group to a carbonyl group by the PLP-dependent aminotransferase MbnN (Figure S2).¹⁷ The Mbn from Ms. sporium NR3K,^5,16 presumably classified as a group I Mbn, contains a pyrazinedione ring in place of the first oxazolone, like three of four structurally characterized group II Mbns from Methylocystis (Mc.) species (Figure S1C–F).¹⁴ The genesis of the pyrazinedione ring has not been established but is hypothesized to involve an FAD-dependent monooxygenase, MbnF,¹² which may act after oxazolone formation by MbnBC (Figure S3). The pyrazinedione ring has been proposed to provide a stabilizing effect at the N-terminus of Mbn, similar to that conferred by transamination.¹⁷

A remarkable feature of the MbnA core peptide is its diversity of amino acid sequence both within methanotroph groups I and II and across all MbnA sequences.^1,5 While the modifiable cysteines are typically followed by a small residue (glycine, alanine, and sometimes serine) and a slightly larger hydrophilic residue (often serine and threonine),¹ other features, including the spacing between the modified cysteines, the presence of additional cysteines, and the overall amino acid composition, are highly variable. Moreover, aside from the genes encoding the MbnBC complex, which are present in all Mbn operons, the operon contents vary within groups (Figure S1H). All currently identified group I operons encode either MbnN or MbnF, while all group II operons encode MbnF along with one of two additional biosynthetic enzymes: MbnS, a sulfotransferase (group IIa), or MbnD, a predicted dioxygenase (group IIb).

The current library of Mbn structures includes two representatives of group I [Ms. trichosporium OB3b¹³ and Ms. sp. LW4¹⁵ (Figure S1A,B)], both of which have operons encoding MbnN, and a presumed member of group I (Ms. sporium NR3K). The Ms. sporium NR3K genome is not available, but its Mbn operon has been predicted to include MbnF.¹⁶ Group II Mbn structures include three representatives and one presumed member of group IIa, all of which have virtually identical sequences and structures (Figure S1D–G).^11,14 Thus, the full diversity of Mbn structures has yet to be revealed. Notably, some methanotroph genomes contain two Mbn operons belonging to different groups, specifically groups I and IIb.^5,12 In these genomes, the group I Mbn operons encode MbnF, not MbnN. Given the paucity of structures of group I Mbns with MbnF in the operon and of group IIb Mbns, we targeted one of these methanotrophs, Ms. sp. LW3, for isolation of new Mbns. Here we report the structural characterization of the Ms. sp. LW3 group I Mbn by mass spectrometry and NMR spectroscopy.

Spent medium from a Ms. sp. LW3 culture grown in a 12 L fermentor under copper-starved conditions (0.1 μM CuSO₄) exhibited ultraviolet–visible (UV–vis) absorption features at 303, 337, and 387 nm, suggesting the production and secretion of a Mbn-like compound (Figure S4). The potential Mbn was isolated from the spent medium using a Diaion HP-20 column followed by high-performance liquid chromatography purification. Like the spent medium, the purified product exhibits prominent UV–vis peaks at 303, 337, and 387 nm (Figure S5A). Upon addition of copper, the absorbance at 387 nm decreased rapidly along with an increase at ~460 nm, consistent with the formation of a copper-loaded species (Figure 1A).¹⁴ The purified compound was then characterized by electrospray ionization mass spectrometry (ESI-MS) and UV–vis spectroscopy (Figure 1B,C and Figure S5B). Negative ion mode MS showed an ion at m/z 1314.649 for the singly charged species and an ion at m/z 656.901 for the doubly charged species (Figure 1B). The theoretical masses of the unmodified MbnA core peptides from Ms. sp. LW3 are 1327.46 Da (group I, Met₁-Cys₂-Ser₃-Ser₄-Cys₅-Pro₆-Met_7-Cys₈-Gly₉-Pro₁₀-Leu₁₁-Cys₁₂-Pro₁₃) and 811.26 Da (group IIb, Asp₁-Cys₂-Gly₃-Thr₄-Ala₅-Cys₆-Trp₇-Gly₈), suggesting that the main product from the spent medium was likely the group I Mbn. Negative ion mode MS of the purified copper-loaded product showed an altered isotopic distribution consistent with the presence of copper in addition to the mass shift for Cu – H (62.107 Da, a loss of a proton in the MS due to the positive charge of Cu⁺) (Figure 1C). These combined data are consistent with isolation of the apo (apoMbn) and copper-loaded (CuMbn) forms of Mbn from Ms. sp. LW3.

Figure 1.

