A Permissive Amide N-Methyltransferase for Dithiolopyrrolones

Abstract

Amide N-methylation is important for the activity and permeability of bioactive compounds but can be challenging to perform selectively. The broad-spectrum antimicrobial natural products thiolutin and holomycin differ only by an N-methyl group at the endocyclic amide of thiolutin, but only thiolutin exhibits antifungal activity. The enzyme responsible for amide N--methylation in thiolutin biosynthesis has remained elusive. Here, we identified and characterized the amide N-methyltransferase DtpM that is encoded >400 kb outside of the thiolutin gene cluster. DtpM catalyzes efficient conversion of holomycin to thiolutin, exhibits broad substrate scope toward dithiolopyrrolones, and has high thermal stability. In addition, sequence similarity network analysis suggests DtpM is more closely related to phenol O-methyltransferases than some amide methyltransferases. This study expands the limited examples of amide N-methyltransferases and may facilitate chemoenzymatic synthesis of diverse dithiolopyrrolone compounds as potential therapeutics.

Graphical Abstract

The N-methylation of biomacromolecules plays an essential role in regulation of gene expression and membrane signal transmission.^1,2 N-methylation, of amides in particular, reduces hydrogen bond donors and promotes hydrophobic interactions, and in turn modulates the potency, specificity, and pharmacokinetics of bioactive compounds.^3,4 For instance, the immunosuppressant cyclosporine contains seven N-methyl amides in its cyclic peptide backbone, which are essential for increased membrane permeability and oral bioavailability.⁵

While methylated amides are important moieties in biomolecules, N-methyltransferases of amide nitrogens are relatively rare.^6,7 The lone pair of electrons of an amide nitrogen is delocalized, thus reducing the nucleophilicity of the nitrogen to attack the methyl donor S-adenosyl-l-methionine (SAM). Because the pK_a of the amide proton is around 15 in water and 25 in DMSO, deprotonation of the amide bond requires a strong base, and selective methylation of the nitrogen by chemical synthesis can be challenging.^7,8 Backbone N-methylation of peptide natural products involves different strategies: in cyclosporine biosynthesis, methyltransferase domains of nonribosomal peptide synthetases (NRPSs) methylate the amine of amino acid thioesters prior to amide bond formation,⁹ whereas in omphalotin A biosynthesis, the methyltransferase OphMA catalyzes N-methylation of amides after amide bond formation.^10,11 Additionally, a few amide N-methyltransferases have been discovered that perform late-stage methylations in other natural product scaffolds, including the polyketide ansamitocin (Asm10),¹² the epipolythiodioxopiperazine gliotoxin (GliN),¹³ doubly methylated 2,5-diketopiperazines (Amir_4628),¹⁴ and the terpenoid N-methyl welwitindolinone C isothiocyanate (WelM).¹⁵ Among these enzymes, Asm10 and Amir_4628 are promiscuous toward non-native substrates,^12,14 indicating potential applications for these amide N-methyltransferases as biocatalysts.

To expand the toolbox of amide N-methyltransferases to include other natural product scaffolds, we studied the N-methylation step in the biosynthesis of dithiolopyrrolones. Dithiolopyrrolones are disulfide-containing bicyclic compounds with broad-spectrum antibiotic and antiangiogenic activities.¹⁶ Studies suggest that dithiolopyrrolones act as prodrugs, and the reduced dithiol form of dithiolopyrrolones chelates metal ions tightly, thus disrupting metal homeostasis in cells and inhibiting some metalloenzymes.^17,18 Thiolutin and holomycin are both dithiolopyrrolones and their structures only differ by an N-methyl group at the endocyclic amide of thiolutin (Figure 1); however, only thiolutin shows antifungal activity.¹⁹ Additionally, synthetic N-alkyl derivatives of dithiolopyrrolones show increased anticancer activity.²⁰ Thus, the N-methyltransferase in thiolutin biosynthesis may be useful for enzymatic diversification of DTPs.

Figure 1.

A) Structures of natural products that contain N-methylated amide groups. B) Comparison of thiolutin and holomycin BGCs. Colored blocks indicate annotated gene functions. C) Genome representation of S. algeriensis NRRL B-24137, which contains 29 predicted BGCs (colored). Positions of dtpM and thiolutin BGC are marked.

