Abstract

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

Introduction

Spermidine is a natural aliphatic amine that belongs to the most abundant polyamines in the cell, including putrescine and spermine. Biosynthesis of spermidine takes place in all organisms from bacteria, thermophilic bacteria, fungi, plants, protozoa and animals. All polyamines have basic chemical properties containing two or more nitrogen atoms that allow them to interact with negatively charged biomolecules, such as DNA and RNA [1]. Moreover, spermidine and other polyamines, such as putrescine or cadaverine and spermine are substrates for amine oxidases, a group of enzymes implicated in cell defence that perform oxidative degradation of polyamines with the production of hydrogen peroxide and corresponding amino aldehyde [2]. As a ubiquitous polyamine, spermidine is involved in a wide range of developmental processes and cell growth [3]. Unbalanced polyamine levels can cause cell arrest or cell death [4], making the enzymes of the spermidine biosynthetic pathway important targets for the development of chemotherapeutic agents to treat cancer and parasitic diseases, such as malaria, Chagas disease, leishmaniasis and sleeping sickness [58].

Spermidine is synthesized by spermidine synthase (SPDS, EC 2.5.1.16) from the substrates putrescine and decarboxylated S-adenosylmethionine (dcAdoMet, also known as dcSAM). SPDSs from most organisms, such as bacteria, plants and mammals, are highly specific for putrescine as the amine acceptor [913]. However, the enzymes from the thermophiles, Pyrococcus furiosus, Thermotoga maritima and Thermus thermophilus, are less specific for putrescine and they can use other polyamines as amine acceptors, like cadaverine and agmatine not found in mammals [1315]. SPDS recently identified from a freshwater unicellular cyanobacterium Synechococcus sp. PCC 7942 (SySPDS) is also highly specific for putrescine with a Km value of 111 µM and does not use other polyamines as substrates [16,17]. In Synechococcus, spermidine biosynthetic gene expression is up-regulated with a concomitant increase in spermidine levels associated with changes of osmotic conditions [17]. This emphasizes the importance of SPDS for Synechococcus in the production of spermidine under stress conditions.

The SPDS crystal structure from T. maritima was the first to be determined [18] and later on over 40 crystal structures from bacteria, protozoan parasites, worms, human and plants have been solved alone or in complex with various ligands (Supplementary Table S1). The structure of SPDS is folded into two domains: an N-terminal β-sheet domain and a C-terminal Rossmann fold-type domain structurally related to S-adenosyl-l-methionine-dependent methyltransferase (SAM MTase) superfamily that uses dcAdoMet as a methyl donor. The general mechanism of SPDS comprises deprotonation of the amino group of putrescine by a highly conserved Asp residue in the active site [13,19]. The reaction consists of the transfer of the aminopropyl group attached to the sulfonium atom of dcAdoMet to putrescine with the generation of spermidine and the by-product 5′-deoxy-5′-methylthioadenosine (MTA). Binding of the substrates induces the ordering process of a flexible loop in SPDS, named the gate-keeping loop, critical for the positioning of the substrates in the active site [13]. This loop is generally observed in the SPDS crystal structures when either the dcAdoMet substrate or MTA product is present, as well as in structures bound to inhibitors that mimic both dcAdoMet and MTA, such as AdoDATO and dcSAH (Supplementary Table S1). However, no crystal structure with only bound putrescine has been reported so far (Supplementary Table S1). Recent inhibitory screening studies with the compound BIPA (5-(1H-benzimidazol-2-yl)pentan-1-amine), designed to bind the putrescine-binding site with the aminopentanyl moiety and the benzimidazole ring to the aminopropyl site [20], also showed ordering of the gate-keeping loop in Plasmodium falciparum SPDS (PDB: 4CWA, [21]). This suggests that reorganization of the gate-keeping loop requires a ligand present in the dcAdoMet-binding site, as proposed earlier. Selectivity for the putrescine substrate has been suggested to result from the presence of a proline residue located in the gate-keeping loop, whose absence in T. maritima SPDS (substituted by glutamine residue) makes the loop more flexible to accommodate other diamines rather than putrescine [13]. On the contrary, either the absence or a longer gate-keeping loop as in Helicobacter pylori SPDS [22] or T. thermophilus SPDS [14], respectively, makes the binding cavity more accessible for other amine substrates than putrescine. Key highly conserved residues involved in the reaction mechanism have been identified by site-directed mutagenesis [13,23] and supported by the analysis of crystal structures alone and with numerous possibilities of bound substrates/products and inhibitors. Our recent study suggested that most of the ligand-binding residues in SySPDS are the same as in the human and protozoan parasite SPDS enzymes [17].