UV–vis and mass spectra of Mbn isolated from Ms. sp. LW3. (A) UV–vis absorption spectra after the addition of copper to apoMbn. Approximately 2 equiv of CuSO₄ was added, and the reaction was monitored for 20 min (starting spectrum colored red, ending spectrum colored purple). Arrows indicate the decrease at 387 nm and the increase at ~460 nm as a function of time. Absorption features corresponding to the oxazolone ring (Oxa) and pyrazinedione ring (Pyr) are labeled in orange and green, respectively. This color coding is used in all of the figures. (B) Negative mode ESI-MS of apoMbn. (C) Negative mode ESI-MS of CuMbn. The inset in panel C shows the effect of copper on the isotopic distribution.

We predicted the structure of this Mbn based on the core peptide sequence and presence of MbnB, MbnC, and MbnF in the Ms. sp. LW3 group I Mbn operon (Figure 2). Unlike Cys₅ and Cys₁₂, Cys₂ and Cys₈ are followed by small hydrophobic residues (Ser₃ and Gly₉), the typical Mbn cysteine modification motif.¹ Therefore, in the predicted structure, Cys₂ and Cys₈ are modified to pyrazinedione/thioamide and oxazolone/thioamide groups, respectively, providing two nitrogen and two sulfur ligands for Cu⁺. Although MbnF’s role has not been established biochemically and there are no characterized group I Mbns with operons encoding MbnF, the presumed group I Mbn from Ms. sporium NR3K (genome not available) possesses an N-terminal pyrazinedione modification (Figure S1C).¹⁶ Additionally, two previously characterized group II Mbns with operons encoding MbnF (Mc. hirsuta CSC1 and Mc. rosea SV97) have the same N-terminal pyrazinedione ring modifications clearly visible in their crystal structures (Figure S1D,F).¹⁴ The one exception is the NMR-derived structure of Mc. str. SB2 Mbn, which was modeled instead with an imidazolone ring (Figure S1G) despite the presence of MbnF in its operon.¹¹ In support of the predicted Ms. sp. LW3 Mbn structure, these previously characterized Mbns exhibit spectral features at ~340 and ~395 nm, corresponding to the oxazolone and pyrazinedione rings, respectively.¹⁴ Thus, it is likely that the 337 and 387 nm spectral features observed for Mbn isolated from Ms. sp. LW3 (Figure 1A) also derive from oxazolone and pyrazinedione rings. Lastly, the two cysteine residues not found in modification motifs, Cys₅ and Cys₁₂, are proposed to form a disulfide bond as observed in the Ms. trichosporium OB3b¹⁸ and Ms. sp. LW4¹⁵ Mbns.

Figure 2.

Predicted structure of the group I CuMbn from Ms. sp. LW3 with its Mbn operon and MbnA sequence (core peptide underlined). The biosynthetic genes and the corresponding modifications are shown in color, while genes involved in regulation or transport are colored gray.

The mass of this predicted structure matches that obtained from negative mode ESI-MS. Modifications of two MbnA cysteines to pyrazinedione/thioamide and oxazolone/thioamide groups correspond to −6H and −4 mass shifts, respectively. These modifications, along with disulfide bond formation and loss of a proton to account for negative ion mode MS, correspond to a total mass shift of −13H. This mass shift matches the difference between the calculated mass of the MbnA core peptide (1327.47 Da) and that initially obtained for apoMbn [m/z 1314.65 (Figure 1B)]. The mass obtained for CuMbn [m/z 1376.76 Da (Figure 1C)] is similarly consistent with the mass of the predicted structure bound to Cu⁺. The observed doubly charged masses also agree with the predicted structure (Figure 1B,C).

We then used LC-tandem mass spectrometry (LC-MS/MS) to localize specific modifications in the structure. In addition to b- and y-type fragment ions (b_n and y_n), internal fragments were detected and are labeled as b_n-X or y_n-X (where X is the amino acid sequence subtracted from either b or y ions) (Figure 3). First, we confirmed the amino acid backbone of apoMbn and CuMbn, verifying that it derives from the Ms. sp. LW3 group I Mbn operon (Figure 3 and Figures S6 and S7). One fragment ion, [PMCG]/[MCGP] (m/z 385.099; Cys₈ is modified to the oxazolone ring), cannot be differentiated by MS because the two fragment ions have the same mass. Second and more importantly, many of the observed apoMbn fragments contain Cys₂ (−6H, −6.0470 Da) and Cys₈ (−4H, −4.0313 Da) with their proposed modifications (Figure 3A and Figure S6), confirming that the modifications take place at these positions. Two particularly revealing fragments from apoMbn are b₃ (m/z 316.042) and [PMCG]/[MCGP] (m/z 385.099) because they contain only the modified cysteine (Cys₂ and Cys₈, respectively), eliminating the possibility that the other two cysteine residues, Cys₅ and Cys₁₂, are modified. Instead, Cys₅ and Cys₁₂ form a disulfide bond. MS² analysis of CuMbn also localizes the modifications to Cys₂ and Cys₈ (Figure 3B). Furthermore, most fragment ions containing a modification bind copper, consistent with these modifications generating the copper-binding ligands (Figure 3B and Figure S7).

