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Structural basis for non-radical catalysis by TsrM, a radical SAM methylase

Nature Chemical Biology volume 17, pages 485–491 (2021)Cite this article

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Abstract

Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of l-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5ʹ-deoxyadenosyl 5ʹ-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor, and a [4Fe–4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CXXXCXXC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating the formation of a C2 carbanion.

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Fig. 1: Structure of thiostrepton A and details of the TsrM reaction.
Fig. 2: The X-ray crystal structure of native KsTsrM with both cofactors (cobalamin and a [4Fe–4S] cluster) bound.
Fig. 3: The X-ray crystal structure of KsTsrM with bound Trp and aza-SAM along with both cofactors.
Fig. 4: Substrate-assisted catalysis by TsrM.

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Data availability

Atomic coordinates and structure factors for the reported crystal structures in this work have been deposited to the Protein Data Bank under accession numbers 6WTE (native KsTsrM) and 6WTF (aza-SAM- and Trp-bound KsTsrM). Source data are provided with this paper.

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Acknowledgements

This work was supported by the NIH (GM-122595 to S.J.B. and GM-126982 to C.L.D.) and the Eberly Family Distinguished Chair in Science (S.J.B.). S.J.B. and C.L.D. are investigators of the Howard Hughes Medical Institute. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Use of GM/CA@APS has been funded in whole or in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The Eiger 16M detector at GM/CA-XSD was funded by NIH grant S10 OD012289. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). This research also used the resources of the Berkeley Center for Structural Biology supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences grant P30 GM124169.

Author information

Author notes

  1. Percival Yang-Ting Chen

    Present address: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA

  2. Anthony J. Blaszczyk

    Present address: Catalent Pharma Solutions, Gaithersburg, MD, USA

  3. Erica L. Schwalm

    Present address: Merck & Co., Inc., Rahway, NJ, USA

Authors and Affiliations

  1. Department of Chemistry, Pennsylvania State University, University Park, PA, USA

    Hayley L. Knox, Arnab Mukherjee, Erica L. Schwalm, Bo Wang & Squire J. Booker

  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Percival Yang-Ting Chen & Catherine L. Drennan

  3. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA

    Anthony J. Blaszczyk & Squire J. Booker

  4. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Tyler L. Grove

  5. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Catherine L. Drennan

  6. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA

    Catherine L. Drennan

  7. Howard Hughes Medical Institute, Pennsylvania State University, University Park, PA, USA

    Squire J. Booker

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Contributions

H.L.K. and S.J.B. developed the research plan and experimental strategy. H.L.K. isolated and crystallized proteins, and H.L.K. and E.L.S. collected the crystallographic data. H.L.K. and A.J.B. performed biochemical experiments. H.L.K., P.Y.-T.C., T.L.G., E.L.S., S.J.B. and C.L.D. analyzed and interpreted the crystallographic data. B.W. synthesized substrates and substrate analogs. A.M. performed computational docking experiments. H.L.K., P.Y.-T.C., S.J.B. and C.L.D. wrote the manuscript, while all other authors reviewed and commented on it.

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Correspondence to Catherine L. Drennan or Squire J. Booker.

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Extended data

Extended Data Fig. 1 Topology diagram of KsTsrM depicting the different domains.

The blue dot represents the conserved Arg residue that occupies the lower axial position of the cobalamin, which is shown as two purple lines. The three yellow spheres on the loop after α1 of the TIM barrel represent the three conserved cysteines that coordinate the [4Fe-4S] cluster. The orange sphere on the loop following β2 of the TIM barrel is a conserved Glu residue that coordinates the unique Fe of the cluster.

Extended Data Fig. 2 Structural comparison of TsrM and OxsB.

