We first constructed an enediyne GNN, including all predicted and experimentally confirmed enediyne biosynthetic gene clusters, to search for candidates conferring self-resistance to the anthraquinone-fused enediynes. The GNN clearly revealed additional homologs of the apoproteins, known for the nine-membered enediyne core subcategory, and the self-sacrifice proteins, known for the ten-membered enediyne CAL, suggesting that both mechanisms are likely shared by many other enediynes; these findings also demonstrated the utility of GNN in identifying candidates conferring enediyne resistance by the known mechanisms ( Figures 1 C and S1 ). Strikingly, GNN analysis failed to identify any candidates for the two known resistance mechanisms from the gene clusters encoding the biosynthesis of the four anthraquinone-fused enediynes, DYN, UCM, TNM, and YPM ( Figure 1 A). Since these producers must have evolved self-resistance elements, close examination of the GNN revealed a distinct family of proteins, consisting of TnmS1, TnmS2, TnmS3, UcmS1, UcmS2, UcmS3, DynE14, DynE15, YpmS1, and YpmS3, from the TNM, UCM, DYN, and YPM biosynthetic machineries, that share 31%–90% amino acid sequence identities ( Figures 1 B, 1C, and S1 Data S1 A), suggesting an unprecedented mechanism of self-resistance for the anthraquinone-fused enediynes. BLASTP analysis revealed that the proteins in this family belong to the glyoxalase superfamily, which are known to perform versatile functions, varying from isomerization (), oxidative cleavage of C-C bonds (), epimerization (), and epoxide hydrolysis (). Selected members in this superfamily are also known to render antibiotics ineffective, such as fosfomycin epoxide hydrolases and thiol transferases (), and bleomycin (BLM) () and mitomycin () binding proteins. With the exception of the antibiotic binding proteins, proteins in this family often possess the conserved amino acid residues His, Glu, and Asp for metal chelating. Since TnmS1, TnmS2, TnmS3, UcmS1, UcmS2, UcmS3, DynE14, DynE15, YpmS1, and YpmS3 lacked these conserved residues, we hypothesized, in the absence of alternative proposals for these proteins to play a role in biosynthesis of the anthraquinone-fused enediynes, that they might be antibiotic binding proteins that confer DYN, UCM, TNM, and YPM resistance by sequestration.

We next decided to use Streptomyces sp. CB03234, the wild-type producer of TNMs, as a model to investigate the self-resistance mechanisms for the anthraquinone-fused enediynes in vivo, confirming that tnmS1, tnmS2, and tnmS3 are necessary to endow Streptomyces sp. CB03234 with high resistance to TNMs. We deleted a 5.5-kb DNA fragment, harboring tnmS1, tnmS2, and tnmS3, as well as three additional genes, tnmT1 and tnmT2 (encoding two putative transporters) and tnmR4 (encoding a putative regulator), which were interspersed among the three predicted resistance genes ( Figure 1 B), in Streptomyces sp. CB03234 to generate the ΔtnmS1-tnmS3 mutant strain SB20003. Its sensitivity to TNMs was first tested by a disk diffusion assay, with the wild-type Streptomyces sp. CB03234 as a positive control. While CB03234 showed high resistance, SB20003 was remarkably sensitive to TNM A, and obvious inhibition zones were observed for SB20003 even at the lowest amount of TNM A (1 ng) tested ( Figure 2 A). We further determined the minimal inhibitory concentrations (MICs) of TNM A against CB03234 and SB20003 by a plate growth assay, with Streptomyces lividans TK24, a naive strain that is sensitive to TNM A, as a negative control. SB20003 was shown to be extremely sensitive to TNM A, with an MIC of 4 ng/mL, which was the same as the MIC for S. lividans TK24 (4 ng/mL) and was minimally 125-fold more sensitive than the MIC for CB03234 (500 ng/mL) ( Table 1 Figure S2 A). These results unambiguously establish that the inherent self-resistant elements for TNMs in Streptomyces sp. CB03234 reside within this six-gene fragment and that there are no other genes, outside the tnm biosynthetic gene cluster, that encode TNM resistance.

(C) E. coli recombinant strains SB20015 to SB20018, expressing the five homologs from the human microbiome, show resistance to TNM A, in comparison with E. coli BL21(DE3)/pET28a as a negative control, revealing that the human microbiome might already possess resistant elements to anthraquinone-fused enediynes. Depicted next to the disks are the amounts of DYN, UCM, TNM A, and YMP, respectively, loaded to each set of the diffusion assays.

(B) E. coli recombinant strains SB20004 to SB20013, expressing tnmS1, tnmS2, tnmS3, and their homologs from the UCM, DYN, and YPM producers, are resistant to TNM A, UCM, DYN, and YPM, respectively, in comparison with E. coli BL21(DE3)/pET28a as a negative control, demonstrating that each of these gene products is sufficient to confer resistance to anthraquinone-fused enediynes in their respective producers.

