Bridging the gap between plant and mammalian polyamine catabolism: a novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis

Abstract

In contrast to animals, where polyamine (PA) catabolism efficiently converts spermine (Spm) to putrescine (Put), plants have been considered to possess a PA catabolic pathway producing 1,3-diaminopropane, Delta(1)-pyrroline, the corresponding aldehyde, and hydrogen peroxide but unable to back-convert Spm to Put. Arabidopsis (Arabidopsis thaliana) genome contains at least five putative PA oxidase (PAO) members with yet-unknown localization and physiological role(s). AtPAO1 was recently identified as an enzyme similar to the mammalian Spm oxidase, which converts Spm to spermidine (Spd). In this work, we have performed in silico analysis of the five Arabidopsis genes and have identified PAO3 (AtPAO3) as a nontypical PAO, in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N(1)-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (agmatine, cadaverine, and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C terminus, and it targets green fluorescence protein to peroxisomes when fused at the N terminus but not at the C terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.

Figure 1.

AtPAO3 sequence alignment with other known plant and mammalian PAOs. AtPAO3 (At3g59050; GenBank accession no. AY143905) amino acid sequence similarity with AtPAO1 (At5g13700; GenBank accession no. NM_121373), AtPAO2 (At2g43020; GenBank accession no. AF364952), AtPAO4 (At1g65840; GenBank accession no. AF364953), AtPAO5 (At4g29720; GenBank accession no. AK118203), maize (ZmPAO; GenBank accession no. NM_001111636), Homo PAO (H. sapiens PAO; GenBank accession no. NM_152911), Mus (GenBank accession no. NM_153783), Homo SMO (H. sapiens SMO; GenBank accession no. NM_175839), and tobacco (NtPAO; GenBank accession no. AB200262) PAOs. Conserved motifs spanning the protein sequences are also indicated (black bars). Alignment and (default) shading were accomplished using ClustalW version 1.8.

Figure 2.

Phylogenetic tree representing the evolutionary relationship of the AtPAO3 gene with other known plant and mammalian PAOs. AtPAO3 (At3g59050; GenBank accession no. AY143905) sequence similarity with AtPAO1 (At5g13700; GenBank accession no. NM_121373), AtPAO2 (At2g43020; GenBank accession no. AF364952), AtPAO4 (At1g65840; GenBank accession no. AF364953), AtPAO5 (At4g29720; GenBank accession no. AK118203), maize (ZmPAO; GenBank accession no. NM_001111636), Homo PAO (H. sapiens PAO; GenBank accession no. NM_152911), mouse (PAO; GenBank accession no. NM_153783), Homo SMO (H. sapiens SMO; GenBank accession no. NM_175839), and tobacco (NtPAO; GenBank accession no. AB200262) PAOs.

Figure 3.

AtPAO3 expression in E. coli and AtPAO3 protein digestion and chromatographic purification. A, The construct used to produce recombinant AtPAO3 in E. coli BL21 cells. B, Time course of AtPAO3 protein accumulation in the total cellular extracts from induced E. coli BL21 cells. C, Detection of the MBP:PAO3 protein fusion in the soluble fraction, in noninduced (−IPTG) and induced cells (+IPTG), using an anti-MBP polyclonal antibody at different temperatures. At higher temperatures (e.g. 37°C), the MBP:PAO3 protein accumulated mostly in the pellet fraction. D, First step protein expression and purification. 1, Cell lysate from noninduced culture; 2, supernatant of the cell lysate from induced culture; 3, pellet of the cell lysate from induced culture; 4, initial flow through the amylose resin; 5, part of the amylose resin before elution of the protein; 6, second flow through the amylose resin; 7, final flow through the amylose resin (10th); 8, elution fractions 4 and 5 from the amylose resin (recombinant MBP:PAO3 eluted between fractions 3–7). E, Digestion of the eluted MBP:PAO3 protein with the specific protease Xa factor for 24 h. As a control, MBP:PAO3 protein was used without addition of Xa factor (left). F, Gel filtration of the MBP:PAO3 protein purified with the amylose resin producing a single MBP:PAO3 band and AtPAO3 protein after digestion and purification with Xa factor. The data presented are from a single representative experiment that was repeated twice with similar results.

Figure 4.

Absorption spectrum of the AtPAO3 purified protein between wavelengths from 300 to 540 from TCA-treated or nontreated protein. AtPAO3 exhibits the characteristic absorbance of FAD-containing enzymes. The data presented are from a single representative experiment that was repeated many times with similar results.

Figure 5.

Biochemical properties of AtPAO3. A, Relative H₂O₂ production (CPM, counts per minute normalized to controls) using Spd and Spm as substrates (10 mm each). B, HPLC analysis of the AtPAO3-dependent Spm conversion to Spd (15 min) and Put (15 and 60 min; AtPAO3+Spm), and Spd conversion to Put (15 and 60 min; AtPAO3+ Spd). A total 1 mm substrate was used and 2 μg of AtPAO3 enzyme. The peaks with retention time varying between approximately 7 to 8 min correspond to traces of benzoyl chloride used as derivatization reagent. C, AtPAO3 dependence from pH and temperature. D, Conversion of Spm to Spd and Put by AtPAO3 as a fraction of time, using 2 μg of AtPAO3 enzyme and Spm as substrate. Data are the means from three independent experiments ± se.

Figure 6.

Radiometric assay for the detection of AtPAO3 reaction products and conversion efficiency using Spm and Spd as substrates. Data are the means from three independent experiments ± se.

Figure 7.

AtPAO3 protein localization and co-localization with plant peroxisomes in N. benthamiana. A, GFP fluorescence localization of the transiently expressed GFP:PAO3 in peroxisomes of N. benthamiana leaves, merged with the localization of chlorophyll fluorescence (left), and with inverse phase microscopy (right). B, GFP fluorescence localization of the transiently expressed PAO3:GFP into the cytoplasm of N. benthamiana leaves (left) and merged with the localization of chlorophyll fluorescence (right). C, GFP fluorescence localization of the GFP: PAO3 expressing binary vector (top left), mCherry fluorescence localization of the mCherry:SKL expressing binary vector (specific for plant peroxisomes; top middle), merged image of the GFP and mCherry fluorescence (top right), in peroxisomes of N. benthamiana leaves transiently expressing the two constructs. GFP fluorescence localization of the GFP:PAO3 expressing binary vector (bottom left), YFP fluorescence localization of the YFP:SKL construct (specific for plant peroxisomes; bottom middle), merged image of the GFP and YFP fluorescence (bottom right), in peroxisomes of N. benthamiana leaves transiently expressing the two constructs. The data presented are from a single representative experiment that was repeated twice with similar results. Data were obtained using a 40× immersion objective.

Figure 8.

AtPAO2, AtPAO3, AtPAO4, and AtPAO5 mRNA abundance under various chemical stimuli 1, 6, and 24 h after the corresponding treatment (each well contains 10 μg of total RNA and exposure time was 48 h). The data presented are from a single representative experiment that was repeated twice with similar results.