FAD-dependent Polyamine Oxidase Research Articles

A Solanum lycopersicum polyamine oxidase contributes to the control of plant growth, xylem differentiation, and drought stress tolerance.

Polyamines are involved in several plant physiological processes. In Arabidopsis thaliana, five FAD-dependent polyamine oxidases (AtPAO1 to AtPAO5) contribute to polyamine homeostasis. AtPAO5 catalyzes the back-conversion of thermospermine (T-Spm) to spermidine and plays a role in plant development, xylem differentiation, and abiotic stress tolerance. In the present study, to verify whether T-Spm metabolism can be exploited as a new route to improve stress tolerance in crops and to investigate the underlying mechanisms, tomato (Solanum lycopersicum) AtPAO5 homologs were identified (SlPAO2, SlPAO3, and SlPAO4) and CRISPR/Cas9-mediated loss-of-function slpao3 mutants were obtained. Morphological, molecular, and physiological analyses showed that slpao3 mutants display increased T-Spm levels and exhibit changes in growth parameters, number and size of xylem elements, and expression levels of auxin- and gibberellin-related genes compared to wild-type plants. The slpao3 mutants are also characterized by improved tolerance to drought stress, which can be attributed to a diminished xylem hydraulic conductivity that limits water loss, as well as to a reduced vulnerability to embolism. Altogether, this study evidences conservation, though with some significant variations, of the T-Spm-mediated regulatory mechanisms controlling plant growth and differentiation across different plant species and highlights the T-Spm role in improving stress tolerance while not constraining growth.

Genome-wide analysis of the polyamine oxidase gene family in wheat (Triticum aestivum L.) reveals involvement in temperature stress response

Amine oxidases (AOs) including copper containing amine oxidases (CuAOs) and FAD-dependent polyamine oxidases (PAOs) are associated with polyamine catabolism in the peroxisome, apoplast and cytoplasm and play an essential role in growth and developmental processes and response to biotic and abiotic stresses. Here, we identified PAO genes in common wheat (Triticum aestivum), T. urartu and Aegilops tauschii and reported the genome organization, evolutionary features and expression profiles of the wheat PAO genes (TaPAO). Expression analysis using publicly available RNASeq data showed that TaPAO genes are expressed redundantly in various tissues and developmental stages. A large percentage of TaPAOs respond significantly to abiotic stresses, especially temperature (i.e. heat and cold stress). Some TaPAOs were also involved in response to other stresses such as powdery mildew, stripe rust and Fusarium infection. Overall, TaPAOs may have various functions in stress tolerances responses, and play vital roles in different tissues and developmental stages. Our results provided a reference for further functional investigation of TaPAO proteins.

Decrease of Arabidopsis PAO activity entails increased RBOH activity, ROS content and altered responses to Pseudomonas

Polyamines (PAs) are small aliphatic amines with important regulatory activities in plants. Biotic stress results in changes in PA levels due to de novo synthesis and PA oxidation. In Arabidopsis thaliana five FAD-dependent polyamine oxidase enzymes (AtPAO1-5) participate in PA back-conversion and degradation. PAO activity generates H2O2, an important molecule involved in cell signaling, elongation, programmed cell death, and defense responses. In this work we analyzed the role of AtPAO genes in the Arabidopsis thaliana-Pseudomonas syringae pathosystem. AtPAO1 and AtPAO2 genes were transcriptionally up-regulated in infected plants. Atpao1-1 and Atpao2-1 single mutant lines displayed altered responses to Pseudomonas, and an increased susceptibility was found in the double mutant Atpao1-1 x Atpao2-1. These polyamine oxidases mutant lines showed disturbed contents of ROS (H2O2 and O2−) and altered activities of RBOH, CAT and SOD enzymes both in infected and control plants. In addition, changes in the expression levels of AtRBOHD, AtRBOHF, AtPRX33, and AtPRX34 genes were also noticed. Our data indicate an important role for polyamine oxidases in plant defense and ROS homeostasis.

Engineering enzyme catalysis: an inverse approach.