(A) The ΔtnmS1-tnmS3 mutant strain SB20003 is sensitive to TNM A, in comparison with the wild-type strain CB03234 as a positive control, establishing that TnmS1, TnmS2, and TnmS3 are necessary and sufficient to confer TNM resistance in its producer.

The tnmS1, tnmS2, tnmS3 Genes, and their Homologs Encoding Resistance to Anthraquinone-Fused Enediynes as Demonstrated by the Disk Diffusion Assays

Figure 2 The tnmS1, tnmS2, tnmS3 Genes, and their Homologs Encoding Resistance to Anthraquinone-Fused Enediynes as Demonstrated by the Disk Diffusion Assays

We then focused on tnmS1, tnmS2, and tnmS3 genes from the tnm cluster, as well as their homologs ucmS1, ucmS2, and ucmS3 genes from the ucm cluster, dynE14 and dynE15 genes from the dyn cluster, and ypmS1and ypmS3 genes from the ypm cluster for comparative studies ( Figure 1 ), and revealed that they encoded self-resistance to the four anthraquinone-fused enediynes TNM, UCM, DYN, and YPM, respectively, capable of conferring cross-resistance. We adopted an in vivo approach to screen if tnmS1, tnmS2, tnmS3, or their homologs could confer resistance to TNMs or the other anthraquinone-fused enediynes UCM, DYN, and YPM, respectively, in E. coli BL21(DE3), which is known to be sensitive to these enediyne natural products. Each of the candidate-resistant genes was cloned into pET28a, affording expression plasmids pBS20007 to pBS20016, and transformed into E. coli BL21(DE3), yielding recombinant strains SB20004 to SB20013 ( Table S2 ). Disk diffusion assays revealed that SB20004−SB20013 are significantly more resistant to the corresponding anthraquinone-fused enediynes, in comparison with the negative control E. coli BL21(DE3)/pET28a ( Figure 2 B). These results provided direct evidence, supporting that tnmS1, tnmS2, tnmS3, or their homologs, individually, was sufficient to endow E. coli BL21(DE3) with resistance to their respective anthraquinone-fused enediynes in vivo. We subsequently determined the MICs of TNM A against SB20004, SB20005, and SB20006 by the plate growth assay for quantitative comparison. Expression of tnmS1, tnmS2, or tnmS3 in E. coli BL21(DE3) revealed a similar level for protein overproduction ( Figure S2 B), and individually endowed E. coli BL21(DE3) with >1,000-fold increase in cellular resistance to TNM A as exemplified by the MICs for SB20004 (2,560 ng/mL), SB20005 (2,560 ng/mL), and SB20006 (20,480 ng/mL) in comparison with the E. coli BL21(DE3) (2.5 ng/mL) ( Table 1 Figure S2 C). Furthermore, SB20004, SB20005, and SB20006 showed cross-resistance to UCM, DYN, and YPM ( Figure S3 A). These results support a common self-resistance mechanism for the anthraquinone-fused enediynes.

To directly measure the binding of TNMs to TnmS1, TnmS2, and TnmS3, we first removed the partially bound riboflavin from TnmS1, TnmS2, and TnmS3 by treatment with activated charcoal. TNM A and TNM C ( Figure 1 A) were used as representative anthraquinone-fused enediynes to measure their direct binding to TnmS1, TnmS2, and TnmS3 in vitro. The purified TnmS1, TnmS2, and TnmS3 were each found to be dimeric in solution upon analysis by size-exclusion chromatography ( Figure S5 A). Fluorescence-quenching analysis revealed that TNM A and TNM C were tightly bound by TnmS1, TnmS2, and TnmS3 with nanomolar Kvalues ( Figure S4 Table 2 ). TNM A, upon recovery from binding to TnmS1, TnmS2, and TnmS3, showed no modification or degradation, further demonstrating the non-covalent, non-destructive, and reversible binding between these proteins and TNMs.

We subsequently characterized the TnmS1, TnmS2, and TnmS3 proteins in vitro, revealing that they bind TNMs with nanomolar affinity. Recombinant TnmS1, TnmS2, and TnmS3 proteins were produced in E. coli and purified to homogeneity. At high concentration (50 mg/mL), the purified TnmS1, TnmS2, and TnmS3 appeared light yellow. To identify the yellow pigment co-purified with TnmS1, TnmS2, and TnmS3, these proteins were denatured by treating with trifluoroacetic acid. The released yellow pigment, which was isolated and subjected to LC-MS analysis, was identified to be riboflavin in comparison with an authentic standard. Given the low occupancy (<20%) and micromolar Kvalues of riboflavin to TnmS1, TnmS2, and TnmS3 ( Figure S4 Table 2 ), we concluded that the co-purified riboflavin resulted from adventitious binding of riboflavin to these proteins upon their overproduction in E. coli and would not interfere with TnmS1, TnmS2, and TnmS3 binding to TNMs, thereby their ability to confer TNM resistance.

