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A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis

Nature volume 544, pages 322–326 (2017)Cite this article

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Abstract

Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, reveals that OXT-A is derived from a 2′-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen atom abstraction from C2′. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme, a family of enzymes with an estimated 7,000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.

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Figure 1: oxsA and oxsB encode OXT-A biosynthetic enzymes.
Figure 2: Structure of OxsB.
Figure 3: A new mode of Cbl binding.
Figure 4: An expanded AdoMet radical domain in OxsB.
Figure 5: Proposed mechanism for generation of OXT-A aldehyde 2.

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References

  1. Shimada, N. et al. Oxetanocin, a novel nucleoside from bacteria. J. Antibiot. (Tokyo) 39, 1623–1625 (1986)

    Article  CAS  Google Scholar 

  2. Izuta, S. et al. Inhibitory effects of triphosphate derivatives of oxetanocin G and related compounds on eukaryotic and viral DNA polymerases and human immunodeficiency virus reverse transcriptase. J. Biochem. 112, 81–87 (1992)

    Article  CAS  PubMed  Google Scholar 

  3. Ueda, K., Tsurimoto, T., Nagahata, T., Chisaka, O. & Matsubara, K. An in vitro system for screening anti-hepatitis B virus drugs. Virology 169, 213–216 (1989)

    Article  CAS  PubMed  Google Scholar 

  4. Morita, M. et al. Cloning of oxetanocin A biosynthetic and resistance genes that reside on a plasmid of Bacillus megaterium strain NK84-0128. Biosci. Biotechnol. Biochem. 63, 563–566 (1999)

    Article  CAS  PubMed  Google Scholar 

  5. Magnusson, O. T., Reed, G. H. & Frey, P. A. Characterization of an allylic analogue of the 5′-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase. Biochemistry 40, 7773–7782 (2001)

    Article  CAS  PubMed  Google Scholar 

  6. Frey, P. A. in Comprehensive Natural Products II. Chemistry and Biology (eds Mander, L . & Liu, H.-W. ) Vol. 7, 501–546 (Elsevier, 2010)

    Google Scholar 

  7. Blodgett, J. A., Zhang, J. K. & Metcalf, W. W. Molecular cloning, sequence analysis, and heterologous expression of the phosphinothricin tripeptide biosynthetic gene cluster from Streptomyces viridochromogenes DSM 40736. Antimicrob. Agents Chemother. 49, 230–240 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kamigiri, K., Hidaka, T., Imai, S., Murakami, T. & Seto, H. Studies on the biosynthesis of bialaphos (SF-1293) 12. C-P bond formation mechanism of bialaphos: discovery of a P-methylation enzyme. J. Antibiot. (Tokyo) 45, 781–787 (1992)

    Article  CAS  Google Scholar 

  9. Kelly, W. L., Pan, L. & Li, C. Thiostrepton biosynthesis: prototype for a new family of bacteriocins. J. Am. Chem. Soc. 131, 4327–4334 (2009)

    Article  CAS  PubMed  Google Scholar 

  10. Kim, H. J. et al. GenK-catalyzed C-6′ methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme. J. Am. Chem. Soc. 135, 8093–8096 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Liao, R. et al. Thiopeptide biosynthesis featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. Chem. Biol. 16, 141–147 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Marous, D. R . et al. Consecutive radical S-adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis. Proc. Natl Acad. Sci. USA 112, 10354–10358 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Werner, W. J. et al. In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea. Biochemistry 50, 8986–8988 (2011)

    Article  CAS  PubMed  Google Scholar 

  14. Westrich, L., Heide, L. & Li, S. M. CloN6, a novel methyltransferase catalysing the methylation of the pyrrole-2-carboxyl moiety of clorobiocin. ChemBioChem 4, 768–773 (2003)

    Article  CAS  PubMed  Google Scholar 

  15. Woodyer, R. D., Li, G., Zhao, H. & van der Donk, W. A. New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin. Chem. Commun. (Camb.) (4) 359–361 (2007)