Here, we describe the SySPDS crystal structure, which is highly conserved with its homologues. Although variation in the position of the flexible gate-keeping loop is well known, the position of the backbone carbonyl of the residue X (any amino acid) next to the catalytic Asp (Asp159 in SySPDS) is conserved among SPDS structures. The residue is followed by the gate-keeping loop that in SySPDS can acquire a conformation similar to other SPDSs from eukaryotes and bacterial counterparts. Hence, the SySPDS structure with both amine substrate and product bound to the active sites provides knowledge on the mechanistic process of spermidine formation in SPDS, where the gate-keeping loop plays a key role in the preparation of the active site of SPDS for catalysis.

Materials and methods

Protein production

The spds gene was amplified from gDNA of Synechococcus sp. PCC 7942 using custom primers listed in Table 1 and subsequently subcloned into NdeI and XhoI restriction sites of the expression pET22b vector [17]. C-terminal hexahistidine-tagged SPDS protein was recombinantly induced for 3 h at 37°C by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside to BL21 Escherichia coli at the logarithmic phase and thereafter purified by affinity chromatography. Briefly, 500 ml cell culture was harvested by centrifugation and the cell pellet was resuspended in lysis buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. Cells were disrupted by sonication followed by centrifugation. The lysate was loaded onto a HisTrap HP 1 ml column (GE Healthcare Life Sciences). Contaminants were removed by washing with lysis buffer followed by one-step wash with 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 20 mM imidazole. SySPDS was eluted with 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 250 mM imidazole. The fractions containing the desired protein were pooled and concentrated using Amicon Ultra filters (Millipore, Ireland). The Synechococcus sp. PCC 7942 SPDS (SySPDS) protein (UniProt accession code Q31QK9) was purified further using a Superdex S200 Increase 10/300 GL (GE Healthcare) in buffer 20 mM Tris–HCl (pH 7.4), 150 mM NaCl. Elution fractions corresponding to SySPDS were pooled and evaluated on SDS–PAGE.

Table 1
Macromolecule production information
Source organism Synechococcus sp. PCC 7942 
DNA source gDNA from Synechococcus sp. PCC 7942 
Forward primer 5′-ggaattccatatgagcgctgacgcacccgttt-3′ 
Reverse primer 5′-ccgctcgaggccttgacgagcgctcaga-3′ 
Expression vector pET22b 
Expression host BL21(DE3) Escherichia coli 
Complete amino acid sequence of the construct produced MSADAPVWIDEVFEDRVRYGLRGQILWEETSPFQKITIVDTEHYGRGLLLDDCWMTAERC 
 EVCYHEYLVHPPLTTAASIARVLVIGGGDGGTVREVLRYAEVEQVDLVEIDGRVVELSQE 
 YLGAIGTAWADPRLNVKIGDGIAFVQTAPDASYDVILVDGSDPAGPAAGLFNREFYENCR 
 RVLKPGGVFASQAESPDSFLAVHLEMIETLSAVFAEAKPYYGWVPMYPSGWWSWLYASDT 
 PGQFQKPQSDRLAAIEPQVEIYNRDIHQAAFAQPNFVRRGLSARQGLERHHHHHH 
Source organism Synechococcus sp. PCC 7942 
DNA source gDNA from Synechococcus sp. PCC 7942 
Forward primer 5′-ggaattccatatgagcgctgacgcacccgttt-3′ 
Reverse primer 5′-ccgctcgaggccttgacgagcgctcaga-3′ 
Expression vector pET22b 
Expression host BL21(DE3) Escherichia coli 
Complete amino acid sequence of the construct produced MSADAPVWIDEVFEDRVRYGLRGQILWEETSPFQKITIVDTEHYGRGLLLDDCWMTAERC 
 EVCYHEYLVHPPLTTAASIARVLVIGGGDGGTVREVLRYAEVEQVDLVEIDGRVVELSQE 
 YLGAIGTAWADPRLNVKIGDGIAFVQTAPDASYDVILVDGSDPAGPAAGLFNREFYENCR 
 RVLKPGGVFASQAESPDSFLAVHLEMIETLSAVFAEAKPYYGWVPMYPSGWWSWLYASDT 
 PGQFQKPQSDRLAAIEPQVEIYNRDIHQAAFAQPNFVRRGLSARQGLERHHHHHH 

Protein crystallization

The SySPDS protein was concentrated to 10–20 mg/ml range and used for initial crystallization trials (Table 2). Crystallization conditions were screened using sitting drop vapour diffusion in 96-well plates. Crystals of SySPDS grew after 2 days using 10 mg/ml of protein under 0.2 M sodium chloride, 0.1 M MES (pH 6.0), 45% v/v pentaerythritol propoxylate (5/4 PO/OH) (PXN) at 21 °C. Crystals were flash-frozen in liquid nitrogen using 10% glycerol in the crystallization buffer.

