Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

Abstract

Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids.

Figure 1

Families with pyrrolizidine alkaloid-containing species within the angiosperms (modified from ref. 34).

Figure 2

Reactions catalyzed by HSS (A) and DHS (B) in their metabolic background. Both enzymes transfer an aminobutyl moiety of spermidine to their individual substrates in an NAD⁺-dependent reaction. (A) HSS catalyzes the first step in PA biosynthesis, generating homospermidine as the precursor of the necine base, the typical backbone structure of PAs. (B) DHS commences the posttranslational activation of eIF5A by modifying a protein-bound lysine residue to deoxyhypusine, which is hydroxylated in a second step to hypusine by deoxyhypusine monooxygenase.

Figure 3

Purification of HSS from roots of S. vernalis. Indian ink stain of an SDS/PAGE on a 12% gel blotted onto a poly(vinylidene difluoride)-membrane. A 10-kDa protein ladder (Life Technologies, Grand Island, NY) (lane 1, the 50-kDa band is indicated by an arrow), crude extract (76.5 μg, lane 2), and the pooled fractions containing HSS activity of DEAE Fractogel (NaCl-elution, 9.5 μg, lane 3), Phenyl Sepharose (1.6 μg, lane 4), DEAE Fractogel (pH-elution, 1.5 μg, lane 5), spermidine-affinity chromatography and gel filtration (0.6 μg, lane 6), and Mono Q (0.6 μg, lane 7).

Figure 4

Alignment of the amino acid sequences of HSS and DHS from S. vernalis (Sv-HSS and Sv-DHS, respectively), DHS from tobacco (Nt-DHS), and DHS from human (Hs-DHS). Identical amino acids in all sequences are shown in black boxes, conservative replacements according to the criteria of BLOSUM matrix (35) are framed. The four peptides sequenced from the purified HSS of S. vernalis are indicated by black bars below the alignment. The amino acid sequence homology of Sv-DHS to Nt-DHS and Hs-DHS is 80% and 61% identity, respectively. The amino acid sequence homology of Sv-HSS to Sv-DHS, Nt-DHS, and Hs-DHS is 79%, 74%, and 61% identity, respectively.