  16. Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F. & Miller, N. E. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 29, 1097–1106 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Allen, K. D. & Wang, S. C. Initial characterization of Fom3 from Streptomyces wedmorensis: the methyltransferase in fosfomycin biosynthesis. Arch. Biochem. Biophys. 543, 67–73 (2014)

    Article  CAS  PubMed  Google Scholar 

  18. Allen, K. D. & Wang, S. C. Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217. Biochim. Biophys. Acta 1844, 2135–2144 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Blaszczyk, A. J. et al. Spectroscopic and electrochemical characterization of the iron-sulfur and cobalamin cofactors of TsrM, an unusual radical S-adenosylmethionine methylase. J. Am. Chem. Soc. 138, 3416–3426 (2016)

    Article  CAS  PubMed  Google Scholar 

  20. Pierre, S. et al. Thiostrepton tryptophan methyltransferase expands the chemistry of radical SAM enzymes. Nat. Chem. Biol. 8, 957–959 (2012)

    Article  CAS  PubMed  Google Scholar 

  21. Chew, A. G. & Bryant, D. A. Chlorophyll biosynthesis in bacteria: the origins of structural and functional diversity. Annu. Rev. Microbiol. 61, 113–129 (2007)

    Article  CAS  PubMed  Google Scholar 

  22. Bridwell-Rabb, J ., Kang, G ., Zhong, A ., Liu, H. W . & Drennan, C. L. An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis. Proc. Natl Acad. Sci. USA 113, 13750–13755 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G. & Ludwig, M. L. How a protein binds B12: A 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266, 1669–1674 (1994)

    Article  ADS  CAS  PubMed  Google Scholar 

  24. Lexa, D. & Saveant, J. M. The electrochemistry of vitamin-B12 . Acc. Chem. Res. 16, 235–243 (1983)

    Article  CAS  Google Scholar 

  25. Vey, J. L . et al. Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme. Proc. Natl Acad. Sci. USA 105, 16137–16141 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Dowling, D. P., Vey, J. L., Croft, A. K. & Drennan, C. L. Structural diversity in the AdoMet radical enzyme superfamily. Biochim. Biophys. Acta 1824, 1178–1195 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Vey, J. L. & Drennan, C. L. Structural insights into radical generation by the radical SAM superfamily. Chem. Rev. 111, 2487–2506 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Goldman, P. J ., Grove, T. L ., Booker, S. J . & Drennan, C. L. X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry. Proc. Natl Acad. Sci. USA 110, 15949–15954 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bandarian, V ., Ludwig, M. L . & Matthews, R. G. Factors modulating conformational equilibria in large modular proteins: a case study with cobalamin-dependent methionine synthase. Proc. Natl Acad. Sci. USA 100, 8156–8163 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Evans, J. C . et al. Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase. Proc. Natl Acad. Sci. USA 101, 3729–3736 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Koutmos, M ., Datta, S ., Pattridge, K. A ., Smith, J. L . & Matthews, R. G. Insights into the reactivation of cobalamin-dependent methionine synthase. Proc. Natl Acad. Sci. USA 106, 18527–18532 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. Banerjee, R. Radical carbon skeleton rearrangements: catalysis by coenzyme B12-dependent mutases. Chem. Rev. 103, 2083–2094 (2003)

    Article  CAS  PubMed  Google Scholar 

  34. Layer, G. et al. The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN. J. Biol. Chem. 281, 15727–15734 (2006)

    Article  CAS  PubMed  Google Scholar 

  35. Ruszczycky, M. W., Choi, S. H. & Liu, H. W. Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical SAM enzyme DesII. J. Am. Chem. Soc. 132, 2359–2369 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shibata, N., Masuda, J., Morimoto, Y., Yasuoka, N. & Toraya, T. Substrate-induced conformational change of a coenzyme B12-dependent enzyme: crystal structure of the substrate-free form of diol dehydratase. Biochemistry 41, 12607–12617 (2002)