Table 2
Crystallization conditions
Method Sitting drop vapour diffusion 
Plate type 96-well (TTP Labtech) 
Temperature (K) 294.15 
Protein concentration 10 mg/ml 
Buffer composition of protein solution 25 mM Tris–HCl (pH 7.4), 150 mM NaCl 
Composition of reservoir solution 0.1 M MES (pH 6.0), 45% v/v pentaerythritol propoxylate (5/4 PO/OH) 
Volume and ratio of drop 2 µl protein:1 µl reservoir 
Volume of reservoir 60 µl 
Method Sitting drop vapour diffusion 
Plate type 96-well (TTP Labtech) 
Temperature (K) 294.15 
Protein concentration 10 mg/ml 
Buffer composition of protein solution 25 mM Tris–HCl (pH 7.4), 150 mM NaCl 
Composition of reservoir solution 0.1 M MES (pH 6.0), 45% v/v pentaerythritol propoxylate (5/4 PO/OH) 
Volume and ratio of drop 2 µl protein:1 µl reservoir 
Volume of reservoir 60 µl 

Data collection and processing

Crystals of SySPDS diffracted to 2.18 Å resolution at the beamline ID30A-3, European Synchrotron Radiation Facility (ESRF), Grenoble, France. Crystals tested without glycerol diffracted to resolution lower than 3.0 Å. Glycerol was used to minimize mosaicity and the best crystals diffracted to 2.18 Å. Datasets were collected and processed using XDS [24]. Data processing statistics are given in Table 3.

Table 3
Data collection and processing

Values for the outer shell are given in parentheses.

Diffraction source ID30A-3/MASSIF-3, ESRF 
Wavelength (Å) 0.9677 
Temperature (K) 100 
Detector EIGER 
Crystal-detector distance (mm) 169.39 
Rotation range per image (°) 0.3 
Total rotation range (°) 210 
Exposure time per image (s) 0.02 
Space group P21 
a, b, c (Å) 57.64, 63.82, 73.50 
α, β, γ (°) 90.00, 92.72, 90.00 
Mosaicity (°) 0.13 
Resolution range (Å) 48.17–2.18 (2.27–2.18) 
Total no. of reflections 115060 (11509) 
No. of unique reflections 27395 (2739) 
Completeness (%) 99 (98.3) 
Redundancy 4.2 (4.1) 
I/σ(I)〉 16.18 (6.67) 
Rmeas 0.083 (0.261) 
Overall B-factor from Wilson plot (Å217.00 
CC1/2 0.997 (0.956) 
Z-score 10.31 
Matthews coefficient 2.11 
Diffraction source ID30A-3/MASSIF-3, ESRF 
Wavelength (Å) 0.9677 
Temperature (K) 100 
Detector EIGER 
Crystal-detector distance (mm) 169.39 
Rotation range per image (°) 0.3 
Total rotation range (°) 210 
Exposure time per image (s) 0.02 
Space group P21 
a, b, c (Å) 57.64, 63.82, 73.50 
α, β, γ (°) 90.00, 92.72, 90.00 
Mosaicity (°) 0.13 
Resolution range (Å) 48.17–2.18 (2.27–2.18) 
Total no. of reflections 115060 (11509) 
No. of unique reflections 27395 (2739) 
Completeness (%) 99 (98.3) 
Redundancy 4.2 (4.1) 
I/σ(I)〉 16.18 (6.67) 
Rmeas 0.083 (0.261) 
Overall B-factor from Wilson plot (Å217.00 
CC1/2 0.997 (0.956) 
Z-score 10.31 
Matthews coefficient 2.11 

Structure solution and refinement

The SySPDS structure was solved by molecular replacement using the programme Phaser [25], searching for Trypanosoma cruzi SPDS (PDB entry 4YUV; 8) as an input model. Iterative model building of the amino acid residues corresponding to SySPDS was manually built into the electron density in Coot [26]. Refinement of the model was carried out by 20 cycles of restrained refinement using Refmac5 from CCP4 suite [27,28]. After a couple of refinement rounds, additional electron densities were observed in the cavities where putrescine, spermidine and MTA are known to bind (Supplementary Table S1). In addition, electron density that matches the PXN shape was present on the surface of chain B. PDB ligand libraries for putrescine, spermidine, MTA and PXN were produced by Grade Web server version 1.2.13 from Global Phasing Ltd. [29]. These ligands were placed into the corresponding electron densities and the SySPDS structure with the bound ligands was subjected to additional rounds of refinement. The final model has putrescine in chain A, spermidine, MTA and PXN in chain B. TLS refinement was included in the last refinement cycle. As a result of crystal contact, the N-terminus of chain A starts from residue 2. The final SySPDS structure has residues 2–161 and 169–285 in chain A but residues 6–285 in chain B. The last C-terminal residue, Gly286, and the His-tag at the C-terminus are disordered in the crystal structure. Details of data refinement statistics are given in Table 4. The structure has been deposited with the PDB ID 6QMM.

