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. 2026 May 22;21(6):1301–1308. doi: 10.1021/acschembio.6c00013

A Cysteine-Dependent Peptide Cyclase with Broad Substrate Tolerance Enables Chemoenzymatic Synthesis of Macolacin Analogs

Miyu Morohashi †, Sho Konno †,*, Koki Yamashita †, Kyoga Kamijo †, Akihiro Taguchi †, Masaki Mishima †, Atsuhiko Taniguchi †, Yoshio Hayashi †,‡,*
PMCID: PMC13288461  PMID: 42172238

Abstract

Thioesterase (TE) domains catalyze peptide release and macrocyclization during nonribosomal peptide biosynthesis, typically via catalytic serine. In contrast, MacB-TE from macolacin biosynthesis employs a rare cysteine nucleophile, raising questions regarding the catalytic requirements of cysteine-dependent peptide cyclases. Here, we report the biochemical and functional characterization of MacB-TE and demonstrate its efficiency, regioselectivity, and exclusive macrocyclization. Using a synthetic thioester substrate, MacB-TE converted >99% of the linear precursor into the cyclic product within minutes. Mutational analysis demonstrated that Cys89 is indispensable for ring formation, whereas His218 and Asp116 support an efficient turnover. Notably, a D116S variant retained substantial cyclization activity, uncovering an unexpected tolerance at the acidic position of the catalytic triad. Comprehensive substrate profiling showed that MacB-TE accommodated extensive side-chain variations and diverse N-terminal lipid groups. The enzyme efficiently macrocyclized all nine alanine-scan variants, with six positions supporting near-quantitative conversion and the remaining three producing more than 80% of the cyclic products. Likewise, lipid-modified substrates, including those with aliphatic and aromatic motifs, were quantitatively converted into their corresponding macrocycles, indicating that lipid variation had a negligible impact on catalysis. Collectively, these findings establish MacB-TE as a highly efficient and broadly tolerant macrocyclase, enabling the chemoenzymatic synthesis of macolacin analogs and expanding the mechanistic understanding of Cys-type TEs.

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Introduction

Multidrug-resistant (MDR) bacteria have emerged as a major global health threat that severely limits the effectiveness of existing antibiotics. − Cyclic lipopeptides, such as colistin, serve as last-resort agents against MDR Gram-negative pathogens; however, the rapid rise of colistin-resistant strains has highlighted the urgent need for new therapeutic strategies. − Recent advances in synthetic bioinformatic natural product approaches have enabled the identification of structurally diverse nonribosomal peptides through genome mining and in silico analysis of nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters. − Among these, macolacin exhibits potent antibacterial activity against Gram-negative bacteria and differs structurally from colistin in its replacement of 2,4-diaminobutyric acid (Dab) at position 3 with d-Ser, Leu at position 7 with Ile, and Thr at position 10 with Leu (Figure ). Macolacin retains activity against Gram-negative strains harboring the mobile colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase that modifies lipid A and confers colistin resistance. Structure–activity relationship studies showed that the presence of d-Ser at position 3 and Leu at position 10 was essential for overcoming mcr-1-mediated resistance to macolacin. The amino acid composition and stereochemistry of macolacin were predicted based on the substrate specificity encoded in its NRPS domain architecture.

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Chemical structures of colistin, polymyxin B, and macolacin.

In the macolacin biosynthetic pathway, three NRPS enzymes (MacA, MacB, and MacE) sequentially assemble the decapeptide backbone (Figure ). In the final step, the C-terminal thioesterase (TE) domain of MacB cleaves the peptidyl carrier protein-tethered linear peptide and promotes macrocyclization (Figure ). Given that macrocyclization in polymyxin-family lipopeptides proceeds through Dab4, macolacin is predicted to undergo macrocyclization via an intramolecular attack of the Dab4 side chain amine. However, because macolacin was originally proposed based on bioinformatic analysis of its biosynthetic gene cluster and subsequently accessed by chemical synthesis rather than isolation, the exact ring-closure position has not yet been experimentally validated. (Figure ). TE domains belong to the α/β-hydrolase superfamily and typically harbor a catalytic serine within a conserved Ser–His–Asp triad. − Owing to their inherent regio- and stereoselective macrocyclization activities, NRPS-TEs represent valuable biocatalysts for the chemoenzymatic synthesis of natural product-like macrocyclic peptides. − In contrast to canonical serine-type TEs, MacB-TE contains a cysteine nucleophile within the Cys–His–Asp catalytic triad, representing a rare subclass that accounts for only ∼2% of known TEs. Although a few cysteine-dependent TEs have been reported, − their catalytic requirements remain poorly understood, and their potential as biocatalysts for synthetic applications has not been systematically explored. Given these mechanistic and functional gaps, studying the catalytic mechanism and substrate selectivity of MacB-TE is important not only for advancing our mechanistic understanding of Cys-type cyclases but also for determining whether this enzyme class can be practically utilized as a platform for chemoenzymatic peptide synthesis.

