Abstract
Spermidine synthases (SPDSs) catalyze the production of the linear triamine, spermidine, from putrescine. They utilize decarboxylated S-adenosylmethionine (dc-SAM), a universal cofactor of aminopropyltransferases, as a donor of the aminopropyl moiety. In this work, we describe crystal structures of two SPDS isoforms from Arabidopsis thaliana (AtSPDS1 and AtSPDS2). AtSPDS1 and AtSPDS2 are dimeric enzymes that share the fold of the polyamine biosynthesis proteins. Subunits of both isoforms present the characteristic two-domain structure. Smaller, N-terminal domain is built of the two β-sheets, while the C-terminal domain has a Rossmann fold-like topology. The catalytic cleft composed of two main compartments, the dc-SAM binding site and the polyamine groove, is created independently in each AtSPDS subunits at the domain interface. We also provide the structural details about the dc-SAM binding mode and the inhibition of SPDS by a potent competitive inhibitor, cyclohexylamine (CHA). CHA occupies the polyamine binding site of AtSPDS where it is bound at the bottom of the active site with the amine group placed analogously to the substrate. The crystallographic snapshots show in detail the structural rearrangements of AtSPDS1 and AtSPDS2 that are required to stabilize ligands within the active site. The concerted movements are observed in both compartments of the catalytic cleft, where three major parts significantly change their conformation. These are (i) the neighborhood of the glycine-rich region where aminopropyl moiety of dc-SAM is bound, (ii) the very flexible gate region with helix η6, which interacts with both, the adenine moiety of dc-SAM and the bound polyamine or inhibitor, and (iii) the N-terminal β-hairpin, that limits the putrescine binding grove at the bottom of the catalytic site.
Highlights
Putrescine (PUT), a linear diamine, is produced by plants from agmatine or ornithine
The Complementary DNA (cDNA) was used as a template for a polymerase chain reaction in order to isolate AtSPDS1 and AtSPDS2 open reading frames (ORF), which are annotated in the GenBank as AJ251296.1 and AJ251297.1, respectively
AtSPDS1 and AtSPDS2 share the fold of polyamine biosynthesis proteins with the characteristic two-domain topology (Figure 1D)
Summary
Putrescine (PUT), a linear diamine, is produced by plants from agmatine or ornithine. Agmatine route, which originated in plants by the horizontal gene transfer from the cyanobacterial ancestor of the chloroplast (Michael, 2017), involves three enzymes: arginine decarboxylase (ADC), agmatine iminohydrolase (AIH), and N-carbamoylputrescine amidohydrolase (CPA). The first enzyme of the pathway, pyridoxal 5 -phosphate-dependent ADC, converts arginine to agmatine. Agmatine is hydrolyzed to N-carbamoylputrescine by AIH, a dimeric enzyme with a 5-bladed propeller fold (Sekula and Dauter, 2019). The alternative route for PUT biosynthesis, ornithine pathway, is carried out by ornithine decarboxylase (ODC). This enzyme is not common for all plant species and some plants lack ODC relying only on agmatine biotransformation (Hanfrey et al, 2001)
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