Molecular evolution of the polyamine oxidase gene family in Metazoa

Abstract

Background: Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived.

Results: We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all the SMOs and APAOs from vertebrates. The two vertebrate monophyletic clades clustered strictly mirroring the organismal phylogeny of fishes, amphibians, reptiles, birds, and mammals. Evidences from comparative genomic analysis, structural evolution and functional divergence in a phylogenetic framework across Metazoa suggested an evolutionary scenario where the ancestor PAO coding sequence, present in invertebrates as an orthologous gene, has been duplicated in the vertebrate branch to originate the paralogous SMO and APAO genes. A further genome evolution event concerns the SMO gene of placental, but not marsupial and monotremate, mammals which increased its functional variation following an alternative splicing (AS) mechanism.

Conclusions: In this study the explicit integration in a phylogenomic framework of phylogenetic tree construction, structure prediction, and biochemical function data/prediction, allowed inferring the molecular evolutionary history of the PAO gene family and to disambiguate paralogous genes related by duplication event (SMO and APAO) and orthologous genes related by speciation events (PAOs, SMOs/APAOs). Further, while in vertebrates experimental data corroborate SMO and APAO molecular function predictions, in invertebrates the finding of a supported phylogenetic clusters of insect PAOs and the co-occurrence of two PAO variants in the amphioxus urgently claim the need for future structure-function studies.

Figure 1

Enzymatic reaction catalyzed by SMO and APAO proteins. SMO oxidises the carbon on the exo-side of the N⁵-nitrogen of Spm, producing Spd, 3-aminopropanal (3-AP) and hydrogen peroxide (H₂O₂). APAO oxidises the carbon on the exo-side of the N⁵-nitrogen of N¹Ac-Spm and N¹Ac-Spd producing Spd and Put respectively, in addition to 3-aceto-aminopropanal (3-aceto-AP) and H₂O₂.

Figure 2

The evolutionaty tree of the the polyamine oxidase proteins. The evolutionary history tree of the SMO, PAO, and APAO polyamine oxidase proteins in Metazoa as inferred by using the Maximum Likelihood method based on the JTT model with a proportion of invariable sites (I) and gamma-distributed rates across sites (G) (Bayesian tree showed identical topology at the main nodes; data not shown). The analysis involved 76 amino acidic sequences with the yeast sequence used as outgroup. In correspondence of the main nodes (black circles) the bootstrap support (BS) and Bayesian posterior probabilities (BPP) values are reported (above: BS; below: BPP). The support for the secondary nodes is reported as white circles (BS = 100; BPP = 1.00) and grey circles (BS ranging from 90 to 99; BPP = 1.00). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Main monophyletic groups are indicated as follow: M = mammals; B = birds; R = reptiles; A = amphibians; F = fishes; I = insects.

Figure 3

Molecular models of the active site region of SMO and PAO. Schematic representation of the active site region of mouse SMO (MmSMO), Drosophila melanogaster PAO (DmPAO) and Saccharomyces cerevisiae PAO (FMS1) in complex with the substrate Spm. MmSMO and DmPAO complexes with Spm have been obtained by molecular modelling ([11] and this work, respectively) while the structure of the FMS1-Spm has been determined experimentally [14]. For the sake of clarity, the FAD cofactor is coloured in purple, backbone atoms in orange and Spm carbon atoms in green.

Figure 4

Three-dimensional view of SMOs and APAOs sequence conservation. Structure-based views of the amino acid sequence conservation in the active site regions of vertebrate SMOs and APAOs. Protein regions coloured in blue correspond to residues conserved in at least 90% of the amino acid sequences analysed. Top panel: Amino acid sequence conservation in the SMO family mapped onto the structural model of mouse SMO [11]. Middle panel: Amino acid sequence conservation in the APAO family mapped onto the structural model of mouse APAO [12]. Bottom panel: Amino acid sequence conservation in the vertebrate PAO family (SMO and APAO sequences combined) mapped onto the structural model of mouse SMO. The green ellipse indicates the location of the polar pocket made up SMOs by residues Glu216 and Ser218 (numbering of the mouse enzyme), which are substituted by aliphatic amino acids in APAOs. The figure was generated using Jalview [32].

Figure 5

Amino acid sequence alignment and structure-based view of SMO isoforms. A) Amino acid sequence alignment of the regions corresponding to Nuclear Domain A (NDA) and B (NDB) of SMO long isoforms. For acronyms and isoform numbering see Table 2. B) Structure-based view of the amino acid sequence conservation in SMOs (left) and in the NDA of placental mammals as opposed to marsupials and monotremates (right). Protein regions coloured in blue correspond to residues conserved in at least 90% of the amino acid sequences analysed.