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Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis

Nature volume 602, pages 343–348 (2022)Cite this article

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Abstract

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of β-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the β-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

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Fig 1: Cbl-dependent radical-mediated methylations in carbapenem biosynthesis.
Fig. 2: TokK binds its carbapenam substrate at the interface of three domains.
Fig. 3: The Cbl- and substrate-binding sites influence overall activity and the relative rates of each TokK methylation step.

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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 (PDB) under accession numbers 7KDX (structure with 5′-dAH + Met) and 7KDY (structure with 5′-dAH + Met + carbapenam substrate).

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) (GM122595 to S.J.B., GM119707 to A.K.B., AI121072 to C.A.T. and GM080189 to E.K.S.) and the Eberly Family Distinguished Chair in Science (S.J.B.). S.J.B. is an investigator 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 Department of Energy Office of Science User Facility under contract no. DE-AC02-05CH11231. The ALS-ENABLE beamlines are supported in part by the NIH, National Institute of General Medical Sciences, grant P30 GM124169. E.K.S. and C.A.T. thank M. S. Lichstrahl for providing a synthetic intermediate, and I. P. Mortimer and J. Tang for their help with ESI-MS and NMR experiments, respectively. We thank D. Iwig for his help in the mass spectrometry analysis of the TokK crystals.

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  1. These authors contributed equally: Hayley L. Knox, Erica K. Sinner

Authors and Affiliations

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

    Hayley L. Knox, Amie K. Boal & Squire J. Booker

  2. Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA

    Erica K. Sinner & Craig A. Townsend

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

    Amie K. Boal & Squire J. Booker

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

    Squire J. Booker

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Contributions

H.L.K., E.K.S., C.A.T. and S.J.B. developed the research plan and experimental strategy. H.L.K. isolated and crystallized proteins and collected crystallographic data. H.L.K. and E.K.S. performed biochemical experiments. H.L.K., E.K.S., C.A.T., A.K.B. and S.J.B. analysed and interpreted crystallographic data. H.L.K., E.K.S., C.A.T., A.K.B. and S.J.B. wrote the manuscript.

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Correspondence to Craig A. Townsend, Amie K. Boal or Squire J. Booker.

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Extended data figures and tables

Extended Data Fig. 1 Comparison to CysS, another class B sequential methylase.

a, CysS performs sequential radical methylations like TokK and ThnK. b, Partial alignment of TokK (Uniprot ID: A0A6B9HEI0), ThnK (Uniprot ID: F8JND9), CysS (Uniprot ID: A0A0H4NV78), TsrM (Uniprot ID: C0JRZ9), Fom3 (Uniprot ID: Q56184), PhpK (Uniprot ID: A0A0M3N271), and GenK (Uniprot ID: Q70KE5). Cysteines that coordinate the iron-sulfur cluster are highlighted in blue. Trp76 (highlighted in red) and Trp215 (highlighted in yellow) in TokK is conserved in ThnK and CysS, but not in other known Cbl-binding RS methylases. Areas of conservation for TokK, ThnK, and CysS around Trp76 are highlighted in grey. The bottom axial amino acid residue for TsrM (Arg69) is highlighted in red. Completely conserved residues are bolded.

Extended Data Fig. 2 Reactions performed by OxsB and TsrM, notable Cbl-binding radical SAM enzymes.

a, Proposed pathway for the biosynthesis of oxetanocin A by OxsB and OxsA. Currently the aldehyde reduction step is unknown. b, Proposed non-radical reaction performed by TsrM. The carboxylate in SAM is implicated in playing a dual role in catalysis, both as the source of the methyl donor and as the base to prime the substrate for nucleophilic attack.

Extended Data Fig. 3 Structural comparisons of Cbl-dependent RS enzymes.

a–c, The overall structures of TokK (a), KsTsrM (PDB ID: 6WTE) (b) and OxsB (PDB ID: 5UL3) (c) are shown as ribbon diagrams and coloured by domain (Cbl-binding domain, teal; RS domain, light blue; and C-terminal domain, pink). OxsB has a fourth domain, an N-terminal domain of an unknown function, shown in gold. All three enzymes share very similar Cbl-binding domains with a characteristic Rossmann fold. However, as shown in Extended Data Fig. 4, the RS domain and C-terminal domain differ in each system.

Extended Data Fig. 4 Comparison of the Cbl-binding, RS, and C-terminal domains of TokK, KsTsrM, and OxsB.

The domains are coloured as in Extended Data Fig. 3. a, The Rossmann fold, in teal, is highly similar among TokK (PDB ID: 7KDY), KsTsrM (PDB ID: 6WTF) and OxsB (PDB ID: 5UL4). b, The core of each of the RS domains is a (β/α)6 motif; however, there are distinct differences. The RS domain of OxsB is more compact than those of TokK or KsTsrM, and all three have unique extra secondary structure features. c, The C-terminal domains for TokK, KsTsrM, and OxsB are vastly different in architecture. d, Comparison of the binding of Met and 5′-dAH, aza-SAM, and SAM for TokK, KsTsrM, and OxsB structures, respectively. Only the relevant binding of SAM to the cluster is shown for OxsB. OxsB has two binding positions of SAM, one to the cluster and one in what is proposed to be an intermediate state towards methylating the Cbl.

