Molecular insights into a distinct class of terpenoid cyclases

Abstract

Terpenoid cyclases (TCs) account for the synthesis of the most widespread and diverse natural compounds. A sesquiterpene cyclase termed BcABA3 from an abscisic acid-producing fungus Botrytis cinerea that yields (2Z,4E)-α-ionylideneethane but lacks signature feature of canonical TCs represents a distinct type of TCs. Here, we report the crystal structures of BcABA3, a closely related RuABA3 from Rutstroemia sp. and a bacterial SkABA3 from Shimazuella kribbensis. These ABA3 proteins adopt an all-α-helix fold and bind pyrophosphate moiety of farnesyl pyrophosphate by Glu-chelated Mg²⁺ ion cluster. We conduct mutagenesis experiments to validate the role of the substrate-binding residues. SkABA3 appears to yield compounds that are distinct from (2Z,4E)-α-ionylideneethane. These results not only provide the molecular insight into ABA3 proteins that serve as an important basis to the future investigation of this class of TCs, but also reveal the existence of more uncharacterized terpenoids synthesized via dedicated machineries.

Fig. 1. Overall structure of ABA3 proteins.

a The overall structures of apo-BcABA3 (PDB ID, 8ZAC), apo-RuABA3 (PDB ID, 8ZAD), and apo-SkABA3 (PDB ID, 8ZAF) in the homodimeric configuration are shown. The dimeric counterpart in each drawing is shown in gray cartoon. The metal ions are displayed in spheres and helix α11’ in chain A of apo-SkABA3 structure is colored in purple. b One polypeptide chain of apo-BcABA3 is displayed with each α-helix labeled numerically. The either end of the missing fragment between helix α11 and α12 are indicated by dashed circles. c The Zn²⁺ ion-coordination residues in each ABA3 structure are displayed in sticks. The Zn²⁺ ions are shown in blue spheres. The dash lines measure the distance between residues and the metal ion and the length is shown in parentheses (unit, Å).

Fig. 2. Enzyme-ligand interaction networks in ABA3 complex structures.

a The overall structure of RuABA3/FSPP (PDB ID, 8ZAE) and SkABA3/PPi (PDB ID, 8ZAG) presented in cartoon models. b The enzyme-ligand interaction networks in the complex structures are depicted, with protein residues shown in lines. The 2F_o-F_c electron density maps of FSPP, PPi, Mg²⁺ ions and coordinating waters contoured at 1.0 σ are shown in mesh. Two views relative at the Y-axis by 180° are presented. Dashed lines indicate distance <3.5 Å. The polder omit maps of the bound ligands are shown in Supplementary Fig. 11. In both panels, the bound ligands, Mg²⁺ ions, Zn²⁺ ions, and water molecules are shown in sticks, green spheres, blue spheres, and red small spheres.

Fig. 3. Substrate-binding pocket of ABA3 proteins.

Apo-BcABA3 (PDB ID, 8ZAC), RuABA3/FSPP (PDB ID, 8ZAE), and SkABA3/PPi (PDB ID, 8ZAG) are superimposed. The overall protein of RuABA3/FSPP is depicted and shown in cartoon model. The residues that comprise substrate-binding pocket are shown in line (green, BcABA3; magenta, RuABA3/FSPP; cyan, SkABA3/PPi). FSPP and PPi are shown in sticks and metal ions are shown in spheres. Two Mg²⁺ ions observed in SkABA3/PPi are displayed as cyan spheres and denoted as Mg²⁺_A and Mg²⁺_B. The Mg²⁺_A-corresponding ion in RuABA3/FSPP is presented as a magenta sphere.

Fig. 4. The roles of the active site residues in BcABA3-catalyzed reaction.

a The PPi release activity of wild type and variant BcABA3. The relative activities of each sample were presented as percentages of the wild type. The individual and average values of each sample in the triplicate assay were presented in dots and bars, respectively. b The reaction mixtures of wild type and variant BcABA3 were analyzed by GC-MS. The GC-MS chromatograms (m/z 133 and 148) of reaction mixture of each enzyme are displayed. The mass spectrum of (E)-β-farnesene is shown in Supplementary Fig. 16. c Two views depicting the substrate interaction network observed in RuABA3/FSPP related at Y-axis by 180° are displayed. Amino acid residues, FSPP, and metal ions are shown in lines, sticks, and spheres. The hydrophilic interactions measured within 3.5 Å are shown in purple dashed lines. The Y96-mediated packing force is noted by a role of short strokes. The corresponding residues in BcABA3 are labeled by green letters. Some carbon atoms of FSPP are labeled by italic Arabic numbers. The numbers shown in parentheses indicate distance (unit, Å). d The proposed BcABA3-catalyzed cyclization process. The substrate and reaction intermediates are colored in black, and the protein residues and pyrophosphate ion are colored in green. WT, wild type. Source data of (a, b) are provided as a Source Data file.