Table 4
Structure solution and refinement for SySPDS

Values for the outer shell are given in parentheses.

Resolution range (Å) 48.17–2.18 (2.27–2.18) 
Completeness (%) 99 (98.3) 
No. of reflections, working set 27,391 (2739) 
No. of reflections, test set 1375 (137) 
Final Rwork 0.142 
Final Rfree 0.191 
Cruickshank DPI 0.2615 
No. of non-H atoms 4728 
Macromolecules 4411 
 Protein residues 557 
 Ligand atoms 61 
 Water 254 
Root-mean-square deviation 
 Bonds (Å) 0.024 
 Angles (°) 2.51 
Average B-factors (Å218.85 
 Protein 18.25 
 Ligand 38.54 
 Water 24.34 
Ramachandran plot 
 Most favoured (%) 94.9 
 Allowed (%) 5.1 
Outliers (%) 
Resolution range (Å) 48.17–2.18 (2.27–2.18) 
Completeness (%) 99 (98.3) 
No. of reflections, working set 27,391 (2739) 
No. of reflections, test set 1375 (137) 
Final Rwork 0.142 
Final Rfree 0.191 
Cruickshank DPI 0.2615 
No. of non-H atoms 4728 
Macromolecules 4411 
 Protein residues 557 
 Ligand atoms 61 
 Water 254 
Root-mean-square deviation 
 Bonds (Å) 0.024 
 Angles (°) 2.51 
Average B-factors (Å218.85 
 Protein 18.25 
 Ligand 38.54 
 Water 24.34 
Ramachandran plot 
 Most favoured (%) 94.9 
 Allowed (%) 5.1 
Outliers (%) 

Mass spectrometry analysis of putrescine

0.1 mM of purified SySPDS was mixed 2:1 ratio with reservoir solution (Table 2) and heated to 95°C for 10 min. The sample was clarified by centrifugation for 30 min at room temperature and the supernatant was used for mass analysis. The supernatant was purified by solid phase extraction before analysis. Cartridge (Sep-Pak® tC18, 100 mg, Waters) was conditioned with 2 ml of methanol and 2 ml of water. 100 µl of supernatant was passed through the cartridge. Analytes were then eluted with 0.6 ml of water:acetic acid (98:2, v/v).

Chromatographic analyses were performed by an Agilent 1100 series liquid chromatography system. Separations were conducted using gradient elution on an Ascentis® Express RP-Amide analytical column (2.1 × 100 mm, particle size 2.7 µm, Supelco). Mobile phases were 5 mM ammonium formate in water (A) and acetonitrile (B). The gradient condition was 80% of B (0–8 min), from 80% to 10% (8–12 min) and 10% (12–30 min). The flow rate was 0.35 ml/min. Mass detection was performed in the selected-ion monitoring (SIM) mode with a single quadrupole mass spectrometer (HP 1100 LC/MSD). Ionization was based on electrospray ionization in the positive mode. The capillary voltage was 3.5 kV and the drying gas temperature was 350°C. The selected ion for putrescine was m/z 89.1 that corresponds to its protonated molecule [M + H]+.

Results and discussion

Dimerization state of SySPDS

SPDS is generally functional as a dimer. Exceptional cases of higher oligomeric states have been reported for thermophilic T. thermophilus and T. maritima SPDS, which are tetrameric [14,18]. In addition, H. pylori SPDS is dimeric and tetrameric in solution [22]. SySPDS has a theoretical molecular mass of 31.851 kDa for the monomeric form. The molecular mass of SySPDS in solution was estimated using size exclusion chromatography, which resulted in a single peak corresponding to a dimer of 67 kDa (Figure 1A). The elution fractions corresponding to SySPDS dimer were evaluated on SDS–PAGE and showed nearly 100% purity (Figure 1B, fractions 1–4).

Dimerization of SySPDS observed using size exclusion chromatography.

Figure 1.
Dimerization of SySPDS observed using size exclusion chromatography.

(A) Elution profile for SySPDS from Superdex 200 Increase 30/100 GL column with maximum absorption corresponding to 67 kDa. Molecular mass markers are shown in the inset figure for horse myoglobulin (17 kDa), chicken ovalbumin (44 kDa), bovine γ-globulin (158 kDa) and bovine thyroglobulin (670 kDa). (B) SDS–PAGE of the elution fractions 1–4 corresponding to the SySPDS dimer.

Figure 1.
Dimerization of SySPDS observed using size exclusion chromatography.

(A) Elution profile for SySPDS from Superdex 200 Increase 30/100 GL column with maximum absorption corresponding to 67 kDa. Molecular mass markers are shown in the inset figure for horse myoglobulin (17 kDa), chicken ovalbumin (44 kDa), bovine γ-globulin (158 kDa) and bovine thyroglobulin (670 kDa). (B) SDS–PAGE of the elution fractions 1–4 corresponding to the SySPDS dimer.

