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 Å 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 Nterminal β-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.The nearly ubiquitous polyamines (putrescine, spermidine and spermine) are polycationic mediators of cell proliferation and differentiation 1 whose functions likely provide both stability and neutralization for nucleic acids. The abundance of polyamines is tightly regulated through biosynthesis, degradation, uptake and efflux 2 . The biosynthesis of polyamines is carried out by three highly conserved polyamine biosynthetic enzymes ( Fig. 1): ornithine decarboxylase, putrescine amino-propyltransferase (PAPT) and spermidine aminopropyltransferase (SAPT). The strong correlation of polyamine synthesis with cell growth renders these aminopropyl transferases (APTs) attractive targets for the development of antiproliferative therapeutics. The APTs are known to be inhibited by their common nucleoside product, as well as other nucleoside analogs. Most inhibitors, such as the nucleoside-polyamine adduct 4 , is also a potent inhibitor of PAPT.The catalytic mechanism of polyamine biosynthesis is unclear, partly because there is no detailed structural information for this class of enzymes. To provide structural insights into the mechanism of spermidine synthesis, the crystal structure of PAPT from Thermotoga maritima (TmPAPT) was determined at high resolution in ligand-free and AdoDATO-bound states. These structures provide insight into the function of the enzyme and its potential mechanism of action. The identification of conserved active site residues should enable accurate models of the mammalian enzymes. The PAPT foldThe structure of TmPAPT was solved using multiwavelength anomalous diffraction (MAD) from selenomethionine (SeMet)-containing crystals. The TmPAPT monomer consists of two domains: an N-terminal domain, composed of six β-strands, and a Rossmann-like C-terminal domain (Fig. 2). The larger C-terminal catalytic core domain (residues 75-296) consists of a seven-stranded β-sheet with a strand order of 9↑, 8↑, 7↑, 10↑, 11↑, 13↓, 12↑ flanked by nine α-helices. This domain resembles a topology observed in...

Characterization of the spermidine synthase‐related gene family in Arabidopsis thaliana

“…Alternatively, it is possible that the major enzymatic function of SPDS3 is not in the synthesis of spermidine in Arabidopsis tissues and that it cannot be reproduced in yeast cells. The recently published crystal structure of a spermidine synthase from Thermotoga maritima revealed deep cavities for binding substrate and cofactor, and a loop that envelops the active site [21]. We note that the Phe residue that is invariant in all known spermidine synthases and likely contributes to the formation of a putrescine-binding cavity [21] is substituted by Val236 in SPDS3 (Fig.…”

Binding and inhibition of human spermidine synthase by decarboxylated S‐adenosylhomocysteine

“…14,16 Assay of aminopropyltransferase activity Activity was measured by following the production of [ 35 S]MTA from [ 35 S]dcAdoMet in 100 mM sodium phosphate buffer (pH 7.5), in the presence of spermidine or spermine as the amine acceptor. 30 Reactions were run in triplicate for 1 h with an amount of enzyme that gave a linear rate of MTA production at 37 C and concentrations of inhibitor that gave from 10 to 90% inhibition.…”

“…Some aminopropyltransferases such as human spermidine synthase (SpdS) (which acts upon putrescine) and spermine synthase (SpmS) (acting on spermidine) are highly specific for their amine acceptors, [12][13][14] while others, such as those from acute thermophiles, which contain a variety of polyamines not found in mammals, are less discriminating. 12,13,[15][16][17] There are now numerous published structures for aminopropyltransferases including those for SpdS from Thermotoga maritima (TmSpdS), 16 Caenorhabditis elegans, 18 Plasmodium falciparum (PfSpdS), 19 Helicobacter pylori, 20 human (hSpdS), 13 Arabidopsis thaliana (PDB code 2Q41), and Trypanosoma cruzi (PDB code 3BWC), aminopropylagmatine/aminopropylcadaverine synthases from Thermus thermophilus (PDB code 1UIR), Pyrococcus horikoshii (PDB code 2ZSU) and Pyrococcus furiosus, 15 and SpmS from humans (hSpmS). 14 A general mechanism for aminopropyl transfer has been proposed based on kinetic studies of hSpdS, TmSpdS, and hSpmS, their structures with bound substrates and inhibitors, and the results of site-directed mutagenesis of key residues (Fig.…”

“…1). 13,16 This mechanism depends on a conserved Asp (residue 173 in hSpdS) in the active site that interacts with the bound amine substrate to deprotonate the attacking nitrogen atom of the amine. This interaction is reinforced by additional interactions of this nitrogen atom with the hydroxyl group of a conserved Tyr (residue 79 in hSpdS) and a backbone carbonyl group (Ser174 in hSpdS).…”

Structural and mechanistic insights into the action of Plasmodium falciparum spermidine synthase

“…Thus, AdoDATO is a compound containing both substrate and product moieties. 29 AdoDATO will be referred to as a substrate analogue from here on. Models were built using Modeller with and without AdoDATO.…”