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Proposed biosynthetic pathway of macolacin.

In this study, we systematically characterized the catalytic properties, active site requirements, and substrate tolerance of MacB-TE using synthetic macolacin precursors. We demonstrated that MacB-mediated macrocyclization proceeds via the intramolecular attack of the Dab4 side chain amine, identifying Dab4 as a ring-closure site. Mutational analysis defined the catalytic contributions of the Cys–His–Asp triad, demonstrating that Cys89 is indispensable for ring formation, whereas His218 and Asp116 are essential for efficient catalysis; notably, a D116S mutant retains its macrocyclic activity. Further comprehensive substrate profiling revealed that MacB-TE tolerated extensive variations in both the peptide side chain composition and the N-terminal lipid moiety. Collectively, these results demonstrated that MacB-TE is a highly efficient and broadly tolerant cysteine-dependent peptide cyclase, providing a practical and robust biocatalyst for chemoenzymatic access to diverse macrocyclic peptide structures.

Results and Discussion

Enzymatic Characterization of MacB-TE

To enable the biochemical characterization of MacB-TE, we first attempted to express the enzyme as an N-terminal His-tagged construct by using a pET28a vector. However, MacB-TE was detected exclusively in inclusion bodies, and extensive optimization of the induction conditions failed to improve its solubility. Therefore, we designed an alternative expression strategy and cloned the gene into a pCold-GST vector that successfully produced MacB-TE as a soluble glutathione S-transferase (GST) fusion protein. After GST removal by NT*-HRV 3C protease, the untagged enzyme remained soluble and was purified using Ni affinity resin (Figures A, S1, and S2).

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In vitro characterization of MacB-TE. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified GST-tagged (a) and untagged (b) MacB-TEs. (B) Design of the substrate MLP-SNAC 1 and the enzymatic conversion by MacB-TE to the cyclic product C8-macolacin 2 and the hydrolyzed product 3. (C) High-performance liquid chromatography (HPLC) analysis of recombinant MacB-TEs. MLP-SNAC 1 (100 μM) was incubated with GST-tagged or untagged MacB-TE (2 μM) in 25 mM Tris–HCl (pH 7.5) for 1 h at 24 °C. (D) Time-course study of the reaction of substrate 1 (100 μM) and GST-tagged MacB-TE (0.5 μM). The product concentrations were calibrated using the standard curves of MLP-SNAC 1 and C8-macolacin 2 (Figure S3). (E) Key heteronuclear multiple bond correlation (HMBC, blue arrows) and nuclear Overhauser effect (NOE, red arrows) correlations observed for C8-macolacin 2, mapped onto the chemical structure.

For enzymatic assays, we prepared a linear macolacin substrate (MLP-SNAC, 1) that mimicked the native peptidyl carrier protein-bound intermediate. The substrate contains an N-terminal lipid and a C-terminal N-acetylcysteamine (SNAC) thioester, which serves as a minimal phosphopantetheine mimic widely used in NRPS-TE studies (Figure B). Although macolacin has been proposed to contain an (S)-6-methyloctanoyl lipid tail, we replaced it with commercially available octanoic acid for synthetic accessibility, anticipating that MacB-TE would tolerate this modification. The peptide was synthesized by Fmoc solid-phase peptide synthesis using a Dawson linker to install the SNAC moiety (Schemes S1 and S2).