Ribbon diagrams of (a) KsTsrM and (b) OxsB with domains colored to highlight structural similarities: Rossman fold (teal), TIM barrel (light blue), and C-terminal domain (purple). The N-terminal domain of OxsB is colored pink. Overlays of the Rossmann fold (c), TIM barrel (d) and C-terminal domain (e) of KsTsrM (teal, light blue, and purple respectively) with OxsB (grey).

Extended Data Fig. 3 Binding of cobalamin in the Rossmann fold of TsrM.

Conserved stabilizing residues among TsrMs are depicted.

Extended Data Fig. 4 Comparison of lower axial ligands of cobalamin.

The different axial ligands in (a) KsTsrM, (b) OxsB, and (c) MetH.

Extended Data Fig. 5 Time-dependent production of MeTrp by wild-type KsTsrM and the Arg69Lys variant.

a, Time-dependent production of MeTrp by KsTsrM (1 µM) using the flavodoxin/flavodoxin reductase/NADPH reducing system. kcat is 14 min−1. b, UV-vis analysis of KsTsrM. KsTsrM was reduced with Ti (III) citrate to form cob(I)alamin. Upon addition of SAM to the reaction, the peak at 390 nm, indicative of cob(I)alamin, decays, while a peak at 520 nm, indicative of MeCbl, grows in. c, Time-dependent production of MeTrp by the KsTsrM R69K variant using either Ti (III) citrate (red) or Fld/Fld Red/NADPH (purple) as reductant. Despite using 75 µM enzyme (indicated by dotted line), not even a full turnover is observed over 20 min by KsTsrM R69K with either reducing system. The inset displays a reduced Y-axis range to allow the small amount of activity in the presence of Ti (III) citrate to be observed. d, UV-vis analysis of KsTsrM R69K. As in (b), KsTsrM R69K was reduced with Ti (III) citrate to generate cob(I)alamin. When SAM is added, cob(I)alamin decays and MeCbl grows in, indicating that MeCbl formation is not significantly diminished with this variant. Error bars represent the standard deviation of reactions conducted in triplicate, with the center point representing the average.

Source data

Extended Data Fig. 6 Different roles of Arg residues that occupy the proximal face of cobalamin.

Comparison of the lower proximal face of cobalamin in (a) KsTsrM and (b) QueG (PDB: 5D08).

Extended Data Fig. 7 Computational docking studies of Trp positioning in active site.

a, Overlay of the two most likely poses for Trp in the computational docking model: pose 1 (lavender) and pose 2 (pink). b, Comparison of computational docking model pose 1 (lavender) to flipped Trp observed in crystal structure (teal). c, Associated distances between carboxylate of aza-SAM to Trp in docking model pose 2. d, Alternative view of pose 2, demonstrating distances from aza-SAM.

Extended Data Fig. 8 Comparison of Tyr308 in native and substrate-bound structures.

a, Overlay of KsTsrM native structure (teal) with substrate bound structure (light blue), showing change in conformation of Tyr308. b, Positioning of Tyr308 in docking model with pose 2, showing proximity to C2 position of Trp.

Extended Data Fig. 9 Analysis of MeCbl formation using dcSAM.

a, LC-MS/MS analysis of MeCbl formation in a reaction containing TsrM (10 µM), flavodoxin/flavodoxin reductase/NADPH, and dcSAM (1.5 mM). b, Time-dependent formation of MeCbl. Error bars represent the standard deviation of triplicate determinations. The dashed line indicates the concentration of enzyme in the reaction, with the central point representing the average.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Table 1.

Reporting Summary

Source data

Source Data Fig. 4

Raw data for Fig. 4d,e.

Source Data Extended Data Fig. 5

Raw data for Extended Data Fig. 5a,c.

Source Data Extended Data Fig. 9

Raw data for Extended Data Fig. 9a,b.

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Knox, H.L., Chen, P.YT., Blaszczyk, A.J. et al. Structural basis for non-radical catalysis by TsrM, a radical SAM methylase. Nat Chem Biol 17, 485–491 (2021). https://doi.org/10.1038/s41589-020-00717-y

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