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Fused radical SAM and αKG-HExxH domain proteins contain a distinct structural fold and catalyse cyclophane formation and β-hydroxylation

Nature Chemistry volume 16, pages 1882–1893 (2024)Cite this article

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Abstract

Two of nature’s recurring binding motifs in metalloproteins are the CxxxCxxC motif in radical SAM enzymes and the 2-His-1-carboxylate motif found both in zincins and α-ketoglutarate and non-haem iron enzymes. Here we show the confluence of these two domains in a single post-translational modifying enzyme containing an N-terminal radical S-adenosylmethionine domain fused to a C-terminal 2-His-1-carboxylate (HExxH) domain. The radical SAM domain catalyses three-residue cyclophane formation and is the signature modification of triceptides, a class of ribosomally synthesized and post-translationally modified peptides. The HExxH domain is a defining feature of zinc metalloproteases. Yet the HExxH motif-containing domain studied here catalyses β-hydroxylation and is an α-ketoglutarate non-haem iron enzyme. We determined the crystal structure for this HExxH protein at 2.8 Å, unveiling a distinct structural fold, thus expanding the family of α-ketoglutarate non-haem iron enzymes with a class that we propose to name αKG-HExxH. αKG-HExxH proteins represent a unique family of ribosomally synthesized and post-translationally modified peptide modifying enzymes that can furnish opportunities for genome mining, synthetic biology and enzymology.

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Fig. 1: Structure and mechanism of a zincin metallopeptidase and an αKG/Fe-dependent oxygenase.
Fig. 2: Fused rSAM αKG-HExxH proteins involved in the biosynthesis of triceptides.
Fig. 3: Functional validation of MscBH and SjiBH, FxsBH family rSAM αKG-HExxH proteins.
Fig. 4: Functional validation of ChlBH.
Fig. 5: In vitro assays with MscH, SjiH and ChlH.
Fig. 6: Crystal structure of ChlH and proposed active site.

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Data availability

The detailed procedures required to duplicate this work are available in Supplementary Information along with full LC–MS and NMR spectra where appropriate. The ChlH atomic coordinates and structure factors are deposited at the Protein Data Bank with ID code 8PP1. Any additional data or unique materials (through a materials transfer agreement) are available from the corresponding authors on reasonable request.

Code availability

The R script used for analysis of αKG-HExxH domain proteins is available at https://github.com/SuzeMa/2022_modHExxH

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program (2018Y F A0900402) to Q.Z., the Innovative research team of high-level local universities in Shanghai and a key laboratory programme of the Education Commission of Shanghai Municipality (ZDSYS14005) to Q.Z., the West Light Foundation of The Chinese Academy of Sciences (xbzg-zdsys-202105) to Q.Z., The National Natural Science Foundation of China (21921003 and U22A20451 to Q.Z. and 223B2701 to S.M.), The Japan Society for Promotion of Science to Y.M. and R.S., The Naito Foundation to Y.M., The Human Frontier Science Program to R.S. and the Ministry of Education Singapore (A-0004623-00-00, A-0008495-00-00 and A-8000449-00-00) to B.I.M. E.D.L.M. and P.A. thank J.C. Fontecilla-Camps for helpful discussions. P.A. appreciates the help from the staff of the computing facility provided by the Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA/DRF/GIPSI), Saclay and CCRT, Bruyères-le-Châtel. Part of this work was supported by the French National Research Agency (ANR-20-CE44-0005) and used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, which is supported by FRISBI (ANR-10-INBS-05-02) and GRAL and financed within the Université Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The authors thank S. Matsunaga (The University of Tokyo) for providing theonellamides as a standard of l-erythro β-OH-Asp. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we would like to thank Christoph Mueller-Dieckmann and Philippe Carpentier for assistance in using beamline ID30B.

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Author notes

  1. Yohei Morishita

    Present address: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

  2. Ryosuke Sugiyama

    Present address: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

  3. These authors contributed equally: Yohei Morishita, Suze Ma, Eugenio De La Mora.

Authors and Affiliations

  1. Department of Pharmacy, National University of Singapore, Singapore, Singapore

    Yohei Morishita, Ryosuke Sugiyama, Yi Wei Tooh, Gregory Gunawan & Brandon I. Morinaka

  2. Department of Chemistry, Fudan University, Shanghai, China

    Suze Ma, He Li, Heng Chen, Xinjian Ji & Qi Zhang

  3. University Grenoble Alpes, CEA, CNRS, IBS, Metalloproteins Unit, Grenoble, France

    Eugenio De La Mora, Anthony Usclat, Patricia Amara & Yvain Nicolet

  4. University Grenoble Alpes, CNRS, CEA, IRIG, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France

    Julien Pérard

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Contributions

B.I.M, Q.Z. and Y.N. designed the research. Y.M., Y.W.T., R.S. and G.G. performed functional and biochemical studies for MscBH, ChlBH and SamBH. S.M., H.L., H.C. and X.J. performed the functional and biochemical studies for SjiBH and SgaBH. E.D.L.M. and A.U. performed crystallography studies for ChlH. P.A. performed in silico modelling. Y.M., S.M., E.D.L.M., Y.N., Q.Z. and B.I.M. wrote the manuscript. All authors analysed the data, discussed the results and commented on the manuscript.

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Correspondence to Yvain Nicolet, Qi Zhang or Brandon I. Morinaka.

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Nature Chemistry thanks Gong-Li Tang and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The hidden sequence space of αKG-HExxH family in Nature.

a, Summary of the protein families that are fused to αKG-HExxH family protein, including standalone αKG-HExxH proteins, and αKG-HExxH proteins that are fused with an rSAM, tetratricopeptide repeat (TPR) or other protein families. b, Unrooted maximum-likelihood phylogenetic tree of the αKG-HExxH domains excised from their native protein sequence. The phylum information and the presence (+) or absence (−) of the HExxH and PWRxxxRP motif are annotated on the tree.

Supplementary information

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Materials and equipment, and Supplementary methods, Figs. 1–63 and Tables 1–10.

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Morishita, Y., Ma, S., De La Mora, E. et al. Fused radical SAM and αKG-HExxH domain proteins contain a distinct structural fold and catalyse cyclophane formation and β-hydroxylation. Nat. Chem. 16, 1882–1893 (2024). https://doi.org/10.1038/s41557-024-01596-9

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