Incubation of MLP-SNAC 1 with either GST-tagged or tag-free MacB-TE yielded a new HPLC peak corresponding to the expected macrocyclic product 2 (Figure C). Because the untagged enzyme was sensitive to freeze–thaw cycles, subsequent analyses were performed using GST-fusion. Time-course analysis revealed that MacB-TE converted more than 99% of the linear substrate into a macrocyclic product within 10 min (Figure D). Notably, MacB-TE generated only the cyclized product, with no detectable hydrolysis, in contrast to many serine-type TEs that typically produce both cyclic and hydrolyzed species. ,− To validate the structure of the enzymatic macrocycle, large-scale reactions were performed to isolate cyclic product 2. Tandem mass spectrometry (MS/MS) analysis, together with HMBC correlations between the Dab4 γ-protons and Leu10 carbonyl group, and additional Nuclear Overhauser Effect (NOE) signals consistent with the Dab4-Leu10 spatial proximity, confirmed macrocyclization at the Dab4-Leu10 linkage (Figures E, S4, and S5). These data provide direct experimental evidence that MacB-TE-mediated macrocyclization proceeds through intramolecular attack of the Dab4 side chain.

Next, we examined the parameters affecting the catalytic efficiency. The enzyme showed maximal conversion at pH 7.5 (Figure S6) and comparable activity at 24 and 37 °C, with moderate attenuation at 16 °C (Figure S7). Product formation was still observed at 4 °C, and the enzyme retained its activity in the presence of 2–20% dimethyl sulfoxide (Figure S8), indicating that MacB-TE tolerates both low temperatures and organic cosolvents. This tolerance to organic solvents is advantageous for preparative chemoenzymatic applications where substrates or intermediates may require a cosolvent for solubility.

Attempts to determine the kinetic parameters revealed that product formation deviated from Michaelis–Menten kinetics. At higher substrate concentrations, the conversion decreased at the same time points, suggesting substrate inhibition (Figure S9). Because the substrate is strongly cationic and MacB-TE possesses a markedly acidic surface enriched with Asp and Glu residues (48 residues; pI 4.6; Figure S10), extensive nonspecific electrostatic interactions may impede turnover.

Although MacB-TE shares 90.6% sequence identity with PmxB-TE from polymyxin B biosynthesis (Figure S11A), the two enzymes display markedly different catalytic efficiencies. Under our assay conditions, MacB-TE achieved near-quantitative macrocyclization, whereas PmxB-TE has been reported to show incomplete conversion under comparable reaction conditions. To visualize the structural context of the sequence differences, the nonidentical residues between MacB-TE and PmxB-TE were mapped onto the AlphaFold model of MacB-TE (Figure S11B). Most substitutions are distributed across surface-exposed regions rather than within the structural core of the enzyme. Given these observations, closely related Cys-type TEs can vary widely in catalytic performance and underscore MacB-TE as a biocatalyst for peptide macrocyclization.

Functional Analysis of Active Site Residues in MacB-TE

MacB-TE contains a cysteine nucleophile (Cys89) instead of serine, which is typically found in NRPS-TEs. Interestingly, despite harboring a Cys nucleophile, MacB-TE clustered phylogenetically with the serine-type peptide cyclases (Figure S12). This raised the possibility that Cys89 might be functionally replaceable with Ser, especially given that certain serine-type TEs such as McyC-TE and Pik-TE gain activity or alter substrate specificity upon Ser-to-Cys substitution. , These observations prompted us to investigate whether the nucleophiles in MacB-TE could similarly tolerate replacement. To test this hypothesis, we generated C89S and C89A mutants and examined their catalytic activities. C89A completely abolished the activity, whereas C89S predominantly produced the hydrolyzed product 3 with no detectable cyclic product (Figures A and S13). Minor Tris-derived adducts were also observed (Figure S13), indicating that Ser89 could form an acyl-enzyme intermediate but could not promote macrocyclization. Together, these results demonstrate that the thiol nucleophile is indispensable for ring formation in MacB-TE.

