The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor

Abstract

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 A resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal beta-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.

Fig. 1

General pathway for the biosynthesis of putrescine, spermidine and spermine.

Fig. 2

Overall structure of TmPAPT monomer and architecture of the tetramer. a, Stereo view of a ribbon diagram representation of TmPAPT subunit D. The α-helixes are lettered, and β-strands are numbered. AdoDATO is shown in ball-and-stick representation in cyan. The ‘gatekeeping’ loop is shown in red. The conformation of the analogous loop in subunit C upon superposition with subunit D is shown in blue. All structural figures, except Fig. 4a, are made with the ICM. b, Architecture of the TmPAPT tetramer. Ribbon diagrams of subunits A, B, C and D are shown in green, yellow, red and blue, respectively. AdoDATO is shown in CPK representation in magenta. The absence of AdoDATO in subunit B can be explained by the presence of additional electron density near the substrate binding site in both structures, which most likely corresponds to a partially ordered PEG molecule important for crystal packing and preventing AdoDATO from binding to subunit B.

Fig. 3

Sequence alignment of TmPAPT with plant and human PAPTs and with PNMT. Color coding of conserved residues by yellow boxes and invariant residues by red boxes is based on alignment of 19 PAPT sequences from various species (data not shown). Out of the >70 putative aminopropyltransferase sequences in the database, only sequences that have been shown to encode a PAPT by enzymatic assay of the expressed protein or by genetic deletion analysis and those derived from species known to contain only spermidine as the higher polyamine were selected for multiple alignment. Accession numbers are shown on the left. Secondary structure elements of TmPAPT are shown on the top. Numbering is shown for the TmPAPT sequence only. The blue number signs are shown on the top of TmPAPT residues interacting with AdoDATO. The bottom sequence is PNMT from Atropa belladona (BAA82264). Residues of PNMT that deviate from conserved residues of PAPT are shown in green boxes. Sequence alignment was generated by CLUSTALW and displayed with ESPrit.

Fig. 4

Interaction of AdoDATO with TmPAPT. a, A stereo view of F_o − F_c electron density map produced by omitting the AdoDATO from the model during simulated annealing refinement and map calculation, and contoured at 2.0 σ level around AdoDATO bound to subunit D (generated with O24). b, Close-up stereo view of substrate binding site. The view is shown from the N-terminal domain toward the C-terminal domain. AdoDATO and selected residues involved in inhibitor binding are shown in stick representation. Nitrogens, oxygens and sulfurs are shown in blue, red and yellow, respectively. Carbons of TmPAPT residues are shown in gray, and carbons of AdoDATO are shown in cyan. The water molecule is shown as an orange sphere. Hydrogen bonds are shown by thin black lines. The C-terminal domain is shown in light yellow transparent ribbon representation. The gatekeeping loop is shown by green worm representation and the corresponding loop of subunit C by transparent blue worm representation. The C11 group of AdoDATO corresponding to attacking amino group of putrescine is highlighted by magenta. The proposed hydrogen bond involved in deprotonation of the corresponding amino group of putrescine is shown by a magenta line. c, Schematic LIGPLOT diagram of the interactions between AdoDATO (bonds shown in violet) and TmPAPT. Protein side chains that form hydrogen bonds with AdoDATO are shown with bonds in orange. Hydrogen bonds are drawn as dashed lines, and the donor-acceptor distances are given. Residues involved in hydrophobic interactions are shown with arcs. Residue names and numbers are highlighted by colors corresponding to colors on structural alignment picture (Fig. 3).