To identify the N-methyltransferase that might be involved in thiolutin biosynthesis, we compared the biosynthetic gene clusters (BGCs) of holomycin and thiolutin. The biosynthesis of holomycin involves condensation of two cysteines and eight-electron oxidation to generate the dithiolopyrrolone core.²¹ The BGC of thiolutin contains the same core biosynthetic genes as that of holomycin.²² Incubating holomycin with the cell extract of the thiolutin producers Saccharothrix algeriensis and Streptomyces thioluteus generated thiolutin, suggesting N-methylation of holomycin is the last step of thiolutin biosynthesis.^22,23 While two putative methyltransferases are encoded in the thiolutin BGC of S. thioluteus and one in that of S. algeriensis, none of these enzymes were found to methylate holomycin in cells or in vitro.^22,23 Such evidence suggests that the methyltransferases that convert holomycin into thiolutin are located outside the thiolutin BGC; however, their identities were unknown.

We explored the S-methylation activity of the methyltransferases encoded in the thiolutin BGCs. The fungal toxin gliotoxin also contains a redox active disulfide and a methylated amide that are essential for bioactivity (Figure 1).²⁴ GliN, a methyltransferase within the gliotoxin BGC, catalyzes the amide N-methylation to activate gliotoxin, whereas TmtA, a methyltransferase outside the BGC, inactivates gliotoxin by S-methylation of reduced gliotoxin.¹³ We have identified a similar back-up protection mechanism for holomycin via S-methylation of reduced holomycin.²⁵ To examine whether the methyltransferases located in the thiolutin BGCs may be involved in S-methylation of reduced thiolutin for self-protection, we cloned and purified these enzymes from both S. thioluteus and S. algeriensis (Figure S1). S-methylation activity of these enzymes was tested toward chemically reduced holomycin and thiolutin in vitro. No methylation was observed toward reduced holomycin, regardless of metal supplementation (Figure S2-S6). We also confirmed that none of these enzymes could convert holomycin to thiolutin (Figure S7). These results suggest that the methyltransferases in the thiolutin BGC do not catalyze N-methylation of holomycin, nor do they catalyze S-methylation of reduced holomycin. None of the methyltransferases in the thiolutin BGCs catalyzed S-methylation of reduced thiolutin, either, except for StMTase_92133 from S. thioluteus, which showed a low level of S-methylating activity toward reduced thiolutin (Figure S2). The functions of these methyltransferases are currently unknown. They may be involved in methylating macromolecules that are relevant to the mode of action of dithiolopyrrolones. As evidence for this possible role, a putative RNA methyltransferase hom12 was found in the holomycin gene cluster from the fish pathogen Yersiniaruckeri.²⁶ A deletion study of hom12 suggested a role of this gene in methylating RNA as a mechanism for self-resistance to holomycin. A hom12 paralog is not present in the holomycin cluster in S. clavuligerus.

We sought to identify the amide N-methyltransferase in thiolutin biosynthesis using other aromatic amide N-methyltransferases as a guide. The structure and mechanistic studies of the amide methyltransferase OphMA^27,28 suggested a general acid–base mechanism that involves proton abstraction; therefore, the pK_a of amide protons might be a critical factor in methylation.²⁸ The bicyclic dithiolopyrrolone core is a highly conjugated aromatic system, and the pK_a of the endocyclic amide proton is estimated to be around 18 in DMSO, lower than the pK_a of nonaromatic amide protons (~25 in DMSO).^8,16 Thus, to identify N-methyltransferases that might be involved in thiolutin biosynthesis, we used as a query the amide methyltransferase WelM, which methylates the aromatic endocyclic amide of welwitindolinone C isothiocyanate.¹⁵ We performed a BLAST search of WelM against the genome of S. algeriensis. A top hit (33% sequence identity) was a SAM-dependent methyltransferase located 423 kb upstream of the thiolutin BGC (Figure 1). We cloned and purified this methyltransferase and named it DtpM (dithiolopyrrolone methyltransferase, Figure S1). Incubating purified DtpM with holomycin and SAM produced a species with the same mass and retention time as the commercial thiolutin standard and thiolutin produced by S. algeriensis as shown by liquid chromatography coupled-high resolution mass spectrometry (LC-MS) analysis (Figure 2A), which confirmed that DtpM catalyzes the conversion of holomycin to thiolutin. BLAST search of WelM against the holomycin producer Streptomyces clavuligerus also gave two hits, ScMTase_10675 and ScMTase_10977, which exhibit 35% and 41% sequence identity with DtpM, respectively. However, ScMTase_10977 was not able to methylate holomycin in vitro, and ScMTase_10675 only showed low levels of activity toward holomycin (Figure S8); ScMTase_10675 was very recently reported to be involved in amide N-methylation of a polycyclic tetramate macrolactam.²⁹ This result shows that not all WelM homologues can catalyze methylation of holomycin.

