Molecular Basis for Sesterterpene Diversity Produced by Plant Terpene Synthases

Abstract

Class I terpene synthase (TPS) generates bioactive terpenoids with diverse backbones. Sesterterpene synthase (sester-TPS, C25), a branch of class I TPSs, was recently identified in Brassicaceae. However, the catalytic mechanisms of sester-TPSs are not fully understood. Here, we first identified three nonclustered functional sester-TPSs (AtTPS06, AtTPS22, and AtTPS29) in Arabidopsis thaliana. AtTPS06 utilizes a type-B cyclization mechanism, whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism. We then determined the crystal structure of the AtTPS18-FSPP complex to explore the cyclization mechanism of plant sester-TPSs. We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism: (1) mainly due to the outward shift of helix G, plant sester-TPSs have a larger catalytic pocket than do mono-, sesqui-, and di-TPSs to accommodate GFPP; (2) type-A sester-TPSs have more aromatic residues (five or six) in their catalytic pocket than classic TPSs (two or three), which also determines whether the type-A or type-B cyclization mechanism is active; and (3) the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs. Altogether, this study improves our understanding of the catalytic mechanism of plant sester-TPS, which ultimately enables the rational engineering of sesterterpenoids for future applications.

Keywords: crystal structure; cyclization mechanism; sesterterpene; terpene synthase; terpenoid.

Figure 1

Sesterterpene Synthases in Arabidopsis and Structures of the Main Products of TPS06 and TPS29. (A) Phylogenetic analyses of 32 TPS proteins from A. thaliana (Columbia-0 ecotype) using the maximum-likelihood method. Bootstrap values (based on 1000 replicates) >75% are shown for the corresponding nodes. The five known Arabidopsis sester-TPSs are marked with black dots, and the functionally identified sester-TPSs are boxed in red (type A and type B). ND, not determined. (B) Scheme for screening sester-TPS genes using the E. coli system. MEP, 2-C-methyl-D-erythritol 4-phosphate pathway; ML, mevalonolactone; MVA, mevalonate pathway. (C) GC–MS analysis (SIM mode, m/z 340 for C₂₅H₄₀) of the sesterterpenes produced in E. coli harboring different TPS-a genes from Arabidopsis: upper panel, AtTPS06; middle panel, AtTPS22; bottom panel, AtTPS29. AtTPS06, a multi-product enzyme, produces more than ten different sesterterpenes from GFPP. (D) Mass spectra and chemical structures of the main sesterterpene products of TPS06 (C1, left) and TPS29 (C2, right).

Figure 2

Proposed Cyclization Scheme for the Synthesis of Sesterterpene Backbones by Arabidopsis sester-TPSs. The chemical structures elucidated in this study are indicated by red numbers. The products of AtTPS18 and its mutants are numbered C3–C7 according to their retention time in the GC–MS experiment carried out in this study (see below). All chemical structure elucidation data in this study are shown in Supplemental Tables 1 and 2 and Supplemental Figures 1–5. IM, intermediate. C3 ((+)-brassitetraene B), C6 ((+)-brassitetraene A), and C9 ((−)-caprutriene) were previously determined by Huang et al., 2017, Huang et al., 2018, whereas C7 ((+)-thalianatriene) and C8 ((−)-retigeranin B) were elucidated by Shao et al., (2017).

Figure 3

Structure of the AtTPS18–FSPP Complex. (A) The overall structure of the AtTPS18–FSPP complex as a ribbon cartoon (NTD and CTD are shown in orange and cyan, respectively). The substrate analog, FSPP, is shown as a yellow stick model, and the two Mg²⁺ atoms are shown as magenta balls. (B) Expansion of the substrate-binding pocket and the docking results showing the substrate binding of GFPP (electron static surface). FSPP and GFPP are shown as yellow and cyan stick models, respectively. (C) Interaction of amino acid residues with FSPP in the binding site. The bound FSPP is depicted as a ball-and-stick model. The 24 residues in the active site of AtTPS18 are shown. Among them, Arg³¹⁹, Arg³²¹, Asp³⁵⁶, Asp³⁵⁷, Asp³⁶⁰, Arg⁴⁹⁷, Asn⁵⁰⁰, Asp⁵¹⁰, Thr⁵⁰⁴, Glu⁵⁰⁸, Arg⁵¹¹, and Glu⁵¹³ were not considered for mutagenesis because they are conserved in the ionization of prenyl diphosphate substrates. (D) Superimposition of the cartoon models of AtTPS18 (magenta), NtEAS (yellow, PDB: 5EAT), taxadiene synthase from Taxus brevifolia (tan, PDB: 3P5R), and limonene synthase from Citrus sinensis (slate blue, PDB: 5UV2) from a top–down perspective. Farnesyl hydroxyphosphonate (FHP) in NtEAS and GFPP in AtTPS18 are shown as yellow and cyan sticks, respectively. The positions of the I–J loop and helix G (G1 and G2) are indicated by red and blue circles, respectively. (E) Close-up view of the helix G region of AtTPS18, 5EAT, 3P5R, and 5UV2 (for details, see the legend of D). The shift distance (Å) of helix G of AtTPS18, relative to that of other plant TPSs, was measured and labeled. (F) Possible interactions between Asn⁴⁹³ (in helix H) and helix G in the AtTPS18–FSPP complex. Dashed lines indicate possible hydrogen bonds.

Figure 4

Effect of Two Amino Acids on the Activity of Two Types of Sester-TPSs. (A) Sequence alignment of nine characterized sester-TPSs and 5EAT (a sesquiterpene synthase). The amino acids marked with stars are located in the catalytic pocket of AtTPS18 (see Figure 3C). Among them, the positions occupied by aromatic residues in sester-TPSs and Gly³²⁸ are boxed in red. (B) GC–MS analysis (SIM mode, m/z 340 for C₂₅H₄₀) of the sesterterpenes produced in E. coli harboring AtTPS06 and its mutants. (−)-Variculatriene, the main product of AtTPS25, was used as the internal standard (I.S.). Notably, the same amount of (−)-variculatriene was used in the experiments shown here and in (C). (C) GC–MS analysis (SIM mode, m/z 340 for C₂₅H₄₀) of the sesterterpenes produced in E. coli harboring AtTPS18 and its mutants.

Figure 5

Conversion between AtTPS18 and Its Close Homologs. C7 ((+)-thalianatriene), C8 ((−)-retigeranin), and C9 ((−)-caprutriene) are the main products of AtTPS18, AtTPS19, and Cru237, respectively. (A) Comparison of the models of AtTPS18 (blue) and AtTPS19 (orange). The different amino acids in the catalytic pockets of AtTPS18 and AtTPS19 are highlighted. (B) GC–MS (SIM mode, m/z 340 for C₂₅H₄₀) analysis of AtTPS18, AtTPS19, and their mutants. The peaks of C7 (the main product of AtTPS18) and C8 (the main product of AtTPS19) are marked with arrows. (C) Comparison of the models of AtTPS18 (blue) and Cru237 (orange). The amino acid at position 353 is highlighted. (D) GC–MS (SIM mode, m/z 340 for C₂₅H₄₀) analysis of AtTPS18, Cru237, and their mutants.