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Enzymatic synthesis of organoselenium compounds via C‒Se bond formation mediated by sulfur carrier proteins

Nature Synthesis volume 3, pages 477–487 (2024)Cite this article

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Abstract

Organoselenium compounds are rare in nature but play important physiological roles by exploiting the distinct features of selenium. However, the ability to explore these compounds and their implications has been hindered by the limited availability of (bio)synthetic tools for the generation of organoselenium molecules, particularly the lack of enzymatic strategies for C‒Se bond formation. Here we develop an enzymatic approach for C‒Se bond formation using sulfur carrier proteins to biosynthesize the isologous selenium counterparts of cysteine, thiamine and a chuangxinmycin derivative. Our results indicate that widespread sulfur-carrier-protein-based biosynthetic systems provide promiscuous and programmable machinery for the production of unnatural Se-containing compounds. We anticipate that the ‘element engineering’ strategy used in this study will provide new opportunities to develop biologically rare molecules or abiological-element-containing chemicals not found in nature.

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Fig. 1: Representative selenium- and sulfur-containing natural products and the SCP-based selenium incorporation system inspired by nature.
Fig. 2: Selenylation of the SCPs.
Fig. 3: Biosynthetic pathways of the organosulfur compounds and their selenium analogues.
Fig. 4: HPLC and HPLC-HRMS analyses of selenium incorporation reactions.
Fig. 5: Recombination of the SCP-based selenium incorporation systems.

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Data availability

Experimental data supporting the conclusions of this study are available within the article and its Supplementary information. Protein sequences are retrieved from the NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/) with the accession numbers in Supplementary Table 1.

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Acknowledgements

We thank J. Qu, G. Lin, J. Zhu, Z. Li and H. Sui from the State Key Laboratory of Microbial Technology at Shandong University for their guidance and help in HPLC-MS and NMR spectroscopy analyses. This work was supported by National Key Research and Development Program of China (2022YFC2804500 to X.Z.), National Natural Science Foundation of China (32025001 to S.L., 22237004 to S.L., 32000039 to X.Z.) and Shandong Provincial Natural Science Foundation (ZR2023ZD50 to X.Z., ZR2019ZD20 to S.L.).

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Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    Xingwang Zhang, Fangyuan Cheng, Jiawei Guo, Shanmin Zheng, Xuan Wang & Shengying Li

  2. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China

    Shengying Li

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Contributions

S.L. designed this study and analysed the results. X.Z. synthesized the substrates, performed the bioassays and analysed the results. F.C., J.G., S.Z. and X.W. cloned the genes, constructed the protein expression vectors and purified the proteins. X.Z. and S.L. wrote and revised the paper.

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Correspondence to Shengying Li.

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Nature Synthesis thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Thomas West, in collaboration with the Nature Synthesis team.

Extended data

Extended Data Fig. 1 Representative sulfur-containing natural products biosynthesized by SCP-involved pathways.

Additional organosulfur structures to Fig. 1.

Extended Data Fig. 2 Enzymatic mechanism of the sulfur incorporation process in cysteine biosynthesis.

Key nucleophilic attack reactions are indicated by orange arrows. The sulfur source for CysO sulfuration can be either HS− (path i) or S2O32− (path ii).

Extended Data Fig. 3 Enzymatic mechanism of the sulfur incorporation process in thiamin biosynthesis.

Key nucleophilic attack reactions are indicated by orange arrows. The sulfur source for ThiS sulfuration can be either HS− (path i) or L-Cys (path ii).

Extended Data Fig. 4 Enzymatic mechanism of the sulfur incorporation process in chuangxinmycin biosynthesis.

Key nucleophilic attack reactions are indicated by orange arrows. The sulfur source for Cxm4* sulfuration can be HS− (path i), L-Cys (path ii) or S2O32− (path iii).

Extended Data Fig. 5 Sulfuration of the SCPs.

a, Schematic ATP-dependent sulfuration of CysO, ThiS, and Cxm4G (the matured form of Cxm4) by MoeZ, ThiF, and CxmM, respectively, with NaSH as the sulfur source. b, Deconvoluted HRESI-MS analysis of the sulfuration efficiency of CysO, ThiS, and Cxm4* under different pH conditions.

Extended Data Fig. 6 HRESI-MS/HPLC analysis of the recombined SCP-based selenium incorporation systems.

a, Deconvoluted HRESI-MS analyses of the selenylation efficiency of CysO (i), ThiS (ii) and Cxm4* (iii) by different activating enzymes. b, HPLC (i and iii) and HPLC-MS (ii) analysis of Se-Cys (i), Se-Thz (ii), and Se-Trp (iii) using different selenylated SCPs (that is, SCP-COSe-) as selenium donors. c, HPLC analysis of Se-Cys (i) and Se-Trp (ii) produced in the reprogrammed pathways.

Extended Data Fig. 7 Recombination in the SCP-based sulfur incorporation systems.

a, Recombination network of SCPs against different activating enzymes MoeZ, ThiF, and CxmM (i) and sulfurtransferases CysM, ThiG, and Cxm3 (ii). The left boxed histograms show the conversion ratios of SCPs (i). The right boxed histograms show the yields of organosulfur products in one-pot reactions (ii) of the three recombined pathways (the yield of Thz was not determined due to unavailability of authentic standard. ‘Positive’ indicates the product was detectable by LC-MS). The colour of each column represents the corresponding same coloured activating enzyme (i) or SCP (ii) supported reaction. b, Deconvoluted HRESI-MS analyses of the selenylation efficiency of CysO (i), ThiS (ii) and Cxm4* (iii) by different activating enzymes. c, HPLC (i and iii) and HPLC-MS (ii) analysis of the sulfur-transfer reactions of Cys (i), Thz (ii) and S-Trp (iii) by different sulfurtransferases.

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Supplementary Methods, Tables 1–3, Schemes 1–5, Figs. 1–41 and References.

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Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

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Zhang, X., Cheng, F., Guo, J. et al. Enzymatic synthesis of organoselenium compounds via C‒Se bond formation mediated by sulfur carrier proteins. Nat. Synth 3, 477–487 (2024). https://doi.org/10.1038/s44160-023-00477-2

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