Crystal structure of thermospermine synthase from Medicago truncatula and substrate discriminatory features of plant aminopropyltransferases

Abstract

Polyamines are linear polycationic compounds that play a crucial role in the growth and development of higher plants. One triamine (spermidine, SPD) and two tetraamine isomers (spermine, SPM, and thermospermine, TSPM) are obtained by the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine and SPD. These reactions are catalyzed by the specialized aminopropyltransferases. In that respect, plants are unique eukaryotes that have independently evolved two enzymes, thermospermine synthase (TSPS), encoded by the gene ACAULIS5, and spermine synthase, which produce TSPM and SPM, respectively. In this work, we structurally characterize the ACAULIS5 gene product, TSPS, from the model legume plant Medicago truncatula (Mt). Six crystal structures of MtTSPS - one without ligands and five in complexes with either reaction substrate (SPD), reaction product (TSPM), or one of three cofactor analogs (5'-methylthioadenosine, S-adenosylthiopropylamine, and adenosine) - give detailed insights into the biosynthesis of TSPM. Combined with small-angle X-ray scattering data, the crystal structures show that MtTSPS is a symmetric homotetramer with an interdomain eight-stranded β-barrel. Such an assembly and the presence of a hinge-like feature between N-terminal and C-terminal domains give the protein additional flexibility which potentially improves loading substrates and discarding products after the catalytic event. We also discuss the sequence and structural features around the active site of the plant aminopropyltransferases that distinguish them from each other and determine their characteristic substrate discrimination.

Keywords: crystallography; enzyme–substrate interactions; polyamines; small-angle scattering.

Figure 1.. SAXS data.

(A) The experimental curve for MtTSPS. (B) Guinier plot (blue dots) of the scattering curve with the best fit shown as a dashed black line. (C) Pair–distance distribution function for MtTSPS SAXS data.

Figure 2.. Monomer of MtTSPS.

(A) Topological diagram of MtTSPS with secondary structure elements. Helices (cylinders) and sheets (arrows) are depicted in red and blue, respectively. (B) Structure of MtTSPS monomer with highlighted active site as solid surface representation. SPD- and dc-SAM-binding sites are shown with light green and purple surfaces, respectively.

Figure 3.. Biological assembly of MtTSPS.

(A) Ab initio averaged envelope (blue mesh) obtained from SAXS data modeling with a superposed crystallographic tetramer. (B) Crystal structure of MtTSPS with transparent surface representation. Chains A and B of the asymmetric unit are shown as cyan and green ribbons, respectively.

Figure 4.. Molecular flexibility of the MtTSPS monomer.

The predicted extent of molecular movements is presented as a superposition of two states (I and II) with a maximal movement amplitude. Monomer of MtTSPS was divided into three regions: core region with lowest flexibility colored in pale cyan (state I) and light pink (state II), α₃-η4/α4 region with moderate flexibility depicted with blue (I) and purple (II) and N-terminal domain with the highest conformational adaptability, cyan (I) and red (II). Hinge residues were marked with black dots. Analysis was made with Hingeprot [70].

Figure 5.. Cofactor binding site.

MTA (A), ADN (B), and ATAM (C) bound in the catalytic site of MtTSPS. 2F_O – F_C electron density maps (blue mesh) are contoured at 1 σ.

Figure 6.. Substrate-binding site.

(A) SPD-binding site. (B) Catalytic site of MtTSPS with superimposed bound ligands: ATAM (purple), SPD (light green), and TSPM (dark green). (C) Bis–Tris propane (BTP) bound in the catalytic site in the MtTSPS–MTA complex. 2F_O – F_C electron density maps (blue mesh) are contoured at 1 σ.

Figure 7.. The comparison of MtTSPS with AtSPDS1.

(A) Superposition of the MtTSPS (cyan) and AtSPDS1 (purple) tetramers. (B) Intersubunit β-barrel created by the N-terminal β-hairpins of the four monomers of MtTSPS (symmetry-related monomers are marked with an asterisk). (C) N-terminal intersubunit β-sheet of AtSPSD1.

Figure 8.. Phylogenetic tree of flowering plant aminopropyltransferases.

The analyzed protein sequences were assigned to the thermospermine synthases (TSPS, 171 sequences), spermine synthases (SPMS, 188), spermidine synthases (SPMS, two subgroups of proteins with 197 and 47 sequences marked in violet and light violet, respectively), and putrescine N-methyltransferases (PMT, 42). The tree was made in MEGA7 [69] using the Neighbor-Joining method [76]. The analysis involved 645 sequences of flowering plant proteins classified to the Polyamine Biosynthesis Domain (IPR030374) by InterPro [77] with the length between 210 and 460 amino acids; significant outliers were manually excluded.

Figure 9.. Sequence conservation around the catalytic site of plant aminopropyltransferases.

Sequences of TSPS, SPMS, and SPDS are aligned and numbers refer to the sequence position in MtTSPS. Representation prepared in WebLogo [71] using multiple sequence alignment of the protein sequences assigned to each group of enzymes by phylogenetic analysis. Each position is presented as stacks of symbols, where the overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position. Violet color denotes residues mentioned in the text, whereas red highlights residues which distinguish TSPS from SPMS and SPDS.

Figure 10.. Architecture of the catalytic site of aminopropyltransferases.

Close-up to the polyamine groove in the structure of MtTSPS in complex with SPD (A), AtSPDS (B), and HsSPMS in complex with SPD (C). For clarity of the comparison, all sequence positions refer to the sequence of MtTSPS.