Figure 3.

MS² fragmentation spectra for (A) apoMbn and (B) CuMbn. All cysteine residues are colored on the basis of their modifications: green for a pyrazinedione ring (−6H, −6.0470 Da), orange for an oxazolone ring (−4H, −4.0313 Da), and gray for a disulfide bond. The b ions contain the N-terminus, and the y ions contain the C-terminus. In panel B, fragment ions that bind copper are colored purple with two m/z representing the two major mass values for the copper isotope. Detailed fragments are shown in Figures S6 and S7.

We then pursued structural characterization of Ms. sp. LW3 CuMbn by a variety of one- and two-dimensional NMR spectroscopies, including ¹H NMR, [¹H–¹H] COSY, [¹H–¹H] TOCSY, [¹H–¹H] ROESY, [¹H–¹³C] HSQC, [¹H–¹³C] HMBC, and [¹H–¹⁵N] HSQC taken in both 10% and 100% D₂O in phosphate buffer (pH 6.5). Because binding copper affords Mbn significantly greater stability and conformational rigidity, CuMbn, rather than apoMbn, was used for all NMR experiments. Peaks were assigned on the basis of the MbnA core sequence and the LC-MS/MS data. With the exception of the modified residues Cys₂ and Cys₈, all carbons, nitrogens, and protons in CuMbn were identified and assigned (Figures S8–S11 and Table S1).

Close analysis of peaks corresponding to residues neighboring Cys₂, Met₁ and Ser₃ in [¹H–¹³C] HMBC provides further support for the proposed pyrazinedione ring replacing Cys₂.First, the H_α and N-terminal amino group that would be present on Met₁ if Cys₂ was either unmodified or modified to an oxazolone ring (Figure S3) are missing. Modification to a pyrazinedione ring incorporates the Met₁-C_α into the pyrazinedione ring as C₂, and the Met₁-H_α is lost. Thus, the chemical shift corresponding to Pyr-C₂ would be shifted downfield into the aromatic region compared to that of a typical C_α and visible only through longer range coupling to hydrogen atoms bound to adjacent carbon atoms in [¹H–¹³C] HMBC. In the [¹H–¹³C] HMBC spectrum, the H_β atoms from Met₁ are coupled to two carbons with chemical shifts at 138.90 and 160.25 ppm (Figure 4A,C, green arrows, and Figures S10D and S11D). These chemical shifts are consistent with Pyr-C₂ and Pyr-C₃ as they are in the aromatic region, and the electron withdrawing character of the alcohol on Pyr-C₃ explains its shift further downfield than that of Pyr-C₂. One H_β atom from Met₁ is coupled to the carbon at 138.90 ppm, but not the carbon at 160.25 ppm, indicating that the carbon at 138.90 ppm is closer to the H_β atoms and is thus Pyr-C₂. These assignments also exclude the possibility of having a carbonyl group next to an imidazolone ring, which was suggested for the Mbn structure from Mc. str. SB2 whose operon also encodes MbnF.¹¹ A carbonyl neighboring an imidazolone would show stronger couplings to the carbon shifted farther downfield because carbonyl shifts are generally further downfield than shifts from N-containing heterocycles. Instead, the opposite is observed. Finally, the H_α of Ser₃ shows multiple ¹H–¹³C couplings, two of which are downfield in the carbonyl regime. The coupling at 172.39 ppm is assigned to the carbonyl next to the H_α, and the one at 191.50 ppm is assigned to the carbon in the thioamide (Figure 4A,C, green arrows). As expected from previous Mbn thioamide assignments,^11,15 this carbon is shifted farther downfield than typical carbonyl groups, likely due to the electron deficient character of thioamides. Additionally, in the [¹H–¹³C] HMBC spectrum recorded in 10% D₂O where the amide protons are present, an amide proton (NH) of Ser₃ shows a coupling to the carbon at 117.89 ppm, corresponding to C₆ of the pyrazinedione ring (Figure S11D).

