Evolution of homospermidine synthase in pyrrolizidine alkaloid biosynthesis of the morning glory (Convolvulaceae) family
Pyrrolizidine alkaloids (PAs) are toxic compounds produced in many different species of flowering plants as a chemical defense against herbivores. The PAs are also economically important as they can cross-contaminate herbals medicines and food products and are harmful if ingested. The biosynthesis of PAs evolved several times independently in different plant lineages, such as Apocynaceae, Asteraceae, Boraginaceae, Convolvulaceae, Fabaceae, Poaceae, and Orchidaceae. Homospermidine synthase (HSS) is the first pathway-specific enzyme of PA biosynthesis. Interestingly, HSS repeatedly evolved by gene duplication events from the primary metabolic gene of deoxyhypusine synthase (DHS) and was successfully recruited into PA metabolism in all the PA producing plant species. DHS is involved in activating the eukaryotic translation initiation factor 5a (eIF5a) by transferring an aminobutyl group from spermidine to a particular lysine residue in eIF5a. The HSS transfers the aminobutyl group from spermidine to putrescine to form homospermidine, which serves as PAs' backbone. In the morning glory (Convolvulaceae) family, the PAs occur in two different clades called Ipomoea (sweet potato clade) and Distimake (wood roses). However, only a single gene duplication event was reported in the morning glory family long before Ipomoea and Distimake diverged. Thus, it was hypothesized that the recruitment of the duplicated copy to PA biosynthesis occurred independently in these two clades, i.e., the functional shift from DHS activity to HSS activity occurred two times. In this thesis, an analytical method was developed to directly measure both DHS and HSS enzymes' activities using reversed-phase high-pressure liquid chromatography (HPLC). The new method uses chromophore to derivatize substrates and products that can be monitored by UV absorbance and fluorescence, thus replacing the old radio-active labeled tracer experiments. The HPLC method proved to be robust, fast, and reliable. This method can also detect various side reactions of both DHS and HSS in addition to their main activities, thus shedding light on the previously unexplored promiscuous activities. Furthermore, an extensive search for hss-like genes in the morning glory family and reconstructing the phylogenetic tree revealed the history of gene loss, neo- and non-functionalization, and duplicate retention. Alkaloid analyses using gas chromatography and mass spectrometry revealed that not all the species that retained the hss-like genes produced PAs. Molecular evolutionary analysis of selective pressure using coding sequences of hss and dhs combined with ancestral sequence reconstruction of ancient HSS enzymes revealed that in both Ipomoea and Distimake clades, the hss genes experienced a similar pattern of selection pressure. The resurrected last common ancestor of hss genes from Ipomoea and Distimake was under strong purifying selection and retained its original ancestral DHS specific activity. However, this last common ancestor also showed high promiscuity in the usage of aminobutyl-acceptors and can produce homospermidine and canavalmine additional to the activation of the eIF5a. In the Ipomoea clade, HSS enzymes of PA producing species were refined towards HSS activity by positive Darwinian selection. In the Distimake clade, relaxed selection pressure resulted in refining the HSS activity in PA producing species. In summary, this work elucidated the independent functional optimization of HSS activity in PA biosynthesis that occurred twice in the morning glory family. This parallel evolution of HSS enzymes occurred predominantly via divergent mutations with few identical amino acid replacements in both genera.