Abstract Detail


Smith, Chelsea R. [1], Kaltenegger, Elisabeth [2], Tasca, Julia A. [3], Teisher, Jordan [4], Straub, Shannon [5], Minbiole, Kevin P. C. [6], Livshultz, Tatyana [4].

Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: inference of ancestral function in the pathway-specific enzyme, homospermidine synthase.

Premise: Orthology is often used to infer homology of function among enzymes. The frequency of parallel evolution of similar function in most orthology groups is unknown.
Background: Fourteen angiosperm families produce pyrrolizidine alkaloids (PAs) as defensive secondary metabolites. The first pathway specific step in PA biosynthesis is catalyzed by homospermidine synthase (HSS), a paralog of the ubiquitous eukaryotic enzyme deoxyhypusine synthase (DHS). In the six PA-producing families in which hss evolution has been studied, there have been 7 independent origins of HSS; each time, a VXXXD amino acid motif evolved from the IXXXN motif in DHS. Previous research found a single origin of hss in the MRCA of all PA-producing Apocynaceae species. The reconstructed ancestral HSS had a VXXXD amino acid motif but some of its descendant loci encoded enzymes with IXXXN and IXXXD motifs.
Objectives: We revisit the reconstruction of the sequence and function of the ancestral HSS with the aim of testing whether PAs are homologous across Apocynaceae.
Methods: We increased species and sequence sampling, obtained novel phenotypic data on PA occurrence, and performed site-directed mutagenesis experiments on the hss of PA-producing Parsonsia alboflavescens to test predictions of enzyme function from amino acid motifs.
Results: A single origin of the hss locus in Apocynaceae is still supported, but the reconstructed HSS-like enzyme has an IXXXD motif. From this IXXXD motif, there was one origin of IXXXN and three origins of VXXXD. None of 7 tested species with an IXXXN motif, 11 of 29 species with a VXXXD motif, and one of 10 species with an IXXXD motif tested positive for PAs. Preliminary data from mutagenesis experiments suggests that the function of an HSS with an IXXXD motif (330 pkat/mg) is much lower than one with VXXXD motif (2,000 pkat/mg) but higher than one with an IXXXN motif (170 pkat/mg).
Conclusions: The ancestral HSS of Apocynaceae is predicted to have low function, but it may have been sufficient for PA biosynthesis since one extant species with an IXXXD motif has PAs.  Whether or not the common ancestor of PA-producing Apocynaceae could produce PAs, it appears that HSS enzymes optimized for PA biosynthesis evolved three times in parallel.  Enzymological investigation of the reconstructed ancestral HSS will allow us to more conclusively determine its function, and identification of additional genes of the PA biosynthetic pathway will allow us to better infer when PAs evolved.

1 - Drexel University, Biodiversity Earth and Environmental Sciences, Academy Of Natural Sciences Of Drexel University, 1900 Benjamin Franklin Parkway, Philadelphia, PA, 19103-1101, United States
2 - Christian-Albrechts-Universität zu Kiel, Botanisches Institut und Bot, Biochemische Ökologie und Molekulare Evolution, Christian-Albrechts-Platz 4, Kiel, 24118, Germany
3 - Villanova University, Department of Chemistry, 800 Lancaster Ave, Villanova, PA, 19085, USA
4 - Academy Of Natural Sciences Of Drexel University, Department Of Botany, 1900 Benjamin Franklin Parkway, Philadelphia, PA, 19103, United States
5 - Hobart And William Smith Colleges, Department Of Biology, 300 Pulteney St., Geneva, NY, 14456, United States
6 - Villanova University, Department of Chemistry, 800 Lancaster Ave, Villanova, PA, 19085, United States

pyrrolizidine alkaloids
Gene duplication
ancestral reconstruction
plant defense
Secondary metabolites.

Presentation Type: Oral Paper
Abstract ID:948
Candidate for Awards:None

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