Figure 2.

DtpM catalyzes N-methylation of diverse dithiolopyrrolones using SAM and analogues. A) Extracted ion chromatograms of thiolutin (m/z 229.0100 [M + H]⁺) from LC-MS analysis of the reaction that contains holomycin, SAM, and DtpM (top), thiolutin standard (middle), and extract of S. algeriensis culture (bottom). B) Substrates modified by DtpM. Conversion rates were calculated based on extracted ion chromatograms with two replicates. C) DtpM accepts SAM, allyl- and propargyl-SAM as cofactors to alkylate dithiolopyrrolones.

We examined the substrate scope of DtpM toward dithiolopyrrolones with various acyl chains (2–9), including octanoyl (2), 5-hexynoyl (3), and trifluoroacetyl (4) groups.^18,30 More than 70% conversion was observed for all dithiolopyrrolone substrates, including thiomarinol A (7), a hybrid antibiotic of holothin and marinolic acid (Figure 2B, Figure S9-S10, Table S3).^31,32 Tandem mass spectrometry and NMR analysis confirmed that methylation occurred on the endocyclic amide of the dithiolopyrrolone of thiomarinol A rather than the polyketide acyl chain that contains four hydroxyl groups (Table S4, Figure S11-S15). NMR analysis also confirmed the structure of the methylated trifluoroacetyl holothin product (Table S5, Figure S16-19). Because S. algeriensis natively produces thiolutin and other dithiolopyrrolones with different acyl groups,³³ the ability of DtpM to accept a wide range of dithiopyrrolones is consistent with methylation being the last step of biosynthesis. Dithiolopyrrolones in the thiosulfonate oxidation state were also well tolerated, as DtpM methylated both oxo-holomycin (5) and thiomarinol B³⁴ (8) with 100% conversion (Figure 2B, Figure S9, S20, S21, Table S3). However, reduced and alkylated holomycin was not methylated by DtpM, likely due to steric hindrance from the S-alkyl groups or the loss of the bicyclic ring. Other nonaromatic amide substrates were not accepted either, including biotin, Glu-Ala dipeptide, and the cyclic dipeptide Ile-Leu (Figure S22-S23). However, DtpM shows modest activity toward other bicyclic aromatic amides, including 2(1H)-quinolinone (6) and 2-oxindole (9), the latter of which is a fragment of welwitindolinone (Figure 2B, Figure S24, Table S3). 6 and 9 tautomerize between the amide and enol form,³⁵ thus methylation could occur on either the nitrogen or the oxygen. Liquid chromatography analysis using commercial standards confirmed that only N-methylated products of 6 and 9 were formed in the DtpM reaction (Figure S25). Thus, DtpM catalyzes selective methylation on the nitrogen of aromatic amides.

We showed that DtpM can also use SAM analogues including allyl-SAM and propargyl-SAM for alkylation of holomycin (Figure 2C, Figure S26-S28, Table S3). Using propargyl-SAM, DtpM installed an alkyne handle on the dithiolopyrrolone core, which could enable further ligation with azide probes via click chemistry.³⁶ Additionally, thermal stability is important for biocatalysts.³⁷ DtpM remained active after boiling for 95 °C for 30 min (Figure S29). Circular dichroism analysis³⁸ suggests that DtpM only partially unfolds at 95 °C and retains some α-helical structure and that the unfolding is fully reversible (Figure S30).

Kinetic analysis of DtpM using a coupled assay yielded a K_M of 2.0 ± 0.2 μM and k_cat of 0.0400 ± 0.0008 s⁻¹ for SAM, and a K_M of 1.0 ± 0.2 μM and k_cat of 0.090 ± 0.005 s⁻¹ for holomycin (Figure 3). Substrate inhibition was observed at higher concentrations of holomycin, with a K_i of 50 ± 8 μM (Figure 3). This inhibition likely contributes to the lower k_cat value measured by varying SAM concentrations compared to the k_cat measured by varying holomycin concentrations. Similar K_M and k_cat were observed for thiomarinol A (Figure S31), indicating that DtpM can accommodate extended and heavily modified acyl chains at the exocyclic amine of dithiolopyrrolones. For thiomarinol B, K_M was similar, but k_cat was 20-fold lower than the k_cat for holomycin. For oxindole, a non-dithiolopyrrolone substrate, the K_M was 590-fold higher and the k_cat was 2000-fold lower compared to holomycin (Figure S31), which indicates the preference of DtpM for dithiopyrrolone substrates. Interestingly, no substrate inhibition was observed when thiomarinol A or B or oxindole were used as substrates, which suggests that substrate inhibition of DtpM is unique to holomycin.