    Article  CAS  PubMed  Google Scholar 

  37. Berkovitch, F., Nicolet, Y., Wan, J. T., Jarrett, J. T. & Drennan, C. L. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303, 76–79 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ewall, R. X. & Bennett, L. E. Reactivity characteristics of cytochrome c(III) adduced from its reduction by hexaammineruthenium(II) ion. J. Am. Chem. Soc. 96, 940–942 (1974)

    Article  CAS  Google Scholar 

  39. Parthasarathy, A. et al. Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS. J. Am. Chem. Soc. 137, 3525–3532 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Broderick, J. B., Duffus, B. R., Duschene, K. S. & Shepard, E. M. Ra dical S-adenosylmethionine enzymes. Chem. Rev. 114, 4229–4317 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Dowling, D. P. et al. Radical SAM enzyme QueE defines a new minimal core fold and metal-dependent mechanism. Nat. Chem. Biol. 10, 106–112 (2014)

    Article  CAS  PubMed  Google Scholar 

  42. Nicolet, Y ., Amara, P ., Mouesca, J. M . & Fontecilla-Camps, J. C. Unexpected electron transfer mechanism upon AdoMet cleavage in radical SAM proteins. Proc. Natl Acad. Sci. USA 106, 14867–14871 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Quitterer, F., List, A., Eisenreich, W., Bacher, A. & Groll, M. Crystal structure of methylornithine synthase (PylB): insights into the pyrrolysine biosynthesis. Angew. Chem. Int. Ed. Engl. 51, 1339–1342 (2012)

    Article  CAS  PubMed  Google Scholar 

  44. Goldman, P. J . et al. X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification. Proc. Natl Acad. Sci. USA 110, 8519–8524 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by National Institute of Health Grants F32-GM108189 (J.B.-R.) and GM035906 (H.-w.L.), and the Welch Foundation grant F-1511 (H.-w.L.). C.L.D. is a Howard Hughes Medical Institute Investigator. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). 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.

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Author notes

  1. Jennifer Bridwell-Rabb and Aoshu Zhong: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Jennifer Bridwell-Rabb & Catherine L. Drennan

  2. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Jennifer Bridwell-Rabb & Catherine L. Drennan

  3. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Jennifer Bridwell-Rabb & Catherine L. Drennan

  4. Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, 78712, Texas, USA

    Aoshu Zhong, He G. Sun & Hung-wen Liu

  5. Department of Chemistry, University of Texas, Austin, 78712, Texas, USA

    Aoshu Zhong, He G. Sun & Hung-wen Liu

Authors
  1. Jennifer Bridwell-Rabb
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  4. Catherine L. Drennan
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Contributions

J.B.-R. performed crystallography, A.Z. performed genetic experiments, A.Z. and H.G.S. performed biochemical assays. J.B.-R., C.L.D., A.Z., H.G.S. and H.-w.L. designed experiments, analysed data, and wrote the manuscript.

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Correspondence to Catherine L. Drennan or Hung-wen Liu.

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Extended data figures and tables

Extended Data Figure 1 Putative substrates tested in the OxsB and OxsA/OxsB reactions.

Reductants used to test these substrates were sodium dithionite, hexa-ammineruthenium(II) chloride, nicotinamide adenine dinucleotide phosphate (NADPH)/methyl viologen, titanium(III) citrate/methyl viologen, and a flavodoxin/flavodoxin–NADPH reductase system35,38,39.