As in solution, the crystal structure of SySPDS solved at 2.18 Å also is a homodimer in the asymmetric unit (Figure 2A,B). Similar to its bacterial and eukaryotic homologues, each monomer of the SySPDS homodimer comprises an N-terminal β-strand domain (residues 1–57) and a C-terminal domain with the Rossmann fold-like topology (residues 58–286). The N-terminal domain is composed of six antiparallel β-strands and the C-terminal domain is structurally related to class I methyltransferases that use dcAdoMet as a methyl donor. The active site lies in the C-terminal domain covered by the N-terminal domain. The C-terminal domain consists of four parallel and three antiparallel β-strands flanked by eight α helices (Figure 2A,B).

Structure of SySPDS with bound substrate and product.

Figure 2.
Structure of SySPDS with bound substrate and product.

Side (A) and top (B) view of the SPDS dimer with the N-terminal β-stranded domain of each monomer shown in light and dark purple, respectively and the C-terminal Rossmann fold-like domain with the β-stranded core (blue) shown in cyan and turquoise, respectively. Putrescine (PUT, olive), spermidine (SPD, orange) and 5′-methylthioadenosine (MTA, green) are shown in sticks.

Figure 2.
Structure of SySPDS with bound substrate and product.

Side (A) and top (B) view of the SPDS dimer with the N-terminal β-stranded domain of each monomer shown in light and dark purple, respectively and the C-terminal Rossmann fold-like domain with the β-stranded core (blue) shown in cyan and turquoise, respectively. Putrescine (PUT, olive), spermidine (SPD, orange) and 5′-methylthioadenosine (MTA, green) are shown in sticks.

Crystal structure of SySPDS with bound substrate and product

Previous structures of SPDS proteins have been solved with either of the amine/dcAdoMet substrates, spermidine/MTA products or one substrate/one product (Supplementary Table S1). The structures show the two interconnected binding cavities for putrescine and dcAdoMet, which are the same cavities where the products spermidine and MTA are formed. None of these structures has bound putrescine substrate alone and spermidine product in the same crystal structure (Supplementary Table S1). In our SySPDS, however, additional electron density was observed in both the polyamine and dcAdoMet-binding pockets after one cycle of rigid refinement without ligands (Figure 3A). Placement of putrescine and spermidine/MTA into the electron density followed by refinement cycles confirmed the presence of putrescine in monomer 1 and spermidine/MTA in monomer 2 (Figure 3A,B). As we did not add any ligand in the crystallization set-up, the ligands observed in the SySPDS complex (Figure 2A,B) originated from the E. coli expression system. We also confirmed the presence of putrescine in the purified protein sample by mass spectrometry (Supplementary Figure S1).

SySPDS in complex with putrescine, spermidine and MTA.

Figure 3.
SySPDS in complex with putrescine, spermidine and MTA.

(A,B) Surface representation of the SySPDS dimer shown in white colour and viewed at 0° and 110°. The cavities I and II are labelled in monomers 1 and 2. (A) The Fo − Fc difference electron density maps (green, 2.2sigma) calculated after one refinement cycle without ligands are shown in each cavity of the monomers 1 and 2. (B) The 2Fo − Fc difference electron density maps calculated with ligands are shown in grey (1.0sigma) with putrescine (PUT), spermidine (SPD) and 5′-methylthioadenosine (MTA) shown in stick. (C) The substrate-binding site in monomer 1. In the absence of dcAdoMet, water molecules (green spheres) occupy its position. Residues involved in interaction with putrescine and waters are represented in sticks. Hydrogen bonds of the residues interacting with putrescine and water molecules are shown as black dashed lines with respective bond distances. (D) The product-binding sites in monomer 2. The same residues as displayed in monomer 1 (panel C) are shown in stick. Pro166 and Ala167 in the gate-keeping loop are shown in stick. Hydrogen bonds with spermidine and MTA are shown as black dashed lines with respective bond distances.

Figure 3.
SySPDS in complex with putrescine, spermidine and MTA.

(A,B) Surface representation of the SySPDS dimer shown in white colour and viewed at 0° and 110°. The cavities I and II are labelled in monomers 1 and 2. (A) The Fo − Fc difference electron density maps (green, 2.2sigma) calculated after one refinement cycle without ligands are shown in each cavity of the monomers 1 and 2. (B) The 2Fo − Fc difference electron density maps calculated with ligands are shown in grey (1.0sigma) with putrescine (PUT), spermidine (SPD) and 5′-methylthioadenosine (MTA) shown in stick. (C) The substrate-binding site in monomer 1. In the absence of dcAdoMet, water molecules (green spheres) occupy its position. Residues involved in interaction with putrescine and waters are represented in sticks. Hydrogen bonds of the residues interacting with putrescine and water molecules are shown as black dashed lines with respective bond distances. (D) The product-binding sites in monomer 2. The same residues as displayed in monomer 1 (panel C) are shown in stick. Pro166 and Ala167 in the gate-keeping loop are shown in stick. Hydrogen bonds with spermidine and MTA are shown as black dashed lines with respective bond distances.