Binding of TnmS1, TnmS2, TnmS3 and the Selected Homologs from the Human Microbiome to TNM A, TNM C, and Riboflavin as Determined by the K D Values

Table 2 Binding of TnmS1, TnmS2, TnmS3 and the Selected Homologs from the Human Microbiome to TNM A, TNM C, and Riboflavin as Determined by the K D Values

Crystal Structures of TnmS1, TnmS2, and TnmS3 Revealing the Molecular Details for Resistance to the Anthraquinone-Fused Enediynes by Sequestration

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Shen B. Genome mining of Micromonospora yangpuensis DSM 45577 as a producer of an anthraquinone-fused enediyne. A and Trp42B via electrostatic interactions of the aromatic rings; (2) the side chains of Gln103A, Gln7A, His39B, and the main chain amine group of Val38B provide hydrogen bond interactions with the hydroxyl and epoxide groups of TNM A; and (3) Gly70A and Leu72A and Leu8B, Trp42B, Trp59B, and Phe60B create a hydrophobic environment to accommodate the enediyne core. Figure 4 The Crystal Structure of TnmS3 in Complex with TNMs Revealing the Molecular Details for Resistance to the Anthraquinone-Fused Enediynes by Sequestration Show full caption (A) The overall structure of TnmS3 in complex with TNM A shown in ribbon diagram. Two molecules of TNM A are bound by one TnmS3 dimer. (B) Local view showing that TnmS3 undergoes a slight conformational change upon TNM A binding, as depicted by the side chains of Trp100 and Gln103 flipping into new conformations to interact with TNM A, with the apo-TnmS3 structure shown in gray. (C) Local view of the enediyne binding cavity highlighting the residues involved in interactions between TnmS3 and TNM A. Hydrogen bonds are shown in red dashed lines. o –F c electron density maps are contoured at 1σ shown as white mesh. The σ-A-weighted difference (mF o –DF c ) omit maps of TnmS3 in complex with TNM A and TNM C, respectively, are shown in (D) The local view of the enediyne binding cavity highlighting the residues involved in interactions between TnmS3 and TNM C. While TNM A and TNM C share many of the same interactions with TnmS3, the side chain at C-26 of TNM C reveals no major interaction. The 2F–Felectron density maps are contoured at 1σ shown as white mesh. The σ-A-weighted difference (mF–DF) omit maps of TnmS3 in complex with TNM A and TNM C, respectively, are shown in Figure S5 The structure of TnmS3 in complex with TNM A shows that each cavity houses one TNM A molecule ( Figure 4 A). TnmS3 undergoes a slight conformational change in β6-loop-β7 upon TNM A binding ( Figure 4 B). The loop between β6 and β7 shifts ∼1.5 Å upon TNM A binding, and the side chains of Trp100 and Gln103 flip into new conformations to interact with TNM A ( Figure 4 B). The structure of TnmS3 in complex with TNM C, a biosynthetic intermediate of TNM A accumulated by the ΔtnmH mutant strain Streptomyces sp. SB20002 (), shows a similar binding environment as TNM A in TnmS3 ( Figures 4 C and 4D). The side chain at C-26 of TNM C reveals no strong interaction with TnmS3 ( Figures 1 A and 4 D). The complex structures of TnmS3 with TNM A and TNM C, therefore, provide direct evidence and reveal the molecular details of how TnmS3 sequesters TNMs. Thus, TNMs are sequestered in the cavity of the β barrel-like structure by three main interactions ( Figure 4 C): (1) the flat anthraquinone moiety of TNM A is sandwiched between the side chains of Trp100and Trp42via electrostatic interactions of the aromatic rings; (2) the side chains of Gln103, Gln7, His39, and the main chain amine group of Val38provide hydrogen bond interactions with the hydroxyl and epoxide groups of TNM A; and (3) Gly70and Leu72and Leu8, Trp42, Trp59, and Phe60create a hydrophobic environment to accommodate the enediyne core.

By superposition of TnmS1 and TnmS2 with the structure of TnmS3 in complex with TNM A, the binding modes of TNM A in TnmS1 and TnmS2 were mapped. The models of TnmS1 and TnmS2 in complex with TNM A revealed that the cavities of the β barrel-like structures provide sufficient space to accommodate TNM A, the binding residues of which are proposed ( Figure 3 B): (1) the anthraquinone moiety interacts electrostatically with Trp41 and Phe104 in TnmS1 and Trp41 and Tyr114 in TnmS2, (2) the epoxide ring is hydrogen bonded by Asn108 in TnmS1 and Glu117 in TnmS2, and (3) the enediyne core interacts hydrophobically with Trp41, Val62, Phe71, and Leu120 and Leu39 in TnmS1, and Trp41, Val55, Val85, and Trp127 in TnmS2.