Extended Data Figure 2 In vivo gene expression and HPLC analysis reveal oxsA and oxsB are required for OXT-A biosynthesis.

a, To probe which genes located within the BglII-D fragment are responsible for OXT-A (chemical structure shown in left panel) production, B. megaterium NRS 269 was transformed using the E. coli–Bacillus shuttle vector pMM1522. b, In vivo product profiles of B. megaterium NRS 269 strains transformed with pMM1522 empty vector (as a control) (i) or transformed with pMM1522 that contains the BglII-D (oxsA, oxsB, oxrA, oxrB) fragment (ii). c, In vivo product profiles of B. megaterium NRS 269 strains carrying only oxsB (i), carrying both oxsA and oxsB (ii), or carrying only oxsA (iii).

Extended Data Figure 3 Enzymatic production of OXT-A.

High-performance liquid chromatography (HPLC) analysis in panel a was performed using a CarboPac PA1 Dionex column whereas a C18 column was used in panels c–e. a, HPLC analysis of the reactions catalysed by OxsA and OxsB using dAMP, dADP, or dATP as substrate. Incubation with reconstituted OxsB and OxsA with dAMP, DTT, AdoMet, HO-Cbl, MgCl2, NADPH, MV (full reaction, see Methods for details) (i); full reaction without OxsB (ii); full reaction without OxsA (iii); full reaction without dAMP (iv); full reaction without AdoMet (v); full reaction without HO-Cbl (vi); full reaction without the reductants NADPH and MV (vii); full reaction substituted with dADP instead of dAMP (viii); full reaction substituted with dATP instead of dAMP (ix). b, Mass spectrometry (MS) (ESI positive) used to confirm the identity of compound 2 as the corresponding aldehyde of OXT-A 5′-monophosphate. MS of the aldehyde compound was performed following treatment of the reaction mixture with CIP and purification by HPLC. c, HPLC analysis confirms that reduction of compound 2 results in formation of OXT-A-P (3). Isolated 2 (i); isolated 2 treated with NaBD4 (ii); co-injection of 2 treated with NaBD4 with a chemically synthesized standard of 3 (iii); standard of 3 (iv). d, Direct formation of OXT-A is observed when cell extract is included in the reaction conditions. HPLC analysis after incubation of reconstituted OxsB with OxsA, dATP, DTT, AdoMet, HO-Cbl, MgCl2, NADPH, MV, and cell extract of B. megaterium NRS 269 (full reaction) (i); full reaction without OxsB (ii); full reaction without OxsA (iii); full reaction without dATP (iv); full reaction without AdoMet (v); full reaction without MgCl2 (vi); full reaction without cell extract (vii); OXT-A (1) standard (viii). e, Direct formation of OXT-A can also be observed when alcohol dehydrogenase is included in the reaction mixture. HPLC analysis after incubation with reconstituted OxsB and OxsA with dATP, DTT, AdoMet, HO-Cbl, MgCl2, NADPH, MV and horse liver alcohol dehydrogenase (full reaction) (i); full reaction without OxsA and OxsB (ii); OXT-A standard (iii).

Extended Data Figure 4 Chemical synthesis of OXT-A-P (3).

Extended Data Figure 5 Characterization of OxsB as an AdoMet radical enzyme.

a, Consistent with its classification as an AdoMet radical enzyme, OxsB catalyses the reductive cleavage of AdoMet to generate 5′-dAdoH. HPLC analysis (C18 column, 2–20% CH3CN in 1% NH4OAc linear gradient elution) of reaction catalysed by OxsA and OxsB. Reaction of reconstituted OxsB with OxsA, dAMP, DTT, AdoMet, HO-Cbl, MgCl2, NADPH, MV (full reaction, see Methods for details) (i); full reaction without dAMP (ii); 5′-dAdoH standard (iii). b, MS spectrum (ESI positive) of 5′-dAdoH generated in the OxsA and OxsB reaction using [2′-2H2]-2′-dAMP as substrate shows incorporation of the deuterium label into 5′-dAdoH and thus indicates hydrogen atom abstraction occurs at C2′. The less than full deuterium incorporation is probably due to the co-occurrence of uncoupled quenching of 5′-dAdo•, a common phenomenon in many AdoMet radical enzymes40. c, MS spectrum (ESI positive) of OXT-A (1) generated in the OxsA and OxsB reaction using [3′-2H]-2′-dAMP as substrate, which shows retention of the deuterium label in product, again consistent with hydrogen atom abstraction at C2′.