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Site-directed mutagenesis and structural modeling of MacB-TEs. (A) HPLC analysis of wild-type and mutant MacB-TEs using substrate 1. MLP-SNAC 1 (150 μM) was incubated with MacB-TE or the mutants (5 μM) in 25 mM Tris–HCl (pH 7.5) for 2 h at 24 °C. From top to bottom: substrate only, wild-type, C89S, C89A, H218A, D116A, D116N, and D116S. The right panels indicate HPLC-derived product distributions based on relative peak areas of substrate 1, cyclic product 2, and hydrolyzed product 3. No product formation was detected in the substrate-only control. Peaks marked with an asterisk (*) correspond to a buffer-derived Tris adduct, as confirmed by LC-MS analysis. Hyd (%) values marked with a hash symbol (#) include the peak area of the Tris adduct, which is indicated by an asterisk (*) in the chromatogram. (B, C) Structural models of the active site of wild-type MacB-TE (B) and the D116S mutant (C) predicted using AlphaFold2. The catalytic triad are shown in green, and hydrogen-bonding interactions are indicated by light-blue dashed lines.

Although sequence and structural analyses suggest the presence of a Cys–His–Asp catalytic triad, previous sequence analyses have reported naturally occurring substitutions of the catalytic acidic residue with Asn or Ser in some Cys-type TE sequences. Based on this observation, we examined whether MacB-TE requires a canonical acid–base architecture by assessing the contributions of His218 and Asp116. Both the H218A and D116A mutants showed reduced macrocyclization activity (Figure A), demonstrating that His218 and Asp116 play important roles in productive turnover.

We next examined whether MacB-TE could tolerate analogous replacements at this position. D116N retained only trace activity, whereas D116S retained substantial macrocyclization, indicating unexpected tolerance at this position, despite the loss of acidity. This result differs from that of classical serine-type TEs, where the acidic residue typically plays a key role in general-base catalysis.

To explore the structural features that may explain this tolerance, we constructed AlphaFold2 models of the wild-type and mutant MacB-TEs. In the wild-type model, the catalytic triad (Cys89–His218–Asp116) displayed the expected hydrogen-bonding arrangement (Figure B). The D116S model retained a similar hydrogen-bond network within the catalytic residues, suggesting that replacement of Asp116 with Ser does not substantially disrupt the overall active-site architecture (Figure C). In contrast, the D116N and D116A models did not maintain this hydrogen-bonding network, which is consistent with their reduced activity observed in the enzymatic assays (Figure S14).

Together, these results establish that MacB-TE strictly requires a cysteine nucleophile to promote macrocyclization, as serine substitution allows acylation but does not facilitate macrocyclization. In contrast, the acidic position showed unexpected tolerance, as the D116S mutant retained substantial macrocyclization activity even though the acidic functionality was replaced by serine. Although the mechanistic basis for the activity retained by D116S remains unclear, this tolerance suggests the catalytic flexibility of Cys-dependent peptide cyclases.

Substrate Tolerance of MacB-TE

After elucidating the catalytic contributions of the active site residues, we investigated the substrate tolerance of MacB-TE. Substrate promiscuity is a key requirement for chemoenzymatic diversification; therefore, we examined the ability of MacB-TE to process substrates bearing alternative electrophiles, side-chain substitutions, and varied lipid tails.

We first tested whether MacB-TE can use an ester electrophile instead of the native thioester. Although certain NRPS-TEs can process ester-linked substrates, thereby simplifying substrate preparation, , the compatibility of Cys-type TEs with such analogs remains unexplored. Incubation of MacB-TE with the ester analog MLP-ONAC 4, synthesized according to Schemes S3 and S4, resulted in no detectable turnover, indicating that a thioester linkage was required for the initial reaction (Figure A). The C89S mutant predominantly generated hydrolyzed product 3, with no detectable cyclic product, as observed for thioester substrate 1. These results are consistent with the mechanistic model depicted in Figure B, in which wild-type MacB-TE cannot form an acyl-enzyme intermediate from the ester substrate, whereas the C89S variant undergoes acylation but fails to promote the final intramolecular aminolysis, resulting in hydrolysis rather than macrocyclization.

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Substrate tolerance of MacB-TE. (A) HPLC traces of the reactions of MLP-ONAC and MacB-TE or C89S mutant. MLP-ONAC 4 (150 μM) was incubated with MacB-TE or C89S mutant (5 μM) in 25 mM Tris–HCl (pH 7.5) for 2 h at 24 °C. (B) Proposed enzymatic reactions of MLP-ONAC and MacB-TEs. (C) Structures of MLP-SNAC analogs 5–18. (D) HPLC traces of the reactions shown in (D). Substrates 1 and 5–18 (200 μM) were incubated with MacB-TE (10 μM) in 25 mM Tris–HCl (pH 7.0) for 2 h at 24 °C. Product distributions were quantified by analytical reversed-phase (RP)-HPLC based on relative peak areas, with values normalized to the total detected peak area.