Figure 4.

Proposed structure of regions in (A) Met₁/Pyr/Ser₃ and (B) Met₇/Oxa/Gly₉ showing couplings on [¹H–¹³C] HMBC taken in 100% D₂O. Couplings to the carbons on either Pyr or Oxa are depicted using arrows (green for Pyr and orange for Oxa). (C) Region of the [¹H–¹³C] HMBC spectrum showing couplings and shifts depicted in panels A and B. Complete [¹H–¹³C] HMBC spectra recorded in 100% and 10% D₂O and their couplings are shown in Figures S10 and S11.

Similarly, the modification of Cys₈ to an oxazolone ring and a thioamide was confirmed by analysis of the neighboring residues Met₇ and Gly₉. In the [¹H–¹³C] HMBC spectrum, the H_α and H_β atoms on Met₇ are coupled to the carbon at 147.89 ppm, which corresponds to C₂ on an oxazolone ring (Figure 4B,C, orange arrows, and Figures S10D and S11D). Additional coupling between the amide proton from Met₇ and the Oxa-C₂ was observed in the [¹H–¹³C] HMBC spectrum taken in 10% D₂O (Figure S11D). The modification of Cys₈ is also supported by the absence of the carbonyl group on Met₇. Moreover, the H_α atoms from Gly₉ are coupled to a carbon at 182.29 ppm (Figure 4B,C), consistent with the downfield shift of a carbon from a thioamide group.

The combined data validate the predicted structure of the Ms. sp. LW3 group I Mbn and reinforce the proposed role of MbnF in generating pyrazinedione rings in Mbns. Pyrazinedione rings have now been observed in three of four characterized Mbns that are known to encode MbnF in their operons.⁵ The one outlier, Mbn from Mc. str. SB2, is widely depicted with an imidazolone ring.^5,19 However, it was structurally characterized via NMR, and as discussed above, differentiating these moieties is more challenging than it is in crystallography, which was used to identify the pyrazinedione groups in the other Mc. Mbns¹⁴ as well as that from Ms. sporium NR3K.¹⁶ On the basis of the content of its Mbn operon, it is likely that the Mc. str. SB2 Mbn contains a pyrazinedione ring instead.

The prevalence of the pyrazinedione ring in Mbns is consistent with the notion that as a more acid-impervious moiety than an oxazolone, it protects the N-terminus of Mbn from degradation.¹⁷ To assess the stability of the pyrazinedione and oxazolone rings in Ms. sp. LW3 Mbn, we performed acid hydrolysis of apoMbn. Concentrated acetic acid was added to an aqueous solution of apoMbn to yield an overall concentration of 0.1% acetic acid. In the presence of acid, shifts of the 337 and 387 nm UV–vis features were observed (Figure S12A), possibly because protonation of the pyrazinedione ring results in the formation of a different tautomer. Acid hydrolysis data have not been reported for any other Mbns confirmed to contain pyrazinediones, although notably Mc. str. SB2 Mbn exhibits a similar spectral change upon acid treatment.^11,20 Spectra of aliquots neutralized with approximately 1 equiv of KOH show that oxazolone hydrolysis as monitored at 337 nm is nearly complete after 1 h, whereas the pyrazinedione absorbance at 387 nm decreases much more slowly and was still present after 24 h (Figure S12B); this is also consistent with the behavior of Mc. str. SB2 Mbn. Negative ion mode MS of the acid-exposed compound showed that the m/z of the resultant intermediate species is 1288.51, consistent with the expected mass of the hydrolyzed product (Figure S13).

The Ms. sp. LW3 group I Mbn structure represents the largest Mbn characterized to date, expands the diversity of known Mbns, and provides support for the proposed biosynthetic role of MbnF in forming a stabilizing pyrazinedione ring. Given the presence of MbnB and MbnC in the operon, and the fact that the pyrazinedione/thioamide pair is derived from a cysteine that is part of an MbnBC modification motif in the MbnA core peptide, MbnF’s substrate may be an oxazolone ring. Biochemical characterization and in vivo characterization of MbnF are likely to add to the emerging diversity in flavoenzyme reactivity and mechanism.²¹ Characterization of the Ms. sp. LW3 group I Mbn also raises the question of Mbn regulation and function in methanotrophs with two Mbn operons. Only the group I Mbn was isolated from copper-starved Ms. sp. LW3, suggesting that other conditions and perhaps additional functions may be associated with production of the group IIb Mbn. Structural and functional characterization of group IIb Mbns remains an important priority for future work.