Extended Data Figure 6 OxsB is organized into four modular domains.

a, A stereoview of the entire (744 amino acids) monomer of OxsB coloured by domain. The N-terminal domain is shown in yellow, and is followed by the Cbl-binding domain displayed in pink, the AdoMet radical domain, which is coloured cyan, and the C-terminal helix bundle domain displayed in blue. b, A topology diagram of OxsB is shown and coloured as in panel a. The yellow sphere in domain II represents the position of Asn186, which is the closest residue to the Co of Cbl. c, The two observed conformations of AdoMet (cyan and wheat), Cbl, and the [4Fe–4S] AdoMet radical cluster (orange and yellow spheres) are shown with simulated annealing composite omit electron density maps contoured at 0.8σ.

Extended Data Figure 7 Cbl interactions in OxsB and comparison to MetH.

a, A stereoview of an overlay of the MetH23 Cbl-binding domain (green) with the Cbl-binding domain of OxsB (pink) shows differences in the positioning of the Cbl cofactor’s corrin ring and the length of the II-β1 loop, which for OxsB lacks a His residue to ligate Cbl. b, Residues from the Cbl-binding domain of OxsB that accommodate or make contact with the DMB tail and corrin ring of Cbl are highlighted and shown as sticks. Residues Gly216 and Ser184 are from the base-off consensus sequence and residues R135–S139 are located on the II-β1 loop. c, Residues from the Cbl-binding domain of MetH23 that interact with Cbl or make room for the DMB tail are highlighted and shown as sticks. d, Residues from panels b and c that make contact with Cbl from the Cbl-binding domains of MetH (top panel) and OxsB (bottom panel) are shown. Residues highlighted in pink are previously identified sequence fingerprints of MetH that have a conserved interaction in OxsB. Residues highlighted in blue are conserved interactions between Cbl and MetH or OxsB, which are not from the standard Cbl-binding motifs. Residues shown in black form interactions with Cbl, but are not conserved between the proteins. e, Stereoview of the contacts that Cbl makes with the Cbl-binding (pink) and AdoMet radical (cyan) domains of OxsB. All of the residues that form interactions with Cbl are highlighted as sticks, but only residues from the AdoMet radical domain are labelled for clarity.

Extended Data Figure 8 Structural comparisons of apo-OxsB, OxsB–Cbl/[4Fe-4S]/AdoMet, and PFL-AE.

a–d, Small conformational changes occur in the reconstituted structures relative to the apo-structure. The r.m.s.d. determined by PyMOL for the OxsB–Cbl/[4Fe–4S]/AdoMet structure compared to the apo structure is 0.98 Å for 4,770 atoms. a, The II-β1 loop of the Cbl-binding domain in the reconstituted OxsB structures swings outward 12.9 Å to avoid steric clashes with the corrin ring of Cbl and now caps the side of the Cbl. b, c, In the absence of a [4Fe-4S] cluster, the Cys residues of the cluster-binding loop in the apo-structure are oriented similarly to those in the reconstituted structure. Cys318 and Cys321 in the apo-structure, however, exhibit a partial occupancy disulfide linkage. At the end of the cluster binding loop, there are more substantial differences between the structures; His325 and Lys326 of OxsB–Cbl/[4Fe–4S]/AdoMet move 8.0 and 8.8 Å, respectively, from their positions in the apo structure to interact with the nucleotide tail of Cbl. d, An overlay of OxsB–Cbl/[4Fe–4S]/AdoMet (light colours) with apo-OxsB (dark colours) shows slight movements in each domain. The arrow indicates closing in of the helix bundle domain towards the cofactors. e, A surface representation of OxsB reveals the open and solvent accessible nature of the active site in OxsB–Cbl/[4Fe–4S]/AdoMet. Water molecules are shown as red spheres. f, Location of polar and aromatic residues near the active site. Presumably, positively charged residues are needed to accommodate the negatively charged phosphate moieties of substrate and an aromatic residue may stack with substrate adenine. g, An overlay of the β-strands from the AdoMet radical domains of OxsB (cyan) with those from a peptide-bound (green) stucture of PFL-AE25 (grey) was used to map the approximate substrate-binding site in OxsB. The yellow sphere, which corresponds to the Cα of the substrate peptide Gly residue, is 3.7 Å away from the 5′ carbon of AdoMet and 3.7 Å away from Co of Cbl.