To evaluate the contribution of individual side chains to the enzymatic macrocyclization, we conducted a systematic alanine scanning study. Nine linear peptide analogs (5–13), in which each residue, except the nucleophilic Dab4, was individually replaced with Ala, were synthesized and subjected to enzymatic analysis with MacB-TE (Figure C and Scheme S5). Under the standard assay conditions, which fully convert the native substrate within 30 min (Figure ), several alanine-substituted substrates were only partially consumed, indicating reduced reaction rates relative to the native substrate (Figure S15). Substitutions at residues located outside the macrocyclic region (e.g., Ala1 (5), Ala2 (6), and Ala3 (7)) retained relatively high conversion, whereas substitutions within the macrocyclic core (Ala5 (8), Ala6 (9), Ala8 (11), Ala9 (12), and Ala10 (13)) generally showed slower turnover. These observations suggest that residues within the macrocyclic portion contribute to productive cyclization, possibly through substrate conformational effects or enzyme–substrate interactions. Consistent with previous studies on NRPS-TEs, residues near the C-terminus appear to play an important role in substrate recognition. , Notably, complete substrate consumption could still be achieved by increasing the enzyme concentration (10 μM) and reaction time (2 h). Six analogs (Ala1 (5), Ala2 (6), Ala3 (7), Ala6 (9), and Ala7 (10), Ala8 (11)) underwent near-quantitative macrocyclization with no detectable hydrolysis (Figures D and S16–S24). In contrast, substitutions at Ala5 (8), Ala9 (12), and Ala10 (13), located near the Dab4–Leu10 ring-forming junction, yielded predominantly cyclized products (≥80% conversion) accompanied by minor hydrolysis. These results indicate that although side-chain substitutions influence catalytic efficiency, MacB-TE remains capable of producing predominantly cyclized products under appropriate reaction conditions.

Finally, we assessed the tolerance toward the N-terminal lipid moiety, which is a major determinant of antibacterial activity in polymyxin-class peptides. , We synthesized and evaluated substrates 14–18 bearing diverse lipid motifs: the native 6-S-methyloctanoyl group (6Me, 14) and a longer C10 decanoyl chain (C10, 15), both representing aliphatic fatty-acid variants; the 4-biphenyl group, previously reported to be the most potent lipid modification in the macolacin analog (4BP, 16); the 3-phenoxybenzoyl group, known to enhance activity in polymyxin derivatives (3PP, 17); and a new 3-biphenyl aromatic group (3BP, 18, Figure C). Remarkably, all substrates were quantitatively converted to their corresponding macrocyclic products in the presence of MacB-TE, indicating that the lipid moiety had negligible influence on MacB-TE catalysis (Figures D and S25–S29).

Taken together, our systematic substrate scope analysis demonstrated that MacB-TE tolerates extensive peptide side chain variations and diverse N-terminal lipid motifs. Coupled with exclusive macrocyclization, MacB-TE serves as a robust Cys-dependent cyclase for the chemoenzymatic synthesis of macolacin analogs.

Conclusion

In this study, we characterized MacB-TE, a rare Cys-dependent macrocyclase that catalyzes rapid, regioselective, and exclusive ring formation. Mutational analysis demonstrated that Cys89 is indispensable for macrocyclization, whereas His218 and Asp116 support an efficient turnover. Notably, the D116S variant retained substantial activity, indicating that MacB-TE tolerates substitution at the acidic position of the catalytic triad. These results demonstrate the unexpected catalytic flexibility of Cys-dependent peptide cyclases.

MacB-TE also displayed extensive substrate tolerance, readily macrocyclizing a broad panel of alanine-substituted peptides and accepting diverse lipid-modified substrates, indicating that both peptide side chains and lipid variations were well accommodated. Intriguingly, the intrinsic ring-closure fidelity of NRPS-TEs indicates that chemoenzymatic assays using NRPS-TEs may offer a practical experimental avenue for validating native ring-closure positions in bioinformatically predicted nonribosomal peptides. This approach provides an emerging methodology for structural elucidation of natural products. Overall, MacB-TE is a representative example that expands our mechanistic and functional understanding of Cys-dependent TEs as well as a versatile biocatalyst that provides a robust platform for the chemoenzymatic synthesis of macolacin analogs and related macrocyclic antibiotics.