Extended Data Fig. 5 Domain architecture of TokK.

a, The three domains of TokK are portrayed in a ribbon diagram. The N-terminal Cbl-binding domain is shown in teal, the RS domain is shown in light blue, and the C-terminal domain is shown in pink. b, A topology diagram of TokK with domains coloured as in a. The Cbl is portrayed in sticks, and the lower axial Trp side chain is shown as a red dot. The iron-sulfur cluster is shown in orange and yellow spheres, and the ligating Cys residues are shown as small yellow spheres. c–e, Zoomed in views of the C-terminal domain (c), RS domain (d) and Cbl-binding domain (e) are shown as ribbon diagrams.

Extended Data Fig. 6 Diagram of carbapenam interactions in the TokK complex with substrate.

Carbapenam substrate 1 shown in blue sticks and coloured by atom type. Polar and hydrophobic interactions were mapped with the LigPlot program and indicated with dashed lines or a starburst symbol.

Extended Data Fig. 7 Comparison of TokK structure with those of enzymes using alternative radical-generating mechanisms.

a–e, Residues that interact with the polar substituents in the β-lactam ring are shown in stick format for TokK (a) and the epimerase CarC (PDB ID: 4OJ8) (b). Although the two enzymes are structurally distinct, they use a similar number and type of functional groups to anchor the β-lactam by using direct and water-mediated contacts to the C7 carbonyl and C3 carboxylate substituents. In addition, comparison of the active site with those of hydroxylases reveals differences in the orientation of hydrogen atom transfer (HAT) intermediates and -OH or -CH3 functionalization moieties. Fe(II)- and 2-oxo-glutarate-dependent (Fe-2OG) oxygenases use a ferryl [Fe(IV)-oxo)] intermediate to abstract an H-atom from an unactivated substrate carbon. The resulting Fe(III)-OH complex then couples with the substrate radical to yield a hydroxylated product. A vanadyl [V(IV)-oxo] mimic of the ferryl intermediate in L-Arg C3 hydroxylase, VioC, reveals that HAT and -OH transfer must occur from the same side of the substrate target carbon (c). The distance between the reactive oxo group and the substrate target carbon (indicated by the arrows) is 3.1 Å in VioC. A structure of the haem-dependent hydroxylase, P450cam, shows a similar phenomenon. A CO-bound mimic of the Fe(IV)-porphyrin radical intermediate, compound I, demands the same arrangement of HAT and -OH transfer components relative to the hydroxylation target on the camphor substrate (d). The distance between a mimic of the reactive group and the substrate target carbon (indicated by the arrows) is 3.1 Å in P450cam. By contrast, RS methylases use separate HAT reagents (5′-dA•) and methyl group donors (Me-Cbl), allowing for more diverse stereochemical outcomes in C-H functionalization reactions. (e) The structure of TokK in complex with carbapenam substrate, 1, shows a ~120° angle between the HAT acceptor (5′C of 5′-dAH), the substrate target carbon (C6), and the Cbl top ligand (-OH of OHCbl, a surrogate for Me-Cbl).

Extended Data Fig. 8 A comparison of the substrate-binding sites in two structurally characterized RS methylases.

a, b, The substrate complexes of TokK (a) and RlmN (PDB ID: 5HR7) (b) are shown. RlmN is an RS methylase that uses a radical-based mechanism to methylate an sp2-hybridized carbon (C2) of an adenine base in transfer or ribosomal RNA. By contrast to TokK and other Cbl-dependent RS enzymes, RlmN uses a 5′-dA• to activate a post-translationally modified methyl-Cys residue on a C-terminal loop in the active site to modify its aromatic substrate via radical addition. Comparison of a structure of RlmN in a cross-linked methylCys-tRNA intermediate state to the TokK substrate complex shows that, despite the differences in reaction mechanism, these systems use a similar orientation of radical initiator (5′-dA•), substrate target carbon (C6, C2), and methyl donor (OH, Me). In the TokK substrate complex, the -OH ligand of the Cbl cofactor serves as a surrogate for the position of the methyl donor.

Extended Data Fig. 9 Solvent accessibility of the bottom axial face of the Cbl in TokK.

a, Nearest residues (~6 Å) to the bottom face of the Cbl. b, View of TokK using space-filling model to show a channel to the active site. As can be seen, only a very small portion of the Cbl (coloured yellow) is solvent accessible, with most of the Cbl being buried within the Rossmann fold. Trp76 is coloured red.

Extended Data Table 1 X-ray crystallographic data collection and refinement statistics
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Knox, H.L., Sinner, E.K., Townsend, C.A. et al. Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis. Nature 602, 343–348 (2022). https://doi.org/10.1038/s41586-021-04392-4

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