The long road of functional recruitment-The evolution of a gene duplicate to pyrrolizidine alkaloid biosynthesis in the morning glories (Convolvulaceae)

Abstract

In plants, homospermidine synthase (HSS) is a pathway-specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA-producing Convolvulaceae ("morning glories"), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs. Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function-the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species.

Keywords: Distimake; Ipomoea; deoxyhypusine synthase; gene duplication; homospermidine synthase; molecular evolution; pyrrolizidine alkaloids.

FIGURE 1

Pyrrolizidine alkaloids (PAs) occurring in the Convolvulaceae. (a) The figure illustrates the phylogenetic tree topology of the Convolvulaceae including the Solanaceae being its sister family. Species analyzed in this study all belong to the Convolvuloideae subfamily, except Cuscuta australis (Cuscutoideae), and Jacquemontia paniculata (Dicranostyloideae). (b) Tree topology of the Convolvuloideae. Groups within the Convolvuloideae, which comprise single species that produce PAs and lolines, are highlighted. Abbreviations: incl. = including. (c) Species included in this study and their affiliation to the phylogenetic groups given in (a) and (b). (d) Structures of PAs and lolines, which have been detected in individual species. The characteristic core structure of PAs, the necine base, is given in red. Ipangulines and Minalobines with a saturated necine base occur in species that belong to the “B2‐1”‐clade within the “Quamoclit” clade (B2) that includes species previously assigned to subgenus Quamoclit, or closely related to it (sensu Austin, 1979), as demonstrated by recent molecular phylogenetic results (Muñoz‐Rodríguez et al., 2019). In Ipomoea meyeri , which belongs to a sister group within this clade (B2‐2), unsaturated turneforcidine derivatives have been found (Eich, ; Tofern, 1999). Lolines have been described to occur in Argyreia mollis (Tofern et al., 1999). And finally, alkaloids that possess a 1,2‐unsaturated necine base occur in Distimake quinquefolius and Distimake cissoides (Eich, ; Mann, 1997). (e) Main reactions catalyzed by homospermidine synthase (HSS) and deoxyhypusine synthase (DHS). While HSS preferentially catalyzes the transfer of an aminobutyl moiety (highlighted in red) from spermidine to putrescine, DHS favors a specific lysine of the eIF5A precursor as aminobutyl acceptor to form deoxyhypusine.

FIGURE 2

Phylogenetic analyses of deoxyhypusine synthase (DHS)‐ and homospermidine synthase (HSS)‐encoding sequences. (a) A maximum likelihood tree was built from the cDNA sequences of HSS and DHS from the Convolvulaceae family and two DHS sequences from Solanaceae family. Percentages of bootstrap support values (for 1000 replicates) are indicated. The ancient duplication is highlighted, and the DHS and HSS clades are indicated. (b) Selected sequences were chosen for biochemical analyses of the encoded enzymes. The calculated specific activity (pkat·mg) of homospermidine production of these enzymes in glycine‐based assay buffer is given (see Table 3). Specific activities marked with an asterisk were taken from a previous study (Kaltenegger et al., 2013) (c) Alignment of the functionally characterized “HSS motif” (H‐V‐D)

FIGURE 3

Heterologously expressed and purified HSS enzymes and their biosynthetic activity. (a) A PageBlue stained SDS‐polyacryl gel of the purified and concentrated CuHSS (41.9 kDa), Dq2HSS (42.1 kDa), Dq1HSS (41.3 kDa), IaHSS (41.5 kDa) in borate (b) and glycine (g) buffer and the Senecio vernalis eIF5A precursor protein (18.3 kDa) is shown. (b) SEC‐MALS/UV confirmed the tetrameric state (~150 kDa) of purified HSS enzymes. Only CuHSS in borate buffer shows a second signal of 92 kDa, indicating misassembly. (c) Potential reactions catalyzed by homospermidine synthase (HSS). If the polyamines cadaverine and N‐methylputrescine are accepted as aminobutyl acceptors from the donor spermidine, (4‐aminobutyl)(5aminopentyl)amine and N‐methylhomospermidine, respectively, would be formed with 1,3‐diaminopropane as byproduct. Potential side reactions include the transfer from aminobutyl to spermidine, yielding canavalmine, and the sole spermidine cleavage, yielding 1,3‐diaminopropane and Δ¹‐Pyrroline. If spermine can be utilized as aminopropyl donor with putrescine as acceptor, spermidine would be formed. (d) The observed specific activities of Dq1HSS and IaHSS in in vitro activity assays with the substrates depicted in (c). Abbreviations: Can, Canavalmine; Dap, 1,3‐Diaminopropane; Spd, Spermidine

FIGURE 4

Homology models of homospermidine synthase (HSS) and deoxyhypusine synthase (DHS). Spermidine and nicotinamide adenine dinucleotide (NAD) are shown as sticks, colored by heteroatom. The N‐terminal “ball‐and‐chain” motifs are omitted. (a) Ribbon representation of the human DHS tetramer (PDB: 6XXJ) with black diamonds indicating the general location of the active sites. The tetramer consists of two dimers (A‐B, A′‐B′), which comprise two antiparallel active sites at their interfaces. (b) Overlay of chain A of all DHS and HSS models in ribbon representation from Ipomoeaalba , Ipomoea neei, Distimake quinquefolius individuum 1 and 2, Camonea umbellata , colored by residue identity from red = low identity to blue = high identity. The catalytic lysine is represented as purple stick. Spermidine and NAD from chain A and B are shown. (c,d) Surface representation of the A‐B dimer and the A monomer from the I. alba HSS model, respectively. Residues that differ between the DHS and HSS pair from I. alba are colored in yellow, and in orange, if they are additionally located in the interface of the A‐B dimer. The interface area of chain A is colored in light gray with the catalytic lysine in purple. The location of the extended HSS motif and the conserved RAM motif is highlighted.