Supplementary Material

cb6c00013_si_001.pdf (23.1MB, pdf)

Acknowledgments

This work was supported by the Uehara Memorial Foundation (S.K.). It was also supported in part by JSPS KAKENHI Grant Number JP23K14375 (S.K.), JP22H02562 and JP24H02094 (M. Mishima), the Takeda Science Foundation (S.K.), Astellas Foundation for Research on Metabolic Disorders (M. Mishima), and JST SPRING JPMJSP2134 (M. Morohashi). The NMR experiments were performed in part using NMR spectrometers with ultrahigh magnetic fields under the Collaborative Research Program of Institute for Protein Research, Osaka University (NMRCR-24-05 and NMRCR-25-05) and MEXT Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence Program (CURE) (JPMXP1323015488, Spin-L program No. spin25XP009). We thank Fukaya and Matsuura (Tokyo University of Pharmacy and Life Sciences) for assistance with MS and MS/MS analysis. We also thank Editage (www.editage.jp) for English language editing.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.6c00013.

  • Synthetic procedures for peptide substrates and analogs; expression, purification, and mutagenesis methods for MacB-TE; detailed enzymatic assay protocols; HPLC, LC-MS, MS/MS, and NMR data supporting experimental results; structural modeling; phylogenetic and computational analyses; and sequence and primer information; references cited in the Supporting Information are included in the main reference list − (PDF)

§.

M.M. and S.K. contributed equally to this work.

The authors declare no competing financial interest.