The general mechanism for putrescine transfer involves a highly conserved Asp residue that deprotonates the nitrogen atom of the bound putrescine substrate [13,19]. Accordingly, the aspartate in SySPDS (Asp159) is in close proximity to the N1 of putrescine in monomer 1 (Figure 3C). The residue Tyr64, the backbone carbonyl of Gly160 and the aromatic ring of Tyr 227 in SySPDS stabilize the aliphatic chain of putrescine (Figure 3C). In human SPDS, the corresponding residues (Tyr79, Ser174 and Tyr 241) are predicted to contribute to the catalytic reaction of N1 of putrescine with the sulfonium atom of dcAdoMet charged with the reacting aminopropyl group [19]. Therefore, they likely have the same function in SySPDS.

The binding cavity I of putrescine connects with cavity II, which is the binding site of dcAdoMet (Figure 3A,B). In monomer 1 of the SySPDS structure, the dcAdoMet-binding pocket is filled with water molecules (Figure 3C), in the same position where the N1 atom of the aminopropyl donor, the ribose ring and nitrogen base bind in other SPDS complexes [8,13,21,31,40]. Interestingly, the same interconnected binding sites are occupied by the products spermidine and MTA in monomer 2 (Figure 3D). Here, residues Gln34, His65, Asp89, Glu109, Asp140 and Asp159 create a charged polar pocket, in which His65, Asp89 and Asp159 interact with the N1 atom of spermidine, and Gln34, Glu109 and Asp140 with the ribose/adenosine of MTA. Pro166 and Ala167 disordered in monomer 1 are visible in monomer 2. They form part of the gate-keeping loop (residues 160–167) (Figure 3D). The gate-keeping loop has been described as the loop forming the active site entrance in SPDS and is highly flexible in the absence of substrates [13]. It is stabilized upon binding of the nucleoside and amine substrates, making the active site inaccessible to solvent. Organization of this loop in SySPDS is dcAdoMet substrate-dependent since no crystal contact is observed with neighbouring asymmetric subunits in the spermidine/MTA monomer. Indeed, the earlier determined structures with bound putrescine also have the MTA product and the gate-keeping loop is visible [13,21]. Likewise, the BIPA inhibitor, which binds to the putrescine-binding site with the aminopentanyl moiety and to the aminopropyl site with its benzimidazole moiety, also stabilizes the gate-keeping loop (PDB: 4CWA, 21). The SySPDS structure shown in this work confirms that binding of the substrate dcAdoMet facilitates arrangement of the gate-keeping loop to close the active site for the catalytic reaction to take place, as proposed earlier [13].

Comparative structural analysis with previously known SPDS structures

The superimposed structures reveal that the overall Synechococcus SPDS structure is similar to its bacterial and eukaryotic homologues. The major difference between SySPDS and SPDS structures is observed in the N-terminal region (Figure 4A). In human, T. cruzi and Caenorhabditis elegans SPDSs, the N-terminus folds in a direction towards the core of the C-terminal domain [8,13,30,31]. Furthermore, the N-terminus can form additional structural elements, i.e. the α-helix in T. maritima SPDS involved in tetramer formation [18] or the N-terminal tail stabilizes the tetrameric form, as in T. thermophilus, Arabidopsis thaliana, H. pylori and Bacillus subtilis SPDSs [22]. Some variations at the C-terminus are seen in T. thermophilus SPDS, which forms two extra β-strands interacting with the gate-keeping loop [14] (Figure 4A, monomer 2). The SySPDS residues at the N- and C-terminus (excluding the first Met and the last Gly) are visible in at least one of the monomers (residues 2–161 and 169–285 in monomer 1, and 6–285 in monomer 2) and do not form extra structural elements that could contribute to higher oligomers (Figure 4A).

Structural differences observed in SPDSs.

Figure 4.
Structural differences observed in SPDSs.