Extended Data Figure 9 AdoMet interactions.

a, A stereoview of the AdoMet radical domain of OxsB. Each of the AdoMet radical motifs26,27 is highlighted including the GGE (E363), ribose (E436), GXIXGXXE (I474), and the β6 (adenine-binding) motif (E545). The GGE motif provides a carbonyl to hydrogen bond with the amino moiety of AdoMet. In OxsB, E363 also contacts a Cbl acetamide. The ribose motif is found at the C-terminal loop following III-β4 where Glu436 forms two hydrogen bonds with the AdoMet ribose hydroxyl moieties. In the same loop, two residues upstream, the backbone amide of Gly434 contributes a hydrogen bond to the carboxyl group of AdoMet. Following a short III-α4a helix that connects III-β4 and III-α4, Lys448 interacts with the AdoMet carboxylate similar to what was previously observed in QueE41, HemN34, HydE42, PylB43, anSMEcpe44, and BtrN28. In terms of the GXIXGXXE motif, Ile474 from a loop following III-β5 provides hydrophobic contacts to the adenine ring of AdoMet as observed previously. However, instead of the backbone of Ile474 being stabilized by a polar residue on III-α5, as found in other AdoMet radical enzymes, the backbone of Ile474 is stabilized through interactions with the side chains of Gln442 and Tyr446 from III-α4a and the backbone of ribose motif residue Glu436. The final motif, the so-called β6 motif, is present although β6 is not. A loop substitutes for β6, with backbone atoms of E545 making hydrogen bonds to the adenine ring of AdoMet. Additional residues F320, M544, I546 and L547 that provide hydrophobic interactions to the adenine ring of AdoMet and a hydrogen bond to N6 are also shown. b, A 2Fo − Fc simulated annealing composite omit electron density map contoured at 1.0σ around the AdoMet radical [4Fe–4S] cluster. c, The radical-competent orientation of AdoMet ligates the unique Fe of the AdoMet radical [4Fe–4S] cluster, which is shown rotated approximately 90° from b. The distance between the unique Fe and the AdoMet amino and carboxylate moieties measure 2.2 Å each. d, The non-radical competent orientation also ligates the AdoMet radical [4Fe–4S] cluster with the amino and carboxylate moieties. These distances measure 2.3 and 2.0 Å, respectively. e, A 2Fo − Fc simulated annealing composite omit electron density map contoured at 1.0σ around the two orientations of AdoMet. f, A simulated annealing composite omit electron density map calculated after the radical-competent orientation of AdoMet was omitted from the refined structure of OxsB–Cbl/[4Fe–4S]/AdoMet. This map is contoured at ± 3.0σ around the radical-competent orientation of AdoMet. g, A similar simulated annealing composite omit electron density map was calculated after the non-radical-competent orientation of AdoMet was omitted from the refined structure of OxsB–Cbl/[4Fe–4S]/AdoMet. This map is also contoured at ± 3.0σ around the observed AdoMet conformation.

Extended Data Table 1 Data collection and refinement statistics of OxsB
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Bridwell-Rabb, J., Zhong, A., Sun, H. et al. A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis. Nature 544, 322–326 (2017). https://doi.org/10.1038/nature21689

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