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LimF is a versatile prenyltransferase for histidine-C-geranylation on diverse non-natural substrates

Nature Catalysis volume 5, pages 682–693 (2022)Cite this article

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Abstract

Prenylation plays an important role in diversifying the structure and function of secondary metabolites. Although several cyanobactin prenyltransferases have been characterized, their chemistries are mainly limited to the modification of electron-rich heteroatoms. Here we report a prenyltransferase, LimF, from Limnothrix sp. CACIAM 69d, geranylating the electron-deficient C2 atom of His imidazole. Interestingly, in addition to its native substrate, LimF also modifies diverse exotic peptides, including thioether-closed macrocycles. We have also serendipitously uncovered Tyr-O-geranylating activity as the secondary function of LimF, providing evolutional insight into the divergent repertoire of prenylated peptides produced by cyanobactin PTases. Crystallographic analysis of LimF complexed with a pentapeptide substrate and a prenyl donor analogue provides the structural basis for its His recognition and its bifunctionality. We also show the prenylation ability of LimF on various bioactive molecules containing an imidazole group, including non-amino acid small molecules, highlighting its potential as a versatile biocatalyst for chemically challenging imidazole C-geranylation.

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Fig. 1: A putative cyanobactin BGC from Limnothrix sp. CACIAM 69d including LimF.
Fig. 2: In vitro LimF reaction confirms the His-C2-forward geranylation in limnothamide.
Fig. 3: Substrate probing by FIT-LimF system validates the broad substrate tolerance of LimF.
Fig. 4: Tyr-O-geranylation occurs as the secondary prenylation mode of LimF.
Fig. 5: Crystallographic characterization of the LimF–GSPP–peptide complex.
Fig. 6: Selective C-geranylation of imidazole-containing bioactive molecules.

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Data availability

SSN analysis results, relevant chromatograms for all the LC–MS and LC–MS/MS experiments, spectra for all the NMR experiments, plots for kinetic analyses of LimF-catalysed geranylation reactions, multiple sequence alignment of LimF and its homologous PTases, accessions for the genes encoded in the lim BGC, data collection and refinement statics for the crystal analysis, primer sequences and DNA template assembly schemes are available in the Supplementary Information. The accession numbers used for bioinformatic analysis are listed in the Supplementary Information. The PDB accession numbers for reference protein structures are shown in Supplementary Fig. 31. The coordinates and structure factors of LimF–GSPP–peptide complex and the LimF–GSPP complex have been deposited in the Protein Data Bank (PDB 7VMW and 7VMY, respectively). Other results are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank H. Nagai (Tokyo University of Marine Science and Technology) and T. Nagashima (RIKEN Center for Biosystems Dynamic Research) for technical assistance with the NMR analysis. We thank all beamline staff at BL32XU (SPring-8) and BL1A (Photon Factory) for their technical support. We also thank A. Vinogradov for stimulating scientific discussions. This work was supported by KAKENHI (JP16H06444 to H.S. and Y.G.; JP17H04762, JP19H01014, JP19K22243, JP20H02866 to Y.G.; JP20H05618 to H.S.; JP21K06051 to K.H.; JP19H02842 and JP21K19056 to M.O.) from the Japan Society for the Promotion of Science, and by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japan Agency for Medical Research and Development (AMED) under grant number JP19am0101070 (support number 1698).

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  1. These authors contributed equally: Yuchen Zhang, Keisuke Hamada.

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  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Yuchen Zhang, Dinh Thanh Nguyen, Sumika Inoue, Masayuki Satake, Yuki Goto & Hiroaki Suga

  2. Department of Biochemistry, Graduate School of Medicine, Yokohama City University, Yokohama, Japan

    Keisuke Hamada, Shunsuke Kobayashi, Chikako Okada, Kazuhiro Ogata & Toru Sengoku

  3. Department of Material and Life Chemistry, Kanagawa University, Yokohama, Japan

    Masahiro Okada

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Contributions

Y.G. and H.S. conceived and supervised the study. Y.Z., K.H., D.T.N., S.I., M.S., M.O., T.S., Y.G. and H.S. designed the experiments. Y.Z., K.H., D.T.N., S.I., S.K. and C.O. prepared recombinant LimF and its mutants. Y.Z., S.I., M.O. and Y.G. performed the bioinformatic analyses. Y.Z., D.T.N., S.I. and Y.G. performed the in vitro LimF reactions and substrate tolerance study. Y.Z., S.I. and M.S. performed the NMR experiments. K.H., S.K., C.O., T.S. and Y.G. performed the crystallographic study. All authors analysed the experimental results. Y.Z., K.H., K.O., M.O., T.S., Y.G. and H.S. wrote the manuscript with input from all authors.

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Correspondence to Toru Sengoku, Yuki Goto or Hiroaki Suga.

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Competing interests

The following authors (H.S., Y.G., Y.Z. and M.O.) are co-inventors on patent application (JP 2021-170768) related to preparation of site-directed geranylated chemical entities with LimF. The other authors declare no competing interests.

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Zhang, Y., Hamada, K., Nguyen, D.T. et al. LimF is a versatile prenyltransferase for histidine-C-geranylation on diverse non-natural substrates. Nat Catal 5, 682–693 (2022). https://doi.org/10.1038/s41929-022-00822-2

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