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. 2025 Jun 6;27(22):5765-5770.
doi: 10.1021/acs.orglett.5c01561. Epub 2025 May 21.

Indolylamide Macrocyclization by a Streptococcus pneumoniae ThiF-like Enzyme Family Member

Anshul Rajput  1 , Keelie S Butler  1 , Daniel A Springer  1 , Jonathan R Chekan  1
Affiliations

Indolylamide Macrocyclization by a Streptococcus pneumoniae ThiF-like Enzyme Family Member

Anshul Rajput et al. Org Lett. .

Abstract

ThiF-like enzymes are present in diverse RiPP biosynthetic pathways and are known to catalyze reactions such as thiolactone and phosphoramidate bond formation. To uncover new chemical space for ThiF-like enzymes, we utilized a global genome mining approach and identified a minimal ind RiPP cluster in the human pathogen Streptococcus pneumoniae. In vitro characterization of IndF demonstrated the first indolylamide (Trp-Ile) linkage in a RiPP pathway and a new reaction type catalyzed by a ThiF-like enzyme.

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Figures

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(A) The ThiF-like enzyme GrcB catalyzes cross-link formation by utilizing ATP to generate an adenylated intermediate. (B) Sequence similarity network of ThiFs with RRE motifs. SSN was generated using an e-value of 90 and displayed as a 90% rep network. Documented ThiF-like enzyme RiPP reactions are indicated. (C) Ind biosynthetic gene cluster from S. pneumoniae. (D) Annotated IndA precursor peptide sequence. (E) UHPLC-HRMS (Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry) analysis of enzymatic assays. Extracted ion chromatograms for IndA alone (top trace), IndF activity assay (middle trace), and SeIndP proteolysis assay (bottom trace); (IndA [M+2H]+2: 1613.7319 m/z, IndA-Modified [M+2H]+2: 1604.7229 m/z, Leader [M+2H]+2: 1028.9822 m/z, Modified Core [M+2H]+2: 585.7591 m/z) using a mass tolerance of 20 ppm.
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(A) Comparison of 1H NMR spectra for linear (Top) and cyclized (Bottom) IndAN5-I9 demonstrates the disappearance of the indole-NH proton (Trp-NH) peak in the cyclized product. (B) The 1H–1H ROESY spectra for IndAN5–I9 demonstrates correlations between the indole-2 proton (Trp-H2) and the Trp-Hβ and Trp-Hα. (C) The 1H–1H ROESY spectra for IndA-CyclicN5–I9 shows correlations between the indole-2 proton (Trp-H2) and the Ile α-proton and Ile β-proton. (D) Selected key ROESY correlation for IndA-CyclicN5–I9.
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(A) AlphaFold 3 model of the dimeric IndA, IndF, ATP, and Mg2+ structure. (B) AlphaFold 3 model of the IndF active site with important residues labeled. (C) Extracted ion chromatograms for IndF mutant assays (IndA substrate [M+2H]+2: 1613.7319 m/z and IndA-Cyclic product [M+2H]+2: 1604.7229 m/z) using a mass tolerance of 20 ppm.
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