Structure of a heterotetrameric geranyl pyrophosphate synthase from mint (Mentha piperita) reveals intersubunit regulation

Abstract

Terpenes (isoprenoids), derived from isoprenyl pyrophosphates, are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. Divergent evolution of homomeric prenyltransferases (PTSs) has allowed PTSs to optimize their active-site pockets to achieve catalytic fidelity and diversity. Little is known about heteromeric PTSs, particularly the mechanisms regulating formation of specific products. Here, we report the crystal structure of the (LSU . SSU)(2)-type (LSU/SSU = large/small subunit) heterotetrameric geranyl pyrophosphate synthase (GPPS) from mint (Mentha piperita). The LSU and SSU of mint GPPS are responsible for catalysis and regulation, respectively, and this SSU lacks the essential catalytic amino acid residues found in LSU and other PTSs. Whereas no activity was detected for individually expressed LSU or SSU, the intact (LSU . SSU)(2) tetramer produced not only C(10)-GPP at the beginning of the reaction but also C(20)-GGPP (geranylgeranyl pyrophosphate) at longer reaction times. The activity for synthesizing C(10)-GPP and C(20)-GGPP, but not C(15)-farnesyl pyrophosphate, reflects a conserved active-site structure of the LSU and the closely related mustard (Sinapis alba) homodimeric GGPPS. Furthermore, using a genetic complementation system, we showed that no C(20)-GGPP is produced by the mint GPPS in vivo. Presumably through protein-protein interactions, the SSU remodels the active-site cavity of LSU for synthesizing C(10)-GPP, the precursor of volatile C(10)-monoterpenes.

Figure 1.

Catalytic Reactions and Multiple Sequence Alignment of Plant PTSs. (A) Schematic diagram of catalytic reactions of PTSs. (B) SSUs from Mp GPPS (Mp SSU), Am GPPS (Am SSU), and Hl GPPS (Hl SSU), Abies grandis GPPS (Ag GPPS), P. abies GPPS (Pa GPPS), Arabidopsis thaliana GGPPS (At GGPPS), S. alba GGPPS (Sa GGPPS), and LSUs from Hl GPPS (Hl LSU), Am GPPS (Am LSU), and Mp GPPS (Mp LSU) are included in the alignment. Regions in the SSU and LSU of Mp GPPS corresponding to their respective structural α-helices are denoted by purple and cyan cylinders. Identical and similar amino acid residues are shaded in black and gray, respectively. The conserved functional motifs, DD(X)_nD, are denoted by yellow boxes. The R loop of SSU is boxed in red. The AC loops 1, 2, and 3 of LSU are boxed in blue, gray, and green, respectively. All sequences presented here have the N-terminal signal peptides omitted.

Figure 2.

Architecture of the (LSU · SSU)₂-Type Heterotetrameric Mp GPPS. One LSU subunit is shown as a filled surface model in cyan, and the other LSU subunit is presented as cylinders in blue. Their associated SSU subunits are shown as magenta cylinders and a wheat-colored surface model, respectively. The conserved DD(X)_nD motifs are shown as sticks in magenta and the Mg²⁺ ions as green balls in the LSU. The bound C₅-DMASPP (a substrate analog) and C₅-IPP are also shown as sticks. In the surface models, the two pyrophosphate groups (PPi) are highlighted in yellow, and the R loop of SSU is colored in red. The extensive interface A (inf-A) associates LSU and SSU into an LSU · SSU dimer. Interface B (inf-B) mediates interactions between two LSU · SSU dimers in an (LSU · SSU)₂ tetramer. The bottom part of the figure shows the model rotated 90°.

Figure 3.

In Vitro Product Analysis of Mp GPPS and Conformations of the LSU. (A) Functional assays (thin layer chromatography) of product synthesis of individual LSU and SSU subunits and complexes of Mp GPPS with three allylic substrates (left column) and C₅-[¹⁴C] IPP. The products of wild-type Sc GGPPS and a mutant (S71Y), synthesizing C₂₀-GGPP and C₁₅-FPP, were used as markers (Chang et al., 2006). (B) Surface representations of the open-form (Mp GPPS-Mg²⁺) and the closed-form [Mp GPPS-Mg²⁺/IPP/DMASPP (I)] of LSU. The C₅-DMASPP (green) and C₅-IPP (yellow) ligands are shown on the surface models and the Mg²⁺ ions as purple balls. AC loops 1, 2, and 3 are highlighted in blue, gray, and green, respectively. Yellow arrows indicate the CP hole for product elongation beyond C₁₀-GPP from the AC (orange dotted circle) into the EC (purple dotted circle). (C) Superposition of the structures of Sa GGPPS and Mp GPPS. It is likely that C₂₀-GGPP (cyan, from Sa GGPPS) extends through the CP hole into the hydrophobic EC of LSU (green surface). C₅-IPP (green, from Mp GPPS-IPP) and C₁₀-GPP (magenta, from Mp GPPS-Mg²⁺/GPP) are represented as sticks.

Figure 4.

Function of AC Loop 3. (A) C-terminal conformational changes of LSU upon C₅-IPP binding. The three conserved terminal residues (RDN) are shown as colored molecular surfaces (blue for Mp GPPS-Mg²⁺, green for Mp GPPS-Mg²⁺/IPP/DMASPP [II], orange for Mp GPPS-IPP, and cyan for Mp GPPS-Mg²⁺/GPP). The Mg²⁺ ions are shown as purple balls, and the C₅-IPP and C₁₀-GPP ligands are shown as sticks. (B) In vitro analysis (thin layer chromatography) of products of [LSU(Δ(293-295)) · SSU]₂, using C₂₀-GGPP and C₁₅-FPP synthesized by Sc GGPPS and the mutant S71Y as markers.

Figure 5.

Schematic Model of the Two-Chamber Architecture for Product Regulation. The cavities for C₅-IPP (misoriented), AC, CP hole, and EC are presented in green, gray, cyan, and purple, respectively. The homoallylic substrate of C₅-IPP, the allylic substrates of C₅-DMAPP, C₁₀-GPP, C₁₅-FPP, and C₂₀-GGPP, and PPi are shown as sticks. The Mg²⁺ ions are presented as purple balls. The AC loop 1 of LSU is depicted as a blue line. The size of the gray arrow indicates the level of catalytic efficiency in the three individual steps of the catalytic reactions (see Figure 1A). The red crosses indicate that the catalytic reactions beyond the first step in vivo are blocked via intersubunit interactions.