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Cited by 5Microbial Natural Products and BiosynthesisEnzyme Structure and FunctionBiochemical and Structural Characterization

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Structure and function of the geldanamycin amide synthase from Streptomyces hygroscopicus

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Ewert, Wiebke, Bartens, Christian, Ongouta, Jekaterina, Holmes, Monika, Heutling, Anja, Kishore, Anusha, Urbansky, Tim, Zeilinger, Carsten, Preller, Matthias, Kirschning, Andreas·2025·Nature Communications
DOI 10.1038/s41467-025-57013-3PMC PMC11903869

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Abstract

Amide synthases catalyze the formation of macrolactam rings from aniline-containing polyketide-derived seco-acids as found in the important class of ansamycin antibiotics. One of these amide synthases is the geldanamycin amide synthase GdmF, which we recombinantly expressed, purified and studied in detail both functionally as well as structurally. Here we show that purified GdmF catalyzes the amide formation using synthetically derived substrates. The atomic structures of the ligand-free enzyme and in complex with simplified substrates reveal distinct structural features of the substrate binding site and a putative role of the flexible interdomain region for the catalysis reaction.

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Figures

Fig. 1 Geldanamycin biosynthesis and modes of amide formation. A Chemical transformation catalyzed by the amide synthase ShGdmF; (B) by the arylamine N-acetyltransferase and (C) in vivo macrolactamization of SNAC ester of ansamitocin seco-acid derivative 8 to ansamitocin P3 derivative 9.
Fig. 1 Geldanamycin biosynthesis and modes of amide formation. A Chemical transformation catalyzed by the amide synthase ShGdmF; (B) by the arylamine N-acetyltransferase and (C) in vivo macrolactamization of SNAC ester of ansamitocin seco-acid derivative 8 to ansamitocin P3 derivative 9.CC BY · PubMed Central
Fig. 2 Structural and mechanistic details of GdmF. a–d Crystal structure of ligand-free enzyme GdmF. e Macrolactam formation of progeldanamycin 2 from activated seco-progeldanamycin 1 by GdmF which corresponds to mechanistic analogy to amide formation established for NATs. a Overview of the ligand-free GdmF crystal structure at 1.4 Å resolution: α-helical bundle (magenta), β-barrel (green), interdomain region (blue), and α/β-lid (orange). Thirteen amino acids located in the interdomain region (dashed line) are not resolved in the crystal structure. b Close-up view of the active site. The catalytic triad and its surrounding hydrophobic residues are shown in stick representation. c Structural differences of the active site residues between GdmF (colored) and MlNAT1 (light gray). d Surface representation of GdmF, showing the active site cleft, and colored according to the electrostatic potential. Color code: red: negative, blue: positive, white, neutral.
Fig. 2 Structural and mechanistic details of GdmF. a–d Crystal structure of ligand-free enzyme GdmF. e Macrolactam formation of progeldanamycin 2 from activated seco-progeldanamycin 1 by GdmF which corresponds to mechanistic analogy to amide formation established for NATs. a Overview of the ligand-free GdmF crystal structure at 1.4 Å resolution: α-helical bundle (magenta), β-barrel (green), interdomain region (blue), and α/β-lid (orange). Thirteen amino acids located in the interdomain region (dashed line) are not resolved in the crystal structure. b Close-up view of the active site. The catalytic triad and its surrounding hydrophobic residues are shown in stick representation. c Structural differences of the active site residues between GdmF (colored) and MlNAT1 (light gray). d Surface representation of GdmF, showing the active site cleft, and colored according to the electrostatic potential. Color code: red: negative, blue: positive, white, neutral.CC BY · PubMed Central
Fig. 3 Syntheses of A. A SNAC esters 11a-c and (B) pantetheine esters 13a-c (further details see supporting information).
Fig. 3 Syntheses of A. A SNAC esters 11a-c and (B) pantetheine esters 13a-c (further details see supporting information).CC BY · PubMed Central
Fig. 4 Syntheses of seco-progeldanamycin derivatives 27b and 29 (further details see supporting information).
Fig. 4 Syntheses of seco-progeldanamycin derivatives 27b and 29 (further details see supporting information).CC BY · PubMed Central
Fig. 5 GdmF-mediated and chemical macrolactamizations of seco-geldanamycin derivatives 27b and 29.
Fig. 5 GdmF-mediated and chemical macrolactamizations of seco-geldanamycin derivatives 27b and 29.CC BY · PubMed Central
Fig. 6 Structurally simplified anilines 32 and 33 and acetyl-CoA 5 used in this study. Bottom: (a) Crystal structure of GdmF with bound pantetheine, and (b) N-acetylcysteamine (SNAC) in the active site cleft (upper panels). The substrates are bound primarily through hydrophobic interactions with aliphatic and aromatic protein residues and a few hydrogen bonds (dashed lines; lower panels). c Steady-state enzymatic assay monitoring amide formation from thioesters 11a-c and 13a-c, and amino 3-aminophenol 32, and (d) 3-amino-5-methylphenol 33 (data are represented as mean ± SD. n = 3 biologically independent replicates). Source data are provided as a Source Data file.
Fig. 6 Structurally simplified anilines 32 and 33 and acetyl-CoA 5 used in this study. Bottom: (a) Crystal structure of GdmF with bound pantetheine, and (b) N-acetylcysteamine (SNAC) in the active site cleft (upper panels). The substrates are bound primarily through hydrophobic interactions with aliphatic and aromatic protein residues and a few hydrogen bonds (dashed lines; lower panels). c Steady-state enzymatic assay monitoring amide formation from thioesters 11a-c and 13a-c, and amino 3-aminophenol 32, and (d) 3-amino-5-methylphenol 33 (data are represented as mean ± SD. n = 3 biologically independent replicates). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 7 Computational all-atom molecular dynamics simulations. a The interdomain region moves 15 Å toward the β-barrel during the 150 ns MD simulations, closing the large active site cleft. Blue arrows show the motion vectors from the initial position (gray) to the equilibrium structure (colored). b The interdomain region (blue) interacts with the β-barrel (green) via a salt bridge between Asp194 and Arg109 and hydrogen bonds between Gln201 and Gln100. c The covalently bound seco-progeldanamycin 1 (line representation) adopts a bent conformation during MD simulations, leading to the formation of a structured α-helical interdomain region (blue). d During MD simulations, the aminophenol moiety of covalently bound seco-progeldanamycin 1 adopts a bent conformation located near the thioester carbon atom. e Free energy landscape of ligand-free GdmF, computed as potential of mean force (PMF) according to the distance between the center of mass of the intermediate loop and cysteine 73 in the active site, and the root mean square deviation (RMSD) of the intermediate loop from the initially modeled loop conformation, derived from two combined 500 ns enhances sampling molecular dynamics simulations. The color bar represents the PMF value in kcal/mol. f Free energy landscape of the intermediate loop in Gdmf in the presence of seco-progeldanamycin. The conformational space is much narrower and shifted toward a more closed substate. The color bar represents the PMF value in kcal/mol. Source data are provided as a Source Data file.
Fig. 7 Computational all-atom molecular dynamics simulations. a The interdomain region moves 15 Å toward the β-barrel during the 150 ns MD simulations, closing the large active site cleft. Blue arrows show the motion vectors from the initial position (gray) to the equilibrium structure (colored). b The interdomain region (blue) interacts with the β-barrel (green) via a salt bridge between Asp194 and Arg109 and hydrogen bonds between Gln201 and Gln100. c The covalently bound seco-progeldanamycin 1 (line representation) adopts a bent conformation during MD simulations, leading to the formation of a structured α-helical interdomain region (blue). d During MD simulations, the aminophenol moiety of covalently bound seco-progeldanamycin 1 adopts a bent conformation located near the thioester carbon atom. e Free energy landscape of ligand-free GdmF, computed as potential of mean force (PMF) according to the distance between the center of mass of the intermediate loop and cysteine 73 in the active site, and the root mean square deviation (RMSD) of the intermediate loop from the initially modeled loop conformation, derived from two combined 500 ns enhances sampling molecular dynamics simulations. The color bar represents the PMF value in kcal/mol. f Free energy landscape of the intermediate loop in Gdmf in the presence of seco-progeldanamycin. The conformational space is much narrower and shifted toward a more closed substate. The color bar represents the PMF value in kcal/mol. Source data are provided as a Source Data file.CC BY · PubMed Central