Figure 3.

Kinetic measurements for methylation of holomycin catalyzed by DtpM.

To assess the similarity of DtpM to other amide methyltransferases, we generated a sequence similarity network (SSN) of several amide methyltransferases and proteins in the same Pfams (Figure 4).³⁹ DtpM is clustered with WelM, but not the other amide methyltransferases such as GliN, Asm10, and OphMA. Instead, DtpM grouped with many O-methyltransferases that methylate phenolic hydroxyls, including CalO6, LaPhzM, ChoMT, and IOMT, all of which are in the same protein family as DtpM (PF00891).^40-42 DtpM also grouped with PhzM, an N-methyltransferase of phenazine-1-carboxylic acid in the biosynthesis of pyocyanin.⁴³ Additionally, size-exclusion chromatography multiangle light scattering analysis suggested DtpM exists exclusively in a dimeric state (Figure S32), consistent with the presence of an N-terminal dimerization domain and similar to some of the other proteins in the protein group.^40-44 DtpM also showed modest O-methylating activity for isoliquiritigenin (Figure S33), the substrate of ChOMT.⁴⁵ These observations suggest that aromatic N-methyltransferases are more closely related to phenolic O-methyltransferases than some amide methyltransferases.

Figure 4.

Sequence similarity network of amide N-methyltransferases and other N- and O-methyltransferases from selected Pfams (alignment score ≥30).

We further identified essential residues for DtpM activity. Sequence alignment of DtpM to other proteins in the same SSN cluster revealed the well-conserved residues in phenolic O-methyltransferases are also present in DtpM, including the catalytic residues, the SAM-binding residues, and some of the substrate-binding residues (Figure S34). Structural and mutagenesis studies on IOMT, an O-methyltransferase in plant phenylpropanoid biosynthesis, suggested that His257 is the catalytic base that deprotonates the substrate while Asp288 and Glu318 sterically constrain His257, and the His257 Nδnitrogen is hydrogen bonding with Glu318.⁴² We mutated the equivalent residues in DtpM—His251, Asp279, and Glu310— to alanines. Mutation of His251 to Ala increased the K_M for holomycin from 1.0 μM to 19 μM and decreased the k_cat from 0.090 s⁻¹ to 0.003 s⁻¹ (Figure S35). The 640-fold decrease in k_cat/K_M of the H251A mutant supports the essential catalytic role of His251 in DtpM. DtpM_E310A showed a more than 20-fold increase in K_M but a modest reduction in k_cat. Mutation of Asp279 to Ala resulted in a slight increase in K_M and a slight decrease in k_cat, but substrate inhibition was no longer observed, thus Asp279 can be mutated to allow rapid catalysis at high substrate concentrations.

In summary, we identified and characterized DtpM, an amide N-methyltransferase for the unique dithiolopyrrolone scaffold. DtpM selectively methylates the endocyclic amide nitrogen of dithiolopyrrolones but accepts varying acyl chains at the exocyclic amine, including the complex polyketide chain in the thiomarinols. While DtpM is related to phenolic O-methyltransferases based on sequence similarity network analysis, it is specific for the amide nitrogen and prefers dithiolopyrrolones over phenolic substrates. The efficient and specific amide N-methylation of dithiolopyrrolones by DtpM suggests that DtpM is likely involved in thiolutin biosynthesis; further gene inactivation studies are needed to validate the involvement.

Our work expands the limited toolbox of amide N-methyltransferases and reveals the potential for DtpM in biocatalytic applications. Its thermal stability and its ability to transfer various alkyl groups from SAM analogues will likely allow chemoenzymatic synthesis and bioactivity studies of dithiolopyrrolone derivatives. Our work also sheds light on the catalytic mechanism of DtpM in amide N-methylation and identifies the D279A mutation that removes substrate inhibition and facilitates catalysis at high substrate concentrations.