(A) The variable N-terminus of the SPDS structures is shown in colour in monomer 1. SySPDS (cyan) is superimposed onto one SPDS dimer from P. horikoshii (Ph, dark grey; PDB: 2ZSU), T. thermophilus (Tt, beige; PDB: 1UIR, 14), T. maritima (Tm, white; PDB 1INL, 18), B. subtilis (Bs, green; PDB: 1IY9), C. elegans (Ce, olive green; PDB: 2B2C), T. cruzi (Tc, light pink; PDB: 4YUW), A. thaliana (At, pink; PDB: 1XJ5) and H. sapiens (Hs, brown; PDB: 2O06, 13). All monomers from monomer 1 are coloured in dark and light grey for the N-terminal and C-terminal domains, respectively. Putrescine (PUT, olive) is shown in sticks. Monomer 2 shows the C-terminal domain in grey and the different conformations for the gate-keeping loop coloured the same as the N-termini of monomer 1, including E. coli (blue; PBD: 3O4F, 32), P. falciparum (yellow; PDB: 2HTE), P. furiosus (orange; PDB: 1MJF, 15) and H. pylori (magenta; PDB: 2CMG). Spermidine (SPD, orange) and 5′-methylthioadenosine (MTA, green) are shown in sticks. (B) Zoomed view of the active site of SySPDS and the gate-keeping loops of the other SPDSs coloured as monomer 2 in panel A and the residues involved in spermidine and MTA binding in stick. The non-conserved residues in the other SPDSs are shown in parenthesis (Hp: Glu58, Ser59, Leu146, Gln101, and Ser206; Hs: Gln80 in human; Tt: Asp108 and Phe230; Tm: Asn152 and Gln178; Ce: Gln95). The conserved hydrogen bond between the backbone carbonyl of the residue next to Asp159 and the N1 of spermidine is shown as black dashed line and highlighted in a black circle.

Figure 4.
Structural differences observed in SPDSs.

(A) The variable N-terminus of the SPDS structures is shown in colour in monomer 1. SySPDS (cyan) is superimposed onto one SPDS dimer from P. horikoshii (Ph, dark grey; PDB: 2ZSU), T. thermophilus (Tt, beige; PDB: 1UIR, 14), T. maritima (Tm, white; PDB 1INL, 18), B. subtilis (Bs, green; PDB: 1IY9), C. elegans (Ce, olive green; PDB: 2B2C), T. cruzi (Tc, light pink; PDB: 4YUW), A. thaliana (At, pink; PDB: 1XJ5) and H. sapiens (Hs, brown; PDB: 2O06, 13). All monomers from monomer 1 are coloured in dark and light grey for the N-terminal and C-terminal domains, respectively. Putrescine (PUT, olive) is shown in sticks. Monomer 2 shows the C-terminal domain in grey and the different conformations for the gate-keeping loop coloured the same as the N-termini of monomer 1, including E. coli (blue; PBD: 3O4F, 32), P. falciparum (yellow; PDB: 2HTE), P. furiosus (orange; PDB: 1MJF, 15) and H. pylori (magenta; PDB: 2CMG). Spermidine (SPD, orange) and 5′-methylthioadenosine (MTA, green) are shown in sticks. (B) Zoomed view of the active site of SySPDS and the gate-keeping loops of the other SPDSs coloured as monomer 2 in panel A and the residues involved in spermidine and MTA binding in stick. The non-conserved residues in the other SPDSs are shown in parenthesis (Hp: Glu58, Ser59, Leu146, Gln101, and Ser206; Hs: Gln80 in human; Tt: Asp108 and Phe230; Tm: Asn152 and Gln178; Ce: Gln95). The conserved hydrogen bond between the backbone carbonyl of the residue next to Asp159 and the N1 of spermidine is shown as black dashed line and highlighted in a black circle.

As described above, the gate-keeping loop (160–166) that covers the active site in the spermidine/MTA-monomer 2 is visible and disordered in the putrescine–monomer 1 complex (Figure 3C,D). Superimposition of the SPDS structures with SySPDS shows that the loop can acquire different conformations, mainly in the MTA bound state (Figure 4B). For E. coli and B. subtilis SPDS lacking a ligand in the dcAdoMet site, the loop has a different orientation [32] and the absence of gate-keeping loop in H. pylori SPDS creates a different environment in the binding pocket [22]. Interestingly, the residue X (any amino acid) next to the catalytic Asp (Asp159 in SySPDS) is visible in the disordered and ordered gate-keeping loop states (Figures 3C,D and 4B). Its backbone carbonyl plays a role in additional interaction with the substrate putrescine [19]. This residue corresponds to Gly160 in SySPDS, and is replaced by Ser (Pyrococcus horikoshii, Pyrococcus furiosus, B. subtilis, P. falciparum, A. thaliana, C. elegans and human), Cys (E. coli), Leu (T. thermophilus) or Thr (T. cruzi) in other SPDSs. Based on the available SPDS structures, the backbone carbonyl is visible in all reported structures in close proximity to the N1 atom of putrescine in monomer 1 and to the same atom of spermidine in monomer 2 in the SySPDS structure (Figures 3C,D and 4B).