References

  1. Laxminarayan R., Duse A., Wattal C., Zaidi A. K. M., Wertheim H. F. L., Sumpradit N., Vlieghe E., Hara G. L., Gould I. M., Goossens H.. et al. Antibiotic resistancethe need for global solutions. Lancet Infect. Dis. 2013;13(12):1057–1098. doi: 10.1016/S1473-3099(13)70318-9. [DOI] [PubMed] [Google Scholar]
  2. Murray C. J. L., Ikuta K. S., Sharara F., Swetschinski L., Robles Aguilar G., Gray A., Han C., Bisignano C., Rao P., Wool E.. et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399(10325):629–655. doi: 10.1016/S0140-6736(21)02724-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ho C. S., Wong C. T. H., Aung T. T., Lakshminarayanan R., Mehta J. S., Rauz S., McNally A., Kintses B., Peacock S. J., de la Fuente-Nunez C.. et al. Antimicrobial resistance: a concise update. Lancet Microbe. 2025;6(1):100947. doi: 10.1016/j.lanmic.2024.07.010. [DOI] [PubMed] [Google Scholar]
  4. Poirel L., Jayol A., Nordmann P.. Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clin. Microbiol. Rev. 2017;30(2):557–596. doi: 10.1128/CMR.00064-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Liu Y.-Y., Wang Y., Walsh T. R., Yi L.-X., Zhang R., Spencer J., Doi Y., Tian G., Dong B., Huang X.. et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect. Dis. 2016;16(2):161–168. doi: 10.1016/S1473-3099(15)00424-7. [DOI] [PubMed] [Google Scholar]
  6. Hinchliffe P., Yang Q. E., Portal E., Young T., Li H., Tooke C. L., Carvalho M. J., Paterson N. G., Brem J., Niumsup P. R.. et al. Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1. Sci. Rep. 2017;7(1):39392. doi: 10.1038/srep39392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. He W., Huan X., Li Y., Deng Q., Chen T., Xiao W., Chen Y., Ma L., Liu N., Shang Z., Wang Z.. A broad-spectrum antibiotic targets multiple-drug-resistant bacteria with dual binding targets and no detectable resistance. Nat. Commun. 2025;16(1):7048. doi: 10.1038/s41467-025-62407-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chu J., Vila-Farres X., Brady S. F.. Bioactive Synthetic-Bioinformatic Natural Product Cyclic Peptides Inspired by Nonribosomal Peptide Synthetase Gene Clusters from the Human Microbiome. J. Am. Chem. Soc. 2019;141(40):15737–15741. doi: 10.1021/jacs.9b07317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chu J., Koirala B., Forelli N., Vila-Farres X., Ternei M. A., Ali T., Colosimo D. A., Brady S. F.. Synthetic-Bioinformatic Natural Product Antibiotics with Diverse Modes of Action. J. Am. Chem. Soc. 2020;142(33):14158–14168. doi: 10.1021/jacs.0c04376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Wang Z., Koirala B., Hernandez Y., Zimmerman M., Park S., Perlin D. S., Brady S. F.. A naturally inspired antibiotic to target multidrug-resistant pathogens. Nature. 2022;601(7894):606–611. doi: 10.1038/s41586-021-04264-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kohli R. M., Walsh C. T.. Enzymology of acyl chain macrocyclization in natural product biosynthesis. Chem. Commun. 2003;(3):297–307. doi: 10.1039/b208333g. [DOI] [PubMed] [Google Scholar]
  12. Nardini M., Dijkstra B. W.. α/β Hydrolase fold enzymes: the family keeps growing. Curr. Opin. Struct. Biol. 1999;9(6):732–737. doi: 10.1016/S0959-440X(99)00037-8. [DOI] [PubMed] [Google Scholar]
  13. Bruner S. D., Weber T., Kohli R. M., Schwarzer D., Marahiel M. A., Walsh C. T., Stubbs M. T.. Structural Basis for the Cyclization of the Lipopeptide Antibiotic Surfactin by the Thioesterase Domain SrfTE. Structure. 2002;10(3):301–310. doi: 10.1016/S0969-2126(02)00716-5. [DOI] [PubMed] [Google Scholar]
  14. Ding Y., Perez-Ortiz G., Butulan A.-G., Sharif H., Barry S. M.. Characterization of RufT Thioesterase Domain Reveals Insights into Rufomycin Cyclization and the Biosynthetic Origin of Rufomyazine. ACS Chem. Biol. 2025;20(3):573–580. doi: 10.1021/acschembio.4c00802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoyer K. M., Mahlert C., Marahiel M. A.. The Iterative Gramicidin S Thioesterase Catalyzes Peptide Ligation and Cyclization. Chem. Biol. 2007;14(1):13–22. doi: 10.1016/j.chembiol.2006.10.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kohli R. M., Walsh C. T., Burkart M. D.. Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature. 2002;418(6898):658–661. doi: 10.1038/nature00907. [DOI] [PubMed] [Google Scholar]
  17. Trauger J. W., Kohli R. M., Mootz H. D., Marahiel M. A., Walsh C. T.. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature. 2000;407(6801):215–218. doi: 10.1038/35025116. [DOI] [PubMed] [Google Scholar]
