Evolution of homospermidine synthase in the convolvulaceae: a story of gene duplication, gene loss, and periods of various selection pressures

Abstract

Homospermidine synthase (HSS), the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, is known to have its origin in the duplication of a gene encoding deoxyhypusine synthase. To study the processes that followed this gene duplication event and gave rise to HSS, we identified sequences encoding HSS and deoxyhypusine synthase from various species of the Convolvulaceae. We show that HSS evolved only once in this lineage. This duplication event was followed by several losses of a functional gene copy attributable to gene loss or pseudogenization. Statistical analyses of sequence data suggest that, in those lineages in which the gene copy was successfully recruited as HSS, the gene duplication event was followed by phases of various selection pressures, including purifying selection, relaxed functional constraints, and possibly positive Darwinian selection. Site-specific mutagenesis experiments have confirmed that the substitution of sites predicted to be under positive Darwinian selection is sufficient to convert a deoxyhypusine synthase into a HSS. In addition, analyses of transcript levels have shown that HSS and deoxyhypusine synthase have also diverged with respect to their regulation. The impact of protein-protein interaction on the evolution of HSS is discussed with respect to current models of enzyme evolution.

Figure 1.

Structural Types of PAs Occurring within the Convolvulaceae. Ipanguline-type PAs of Ipomoea species (section Mina) characterized by the saturated necine base platynecine forming mono- and diesters with aromatic and aliphatic necic acids (A). Triangularine-type PAs of I. meyeri (section Pharbitis) with saturated necine bases, predominantly turneforcidine, and aliphatic necic acids (B). Lycopsamine-type PAs of M. quinquefolia with the 1,2-unsaturated necine base retronecine and unique C₇ necic acids (C). Loline-type PAs of A. mollis characterized by a 2,7 ether bridge and the replacement of C-9 by an amino substituent (D). The studied species in which the PA type was detected (with the exception of the loline-type PAs) are given.

Figure 2.

Unrooted Maximum Likelihood Tree of HSS- and DHS-Encoding cDNA Sequences of Various Angiosperm Species. Sequences coding for HSS and DHS are shown in red and black, respectively. (A) The branching pattern supports five independent gene duplication events that give rise to HSS-encoding sequences in the various lineages (i.e., two in the Asteraceae and one in the lineages of the Boraginaceae, the Monocots, and the Convolvulaceae). (B) Detail of (A) showing the sequence cluster of the Convolvulaceae. Sequences that have been biochemically characterized are given in bold. Bootstrap proportions resulted from 1000 replicates and are given only for values ≥ 50.

Figure 3.

Branch-Specific Analyses of ω Ratios of DHS- and HSS-Coding Sequences of the Solanales. Colored lines indicate branches that were defined a priori as foreground branches in separate calculations with the branch-site model. Furthermore, the whole HSS clade, marked with a gray box, was defined as a foreground clade in one calculation using the branch-site model. In the branch model, ω ratios for five categories of branches were calculated: ω₀ for branches in the DHS clade, ω_a for branch “a” within the HSS clade, and ω_B, ω_C, and ω_D for branches B, C, and D, respectively. The table summarizes the calculations of the branch model and the branch-site model. Note, in the branch-site model, ω ratios (ω₂) were calculated for individual codon sites along the prespecified lineages. The number of sites is given in parentheses and is one of the calculated variables of the branch-site model. In the branch model, ω ratios were averaged over all sites in the protein. (A) Tree topology and summarized results of the analyses with data set A. (B) Tree topology of the HSS clade and summarized results of the analyses with data set B, which includes the partial exon sequences of two pseudogenes from C. arvensis (carv-2 and carv-3).

Figure 4.

Influence of Sites Predicted to Be under Positive Selection on Substrate Specificity. (A) Amino acid polymorphisms between DHS and HSS of the Solanales. Shown are amino acids from position 220 to 290. Numbering of the amino acid positions is based on the amino acid sequence of the DHS from I. neei. Position 1 is defined as the start codon in the ORF. Identical amino acids are shaded gray. Sites that are calculated to be under positive selection with the branch-site model are boxed when clade a was tested and marked with an arrow when branch C was tested (Asn-281 with data set A, and Asn-266, Ile-277, and Asn-281 with data set B). (B) Relative activities of modified DHS proteins from I. neei compared with wild-type DHS (wt). DHS activity is determined by product formation with eIF5A precursor protein as substrate (blue), whereas HSS activity is determined with putrescine as substrate (red). *Significant differences from the activity of the wild type. [See online article for color version of this figure.]