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Metadata

Authors
Ewert, W., Bartens, C., Ongouta, J., Holmes, M., Heutling, A., Kishore, A., Urbansky, T., Zeilinger, C., Preller, M., & Kirschning, A.
Affiliations
  • Medizinische Hochschule Hannover (DE)
  • Leibniz University Hannover (DE)
  • Hochschule Bonn-Rhein-Sieg (DE)
Corresponding author
  • Zeilinger, Carsten
  • Preller, Matthias
  • Kirschning, Andreas
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-03-12
Volume
16
Issue
1
Pages
2464-2464
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-57013-3
PMC
PMC11903869
OpenAlex
W4408348578
Cited by
5
References
1
Citations by year
2026: 3 · 2025: 2
FWCI
2.83
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Publication Server of Bonn-Rhein-Sieg University of Applied Sciences (Bonn-Rhein-Sieg University of Applied Sciences) (publishedVersion, cc-by)
  • Uppsala University Publications (Uppsala University) (submittedVersion, cc-by)
  • Institutional Repository of Leibniz Universität Hannover (Leibniz Universität Hannover) (submittedVersion, cc-by-nc-nd)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Leibniz Universität Hannover (cc-by)
Funders
Deutsche Forschungsgemeinschaft (Ki397/13-1)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
Streptomyces hygroscopicus, ansamycin antibiotics, amide synthesis, enzyme structure, substrate binding site, interdomain flexibility, enzyme catalysis
MeSH terms
Bacterial Proteins, Binding Sites, Models, Molecular, Recombinant Proteins, Streptomyces, Substrate Specificity, Benzoquinones, Crystallography, X-Ray, Amide Synthases, Lactams, Macrocyclic

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