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Structure–function and mechanistic analyses of nickel-dependent sulfonamide synthase

Nature Catalysis (2026)Cite this article

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Abstract

The sulfonamide group is an important functional group in biology and medicine that shows unique chemical properties and functions and is widely used in many marketed drugs. However, insertion of a sulfonamide group into a chemical scaffold is challenging and little is known about enzymatic sulfonamide biosynthesis. Here we show the structure, function and mechanism of the sulfonamide synthase SbzM, which is involved in the biosynthesis of altemicidin. Biochemical investigations established that SbzM strictly utilizes Ni2+ to convert L-cysteine into 2-sulfamoylacetic aldehyde via an initial decarboxylation followed by sulfur oxidation, representing a cysteine metabolism pathway distinct from those of canonical Fe2+-dependent cysteine dioxygenases. Further mechanistic studies, including site-directed mutagenesis, 18O-labelling, oxygen stoichiometry and computational studies, provided detailed insights into the SbzM-catalysed sulfonamide-formation reaction, which uses two dioxygen molecules and a Ni2+/Ni3+ redox cycle during catalysis. This study proposes a sulfonamide biosynthesis pathway with potential applications in biotechnology for sulfonamide synthesis through an environmentally benign process.

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Fig. 1: Enzyme reaction of the cupin superfamily sulfonamide synthase SbzM.
Fig. 2: Distribution of the cupin superfamily sulfonamide synthase SbzM.
Fig. 3: Structure of AmSbzM.
Fig. 4: Analysis of the products of the AmSbzM H107A variant.
Fig. 5: Mechanistic analysis of AmSbzM.
Fig. 6: Calculations on the proposed mechanism of SbzM.

Data availability

The data that support the conclusions of this study are provided in the main text, the Supplementary Information and the associated source files. Crystallographic data have been deposited in the Protein Data Bank (http://www.rcsb.org). The coordinates and structure factor amplitudes for AmSbzM are available under the PDB accession code 9UG7. Source data are provided with this paper.

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Acknowledgements

The synchrotron radiation experiments were performed at BL-1A of the Photon Factory. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI grant numbers 22H05123, 23H00393, 23K27332, 25K02420 and 25H01417), the New Energy and Industrial Technology Development Organization (NEDO, grant number JPNP20011), AMED (grant number JP21ak0101164), the PRESTO programme from the Japan Science and Technology Agency (JPMJPR20DA), the FOREST programme from the Japan Science and Technology Agency (JPMJFR226I and JPMJFR2301), the Kobayashi Foundation and the Senri Life Science Foundation. H.P.H.W. thanks the University of Manchester for a Postgraduate Research and Teaching Award and the JSPS for a travel grant (SP23018). The Computational Shared Facilities at the University of Manchester and the RIKEN FUGAKU are acknowledged for providing support and access to CPU and GPU time for this work. The Daiwa Anglo-Japanese Foundation is acknowledged for a collaborative grant between the University of Manchester, the University of Tokyo and RIKEN. The Turing Scheme and the Department of Chemical Engineering at the University of Manchester are acknowledged for travel bursaries to H.P.H.W.

Author information

Author notes

  1. These authors contributed equally: Yuhao Zhu, Takahiro Mori, Henrik P. H. Wong.

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Yuhao Zhu, Takahiro Mori & Ikuro Abe

  2. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan

    Takahiro Mori & Ikuro Abe

  3. FOREST, Japan Science and Technology Agency, Saitama, Japan

    Takahiro Mori

  4. Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK

    Henrik P. H. Wong & Sam P. de Visser

  5. Department of Chemical Engineering, The University of Manchester, Manchester, UK

    Henrik P. H. Wong & Sam P. de Visser

  6. RIKEN Center for Sustainable Resource Science, Wako, Saitama, Japan

    Takayoshi Awakawa

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Contributions

T.M., I.A. and S.P.d.V. designed the experiments and simulations. Y.Z. and T.M. performed in vitro analysis and crystallization experiments. Y.Z. and H.P.H.W. performed docking studies. H.P.H.W. ran the MD and quantum mechanics calculations. Y.Z., T.M., H.P.H.W., T.A., S.P.d.V. and I.A. analysed the data. Y.Z., T.M., H.P.H.W, S.P.d.V. and I.A. wrote the paper.

Corresponding authors

Correspondence to Takahiro Mori, Sam P. de Visser or Ikuro Abe.

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The authors declare no competing interests.