  18. Galea C. A., Roberts K. D., Zhu Y., Thompson P. E., Li J., Velkov T.. Functional Characterization of the Unique Terminal Thioesterase Domain from Polymyxin Synthetase. Biochemistry. 2017;56(4):657–668. doi: 10.1021/acs.biochem.6b01139. [DOI] [PubMed] [Google Scholar]
  19. Maruyama H., Yamada Y., Igarashi Y., Matsuda K., Wakimoto T.. Enzymatic peptide macrocyclization via indole-N-acylation. Chem. Sci. 2025;16(9):3872–3877. doi: 10.1039/D4SC07839J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Zhong W., Budimir Z. L., Johnson L. O., Parkinson E. I., Agarwal V.. Activity and Biocatalytic Potential of an Indolylamide Generating Thioesterase. Org. Lett. 2024;26(43):9378–9382. doi: 10.1021/acs.orglett.4c03648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Abdelkader E. H., Otting G.. NT*-HRV3CP: An optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage. J. Biotechnol. 2021;325:145–151. doi: 10.1016/j.jbiotec.2020.11.005. [DOI] [PubMed] [Google Scholar]
  22. Blanco-Canosa J. B., Nardone B., Albericio F., Dawson P. E.. Chemical Protein Synthesis Using a Second-Generation N-Acylurea Linker for the Preparation of Peptide-Thioester Precursors. J. Am. Chem. Soc. 2015;137(22):7197–7209. doi: 10.1021/jacs.5b03504. [DOI] [PubMed] [Google Scholar]
  23. Tseng C. C., Bruner S. D., Kohli R. M., Marahiel M. A., Walsh C. T., Sieber S. A.. Characterization of the Surfactin Synthetase C-Terminal Thioesterase Domain as a Cyclic Depsipeptide Synthase. Biochemistry. 2002;41(45):13350–13359. doi: 10.1021/bi026592a. [DOI] [PubMed] [Google Scholar]
  24. Grünewald J., Sieber S. A., Mahlert C., Linne U., Marahiel M. A.. Synthesis and Derivatization of Daptomycin: A Chemoenzymatic Route to Acidic Lipopeptide Antibiotics. J. Am. Chem. Soc. 2004;126(51):17025–17031. doi: 10.1021/ja045455t. [DOI] [PubMed] [Google Scholar]
  25. Koketsu K., Oguri H., Watanabe K., Oikawa H.. Enzymatic macrolactonization in the presence of DNA leading to triostin A analogs. Chem. Biol. 2008;15(8):818–828. doi: 10.1016/j.chembiol.2008.05.022. [DOI] [PubMed] [Google Scholar]
  26. Qiao L., Fang J., Zhu P., Huang H., Dang C., Pang J., Gao W., Qiu X., Huang L., Li Y.. A Novel Chemoenzymatic Approach to Produce Cilengitide Using the Thioesterase Domain from Microcystis aeruginosa Microcystin Synthetase C. Protein J. 2019;38(6):658–666. doi: 10.1007/s10930-019-09864-1. [DOI] [PubMed] [Google Scholar]
  27. Koch A. A., Hansen D. A., Shende V. V., Furan L. R., Houk K. N., Jiménez-Osés G., Sherman D. H.. A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst. J. Am. Chem. Soc. 2017;139(38):13456–13465. doi: 10.1021/jacs.7b06436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Žídek A., Potapenko A.. et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–589. doi: 10.1038/s41586-021-03819-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kobayashi M., Fujita K., Matsuda K., Wakimoto T.. Streamlined Chemoenzymatic Synthesis of Cyclic Peptides by Non-ribosomal Peptide Cyclases. J. Am. Chem. Soc. 2023;145(6):3270–3275. doi: 10.1021/jacs.2c11082. [DOI] [PubMed] [Google Scholar]
  30. Slingerland C. J., Martin N. I.. Recent Advances in the Development of Polymyxin Antibiotics: 2010–2023. ACS Infect. Dis. 2024;10(4):1056–1079. doi: 10.1021/acsinfecdis.3c00630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Gallardo-Godoy A., Muldoon C., Becker B., Elliott A. G., Lash L. H., Huang J. X., Butler M. S., Pelingon R., Kavanagh A. M., Ramu S.. et al. Activity and Predicted Nephrotoxicity of Synthetic Antibiotics Based on Polymyxin B. J. Med. Chem. 2016;59(3):1068–1077. doi: 10.1021/acs.jmedchem.5b01593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Meng E. C., Goddard T. D., Pettersen E. F., Couch G. S., Pearson Z. J., Morris J. H., Ferrin T. E.. UCSF ChimeraX: Tools for structure building and analysis. Protein Sci. 2023;32(11):e4792. doi: 10.1002/pro.4792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Trost B. M., Biannic B., Brindle C. S., O’Keefe B. M., Hunter T. J., Ngai M.-Y.. A Highly Convergent Total Synthesis of Leustroducsin B. J. Am. Chem. Soc. 2015;137(36):11594–11597. doi: 10.1021/jacs.5b07438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lee W., Rahimi M., Lee Y., Chiu A.. POKY: a software suite for multidimensional NMR and 3D structure calculation of biomolecules. Bioinformatics. 2021;37(18):3041–3042. doi: 10.1093/bioinformatics/btab180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Konno S., Tanaka M., Mizuguchi T., Toyokai H., Taguchi A., Taniguchi A., Hayashi Y.. Peptide mixed phosphonates for covalent complex formation with thioesterases in nonribosomal peptide synthetases. J. Pept. Sci. 2024;30:e3532. doi: 10.1002/psc.3532. [DOI] [PubMed] [Google Scholar]

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