To find out an explanation regarding the presence of putrescine and spermidine ligands in the SySPDS structure, we discuss the regulation of the polyamine biosynthetic pathways briefly. Kinetic studies show that SySPDS and other SPDSs have a higher affinity for dcAdoMet than putrescine [9,11,13,17], which suggests SPDS activity is likely regulated by the availability of dcAdoMet substrate. A single displacement reaction [13,33] has been proposed for SPDS, but it is not yet determined whether SPDS follows an ordered or random mechanism. The concentration of dcAdoMet substrate in the cell is highly regulated by the presence of putrescine [3437]. dcAdoMet is synthesized from the decarboxylation of S-adenosylmethionine by S-adenosylmethionine decarboxylase (AdoMetDC), an enzyme with a short half-life controlled and stimulated by putrescine [34,37]. The overall concentrations of polyamines can be found in the micromolar and few millimolar range with putrescine in lower concentrations than spermidine and spermine in mammalian cells [1,34,36] and higher than spermidine in microorganisms like E. coli [38]. The formation of putrescine takes place from decarboxylation of ornithine by the action of ornithine decarboxylase (ODC), another highly regulated enzyme of the polyamine biosynthetic pathway that maintains putrescine levels for its use mainly in the synthesis of spermidine [1,34,39]. When the activity of ODC is elevated, the product putrescine would be able to bind to SPDS prior to dcAdoMet. The subsequent binding of dcAdoMet could rely on the concentrations of intracellular putrescine that activates AdoMetDC to supply dcAdoMet and further activation of SPDS. As putrescine and spermidine are found in complex with SySPDS in this work, we speculate that overexpression of SySPDS in E. coli may have induced production of putrescine and activated the synthesis of dcAdoMet by the putrescine-dependent enzyme AdoMetDC.

To date, most of the SPDS structures solved in the presence of substrates or products have bound dcAdoMet or MTA in the active site (Supplementary Table S1). Only two structures have been reported in complex with both putrescine and MTA, provided in the crystallization condition or soaking experiments [13,21] (Supplementary Table S1). The concentrations of putrescine:protein used in the crystallization conditions are five to ten molar excess, to allow binding of the substrate in the active site [13,21]. One of the monomers in the structure of SySPDS reported here is bound to putrescine taken from the expression host, as confirmed by MS analysis with the purified protein (Supplementary Figure S1). This suggests that putrescine-dependent SPDS follows a single displacement reaction with the random mechanism, in which putrescine is able to bind first to the polyamine active site, leaving the interconnected binding site open for dcAdoMet entrance (Figure 5). However, a single displacement reaction with ordered mechanism cannot be ruled out as the kinetic studies suggest dcAdoMet to be the preferable first substrate and the subsequent binding of putrescine might rely on the intracellular conditions of dcAdoMet produced by dcAdoMetDC. The backbone carbonyl group of the residue next to the conserved catalytic Asp plays a role in positioning putrescine and in stabilizing the backbone of the gate-keeping loop near it. This mechanism is conserved among the structures. Binding of dcAdoMet induces ordering of the gate-keeping loop, preparing the active site for the transfer of the aminopropyl molecule when putrescine is present in the active site.

General reaction catalysed by SPDS.

Figure 5.
General reaction catalysed by SPDS.

The ternary complex of Put-SPDS-DcAdoMet is formed when either of the substrates (Put or DcAdoMet) can bind to SPDS first.

Figure 5.
General reaction catalysed by SPDS.

The ternary complex of Put-SPDS-DcAdoMet is formed when either of the substrates (Put or DcAdoMet) can bind to SPDS first.

Abbreviations

     
  • AdoMetDC

    S-adenosylmethionine decarboxylase

  •  
  • BIPA

    5-(1H-benzimidazol-2-yl)pentan-1-amine

  •  
  • ESRF

    European Synchrotron Radiation Facility

  •  
  • MTA

    5′-deoxy-5′-methylthioadenosine

  •  
  • ODC

    ornithine decarboxylase

  •  
  • SPDS

    spermidine synthase

Author Contribution

A.P., G.G. and S.S. performed the experiments. G.G., A.P., S.S., A.L., S.J., T.A.S. and A.I. analysed the results. G.G. and T.A.S. wrote the paper with contribution from all authors. T.A.S., A.L. and A.I. provided funding.

Funding

This work was supported by Sigrid Jusélius Foundation (T.A.S.), Tor, Joe, and Pentti Borg's Foundation (T.A.S.), Thailand Research Fund, IRG5780008 (A.I.) and the National Doctoral Programme in Informational and Structural Biology, Åbo Akademi University (S.S.).

Acknowledgements

We thank the scientists of the beamline ID30A-3 at the ESRF for the provision of beam time access and user support. Markus Vehniäinen from the University of Turku is acknowledged for additional access to the Äkta purification facility. We thank the bioinformatics (J.V. Lehtonen), translational activities and structural biology infrastructure support from Biocenter Finland and Instruct-FI, and CSC IT Center for Science for computational infrastructure support at the Structural Bioinformatics Laboratory, Åbo Akademi University.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.