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Nature Catalysis thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Metal dependencies of SbzM family enzyme reactions.

a, HPLC detection of Dns-L-Cys and Dns-2 produced by SsSbzM, AmSbzM, and SpSbzM. b, LC-MS detection of the reaction products of SsSbzM, AmSbzM, and SpSbzM derivatized with 2,4-dinitrophenylhydrazine (DNPH). c,d Metal dependencies of c, SsSbzM and d, SpSbzM.

Extended Data Fig. 2 ICPMS for wild-type AmSbzM and its variants.

a, Ni and Fe atom amounts detected with or without the incubation of Ni or Fe. b, Ni and Zn atom amounts detected with or without the incubation of 20 equivalents of Ni2+. All measurements were performed in triplicate. Data are presented as mean values, and error bars indicate standard deviations (SD).

Extended Data Fig. 3 ITC assays for wild-type AmSbzM and different metals.

ITC assays for a, NiCl2. b, CaCl2. c, MnCl2, d, FeCl3, e, CoCl2, f, MgCl2. g, CuCl2, h, ZnCl2. i, Interaction parameters between AmSbzM and Ni. All metal solutions were fully soluble, and no precipitation was observed during the ITC measurements.

Extended Data Fig. 4 XAS analysis of Ni and Zn in AmSbzM.

a, Ni signal detected from resting state AmSbzM. b, Ni signal detected from EDTA-Ni2+ standard. c, Ni signal detected from AmSbzM incubated with L-Cys. d, Ni signal detected from TBA-Ni3+ standard. e, Zn signal detected from AmSbzM. f, Zn signal detected from standard compound ZnS.

Extended Data Fig. 5 Active site architecture of AmSbzM and its mutagenesis experiments.

a, b, Comparison of the active site structures of a, docking model of AmSbzM with L-Cys and b, CDO from Homo sapiens (PDB: 2IC1). c, Relative activities of AmSbzM wild-type and its variants. d, Comparison of L-Cys consumption rates and 1 formation rates by wild-type AmSbzM, and its H107A and H107F variants. Gray and red bars indicate the relative peak area of remaining L-Cys and the produced 1, respectively. e,f, Michaelis Menten plot of e, wild-type reaction and f, H107A reaction. g. Kinetic values of wild-type AmSbzM and the H107A variant. All mutant proteins were expressed and purified in soluble form, as confirmed by SDS-PAGE (Supplementary Fig. 45). All reactions were performed in triplicate. Data are presented as mean values, and error bars indicate standard deviations (SD).

Source data

Extended Data Fig. 6 Stoichiometric analysis of oxygen molecule in the reactions.

a, HPLC profiles of the wild-type reaction under varying oxygen concentrations. b, HPLC profiles of the H107A variant reaction under varying oxygen concentrations. c, O2 stoichiometry curves for the AmSbzM wild-type and H107A reactions. d, Scheme for the O2 consumption in the reactions catalyzed by wild-type AmSbzM and the H107A variant. All reactions were performed in triplicate. Data are presented as mean values, and error bars indicate standard deviations (SD).

Source data

Extended Data Fig. 7 Enzyme reaction of AmSbzM H107A variant with L-Cys-3,3-D2.

a, Detection of DNPH-3 in the H107A reaction with L-Cys and L-Cys-3,3-D2. b, Scheme for the reaction of AmSbzM H107A with L-Cys-3,3-D2.

Extended Data Fig. 8 Proposed reaction mechanism of SbzM.

Schematic representation of the proposed mechanism for the SbzM-catalyzed 1-forming reaction.

Supplementary information

Supplementary Information

Supplementary Methods, Tables 1–5, Figs. 1–7 and references.

Reporting Summary

Supplementary Data 1

Lists the sequences used in the SSN network analysis.

Supplementary Data 2

Initial structure in MD simulation.

Supplementary Data 3

Final structure in MD simulation.

Supplementary Data 4

Atomic coordinates of the optimized computational models.

Supplementary Data 5

Validation report of SbzM structure.

Supplementary Data 6

Uncropped scans of gels.

Supplementary Data 7

Primer sequences.

Source data

Source Data Extended Data Figs. 5 and 6

Statistical source data and kinetic source data.

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Zhu, Y., Mori, T., Wong, H.P.H. et al. Structure–function and mechanistic analyses of nickel-dependent sulfonamide synthase. Nat Catal (2026). https://doi.org/10.1038/s41929-026-01493-z

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