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A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds

Nature Chemistry volume 12, pages 968–972 (2020)Cite this article

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Abstract

Class II terpene cyclases, such as oxidosqualene and squalene-hopene cyclases, catalyse some of the most complex polycyclization reactions. They minimally exhibit a β,γ-didomain architecture that has been evolutionarily repurposed in a wide range of terpene-processing enzymes and likely resulted from a fusion of unidentified monodomain proteins. Although single domain class I terpene cyclases have already been identified, the corresponding class II counterparts have not been previously reported. Here we present high-resolution X-ray structures of a monodomain class II cyclase, merosterolic acid synthase (MstE). With a minimalistic β-domain architecture, this cyanobacterial enzyme is able to construct four rings in cytotoxic meroterpenoids with a sterol-like topology. The structures with bound substrate, product, and inhibitor provide detailed snapshots of a cyclization mechanism largely governed by residues located in a noncanonical enzyme region. Our results complement the few known class II cyclase crystal structures, while also indicating that archaic monodomain cyclases might have already catalyzed complex reaction cascades.

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Fig. 1: Architectural variants of class I and class II terpene cyclases.
Fig. 2: Merosterol biosynthesis and comparison with the lanosterol and hopene cyclization pathways.
Fig. 3: The structure of MstE.
Fig. 4: Biosynthesis of new product by mutant MstE_Y157A.

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Data availability

Crystallographic data were deposited in the RCSB PDB with the following IDs: 6SBB, 6SBC, 6SBD, 6SBE, 6SBF, and 6SBG. All other data are available in the text or in the Supplementary Information.

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Acknowledgements

We thank R. Ueoka for support with NMR and the staff of beamline X06SA at the Paul Scherrer Institute for assistance during data collection. We acknowledge funding by the Swiss National Science Foundation (contract numbers 205321-165695 and 205320-185077) and by the Helmut Horten Foundation (to J.P.).

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Author notes

  1. Philipp Moosmann

    Present address: Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

  2. These authors contributed equally: Philipp Moosmann, Felix Ecker.

Authors and Affiliations

  1. Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland

    Philipp Moosmann, Stefan Leopold-Messer, Jackson K. B. Cahn, Cora L. Dieterich & Jörn Piel

  2. Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Chair of Biochemistry, Technische Universität München, Garching, Germany

    Felix Ecker & Michael Groll

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Contributions

P.M., F.E., M.G., and J.P. designed, conducted, and analysed the experiments. S.L.-M. and C.L.D. conducted organic syntheses. J.K.B.C. performed phylogenetic analyses. J.P., M.G, F.E., and P.M. wrote the paper. All authors edited the paper.

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Correspondence to Michael Groll or Jörn Piel.

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Extended data

Extended Data Fig. 1 Protein alignment.

Structure-guided alignment of merosterolic acid synthase (MstE) with squalene-hopene cyclase (SHC; PDB 2SQC; 15.91 % identity*) and oxidosqualene cyclase (OSC; PDB 1W6K; 15.26 % identity*). Alpha helices are indicated with cylinders under the sequences, beta strands with arrows. These secondary structural elements are colored yellow if they are part of the core scaffold of the γ domain and blue if they are part of β domain. Non-conserved secondary structures are depicted in grey. The catalytic aspartate residue is indicated with a red asterisk. *Identity percentages were calculated without the γ-domains of SHC and OSC.

Extended Data Fig. 2 Phylogenetic tree of selected terpene cyclase β domains.

Maximum-likelihood tree of β domains from functionally characterized and unknown terpene cyclases. Organisms are colored according to the phylogeny of their origin: Protists grey, animals gold, fungi red, plants green, and bacteria blue. Terpene enzyme class, domain architecture, and product are indicated.

Extended Data Fig. 3 Structures of MstE in complex with different ligands.

Left: Side- and top-views (rotated by 90°) of MstE (a), MstE:FS-DHB (b), MstE_D109N:GG-DHB (c), MstE_D109A:MA (d), MstE_R337A:GG-DHB (e) and MstE_Y157F (f) as cartoons with helices and β-sheets colored in pink and blue, respectively. Dashed loops indicate disordered regions in the crystal structures and are labeled accordingly. Carbon atoms of ligands, if present, are shown as sticks in gold, with the DHB moieties in black. Double bonds and newly formed C-C bonds are highlighted in black. Right: Close-up views of the active sites. The 2FO-FC electron density maps (gray mesh, contoured to 1.0 σ) of ligands are shown together with residues engaged in binding. Side-views of all compounds (rotated by 90°) are depicted on the far right. Dashed lines represent important interactions. Mutated residues are colored green. Diffuse electron density in F indicates the presence of a ligand at low occupancy.

Extended Data Fig. 4 A proton relay conveys protonation of GG-DHB.

(a) The structure of MstE:FS-DHB superpositioned with MstE_D109N:GG-DHB (RMSD-Cα: 0.12 Å) reveals that Asp109 adopts anti-position for protonation of the terminal C = C in GG-DHB. Two water molecules align between Tyr216 and Asp109 to activate the Brønsted acid and replace the proton after reaction. Oxygen atoms are highlighted in red; the protons (light blue) were added with PyMol (37) and oriented according to potential interactions represented as dashed lines (distance given in Å). (b) The primary amine of Asn109 in MstE_D109N:GG-DHB displaces one of the water molecules by 0.6 Å, flipping the orientation of all protons in the cluster.

Extended Data Fig. 5 A charge relay supports deprotonation of GG-DHB.

The structure of MstE_D109N:GG-DHB reveals the formation of a triad between Glu339, Arg337 and Asp162. Asp162 forms a salt bridge with the guanidino group of Arg337. This pushes electrons towards the secondary amine which in turn polarizes the carboxylate of Glu339, strengthening the H-bond with the meta-hydroxy group of DHB and increasing basicity. After deprotonation, this cluster acts as a proton-relay, before it is promptly broken and reordered to accommodate the product. Heteroatoms are highlighted in red (O) and blue (N); the proton (light blue) was added with PyMol. Interactions are represented as dashed lines (distance given in Å).

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Supplementary Methods, Supplementary Figs. 1–36, and Supplementary Tables 1–4.

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Moosmann, P., Ecker, F., Leopold-Messer, S. et al. A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds. Nat. Chem. 12, 968–972 (2020). https://doi.org/10.1038/s41557-020-0515-3

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