Enzymes’ inherent chirality confers their exquisite enantiomeric specificity and makes their use as green alternatives to chiral metal complexes or chiral organocatalysts invaluable to the fine chemical industry. The most prevalent way to alter enzyme activity in terms of regioselectivity and stereoselectivity for both industry and fundamental research is to engineer the enzyme. In a recent article by Keinänen et al., published in Bioscience Reports 2018, ‘Controlling the regioselectivity and stereoselectivity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators’, an inverse approach was presented that focuses on the manipulation of the enzyme substrate rather than the enzyme. This approach not only uncovered dormant enantioselectivity in related enzymes but allowed for its control by the use of guide molecules simply added to the reaction solution or covalently linked to an achiral scaffold molecule.

Controlling the regioselectivity and stereospecificity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators.

Enzymes generally display strict stereospecificity and regioselectivity for their substrates. Here by using FAD-dependent human acetylpolyamine oxidase (APAO), human spermine (Spm) oxidase (SMOX) and yeast polyamine oxidase (Fms1), we demonstrate that these fundamental properties of the enzymes may be regulated using simple guide molecules, being either covalently attached to polyamines or used as a supplement to the substrate mixtures. APAO, which naturally metabolizes achiral N1-acetylated polyamines, displays aldehyde-controllable stereospecificity with chiral 1-methylated polyamines, like (R)- and (S)-1-methylspermidine (1,8-diamino-5-azanonane) (1-MeSpd). Among the novel N1-acyl derivatives of MeSpd, isonicotinic acid (P4) or benzoic acid (Bz) with (R)-MeSpd had Km of 3.6 ± 0.6/1.2 ± 0.7 µM and kcat of 5.2 ± 0.6/4.6 ± 0.7 s−1 respectively, while N1-AcSpd had Km 8.2 ± 0.4 µM and kcat 2.7 ± 0.0 s−1. On the contrary, corresponding (S)-MeSpd amides were practically inactive (kcat < 0.03 s−1) but they retained micromole level Km for APAO. SMOX did not metabolize any of the tested compounds (kcat < 0.05 s−1) that acted as non-competitive inhibitors having Ki ≥ 155 µM for SMOX. In addition, we tested (R,R)-1,12-bis-methylspermine (2,13-diamino-5,10-diazatetradecane) (R,R)-(Me2Spm) and (S,S)-Me2Spm as substrates for Fms1. Fms1 preferred (S,S)- to (R,R)-diastereoisomer, but with notably lower kcat in comparison with spermine. Interestingly, Fms1 was prone to aldehyde supplementation in its regioselectivity, i.e. the cleavage site of spermidine. Thus, aldehyde supplementation to generate aldimines or N-terminal substituents in polyamines, i.e. attachment of guide molecule, generates novel ligands with altered charge distribution changing the binding and catalytic properties with polyamine oxidases. This provides means for exploiting hidden capabilities of polyamine oxidases for controlling their regioselectivity and stereospecificity.

Biochemical characterization and homology modeling of polyamine oxidase from cyanobacterium Synechocystis sp. PCC 6803

The intracellular polyamine contents are regulated not only by polyamine biosynthesis and transport but also by polyamine degradation catalyzed by copper-dependent amine oxidase (DAO) and FAD-dependent polyamine oxidase (PAO). The genome sequence of Synechocystis sp. PCC 6803 reveals the presence of at least one putative polyamine oxidase gene, slr5093. The open reading frame of slr5093 encoding Synechocystis polyamine oxidase (SynPAO, E.C. 1.5.3.17) was expressed in Escherichia coli. The purified recombinant enzyme had the characteristic absorption spectrum of a flavoprotein with absorbance peaks at 380 and 450 nm. The optimum pH and temperature for the oxidation of both spermidine and spermine are 8.5 and 30 °C, respectively. The enzyme catalyzed the conversion of spermine and spermidine to spermidine and putrescine, respectively, with higher catalytic efficiency when spermine served as substrate. These results suggest that SynPAO is a polyamine oxidase involved in a polyamine back-conversion pathway. Based on the structural analysis, Gln94, Tyr403 and Thr440 in SynPAO are predicted to be important residues in the active site.

Copper-Containing Amine Oxidases and FAD-Dependent Polyamine Oxidases Are Key Players in Plant Tissue Differentiation and Organ Development.

Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell-wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H2O2 in light- or developmentally-regulated differentiation events, thus influencing cell-wall architecture and maturation as well as PCD. Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and apoplastic metabolic pathways, in tissue differentiation and organ development.

Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants

Metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, has been a target for antineoplastic therapy since these naturally occurring alkyl amines were found essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. A correlation between regulation of cell proliferation and polyamine metabolism is described. In particular, polyamine catabolism involves copper-containing amine oxidases and FAD-dependent polyamine oxidases. Several studies showed an important role of these enzymes in several developmental and disease-related processes in both animals and plants through a control on polyamine homeostasis in response to normal cellular signals, drug treatment, environmental and/or cellular stressors. The production of toxic aldehydes and reactive oxygen species, H(2)O(2) in particular, by these oxidases using extracellular and intracellular polyamines as substrates, suggests a mechanism by which the oxidases can be exploited as antineoplastic drug targets. This minireview summarizes recent advances on the physiological roles of polyamine catabolism in animals and plants in an attempt to highlight differences and similarities that may contribute to determine in detail the underlined mechanisms involved. This information could be useful in evaluating the possibility of this metabolic pathway as a target for new antiproliferative therapies in animals and stress tolerance strategies in plants.

Role of the FAD-dependent polyamine oxidase in the selective formation of N1,N8-bis(γ- glutamyl)spermidine protein cross-links

Protein-bound gamma-glutamylpolyamines have highlighted a new pathway in polyamine metabolism. Human foreskin keratinocytes offer a suitable model for this study. Indeed, they develop polymerized envelopes, as they differentiate, rich in epsilon-(gamma-glutamyl)lysine and N(1),N(8)-bis(gamma-glutamyl)spermidine cross-links. We have found that the selective oxidation of N(1)-(gamma-glutamyl)spermidine and N-(gamma-glutamyl)spermine by FAD-dependent polyamine oxidase (PAO) may be one of the cellular mechanisms regulating the preferential formation of a sterically defined bis(gamma-glutamyl)spermidine cross-link. The significance of this finding is unknown, but it suggests that the target of this PAO-modulation is to achieve the biochemical prerequisite for production of a normal epidermal stratum corneum.

Guide Molecule-driven Stereospecific Degradation of α-Methylpolyamines by Polyamine Oxidase

FAD-dependent polyamine oxidase (PAO; EC 1.5.3.11) is one of the key enzymes in the catabolism of polyamines spermidine and spermine. The natural substrates for the enzyme are N1-acetylspermidine, N1-acetylspermine, and N1,N12-diacetylspermine. Here we report that PAO, which normally metabolizes achiral substrates, oxidized (R)-isomer of 1-amino-8-acetamido-5-azanonane and N1-acetylspermidine as efficiently while (S)-1-amino-8-acetamido-5-azanonane was a much less preferred substrate. It has been shown that in the presence of certain aldehydes, the substrate specificity of PAO and the kinetics of the reaction are changed to favor spermine and spermidine as substrates. Therefore, we examined the effect of several aldehydes on the ability of PAO to oxidize different enantiomers of alpha-methylated polyamines. PAO supplemented with benzaldehyde predominantly catalyzed the cleavage of (R)-isomer of alpha-methylspermidine, whereas in the presence of pyridoxal the (S)-alpha-methylspermidine was preferred. PAO displayed the same stereospecificity with both singly and doubly alpha-methylated spermine derivatives when supplemented with the same aldehydes. Structurally related ketones proved to be ineffective. This is the first time that the stereospecificity of FAD-dependent oxidase has been successfully regulated by changing the supplementary aldehyde. These findings might facilitate the chemical regulation of stereospecificity of the enzymes.

Interaction of bovine serum amine oxidase with the polyamine oxidase inactivator MDL 72527

MDL 72527 was considered a selective inhibitor of FAD-dependent polyamine oxidases. In the present communication, we demonstrate that MDL 72527 inactivates bovine serum amine oxidase, a copper-containing, TPQ-enzyme, time-dependently at 25 °C. In striking contrast, the enzyme remained active after incubation with excessive MDL 72527 at 37 °C, even after 70 h of incubation. Inactivation of BSAO with MDL 72527 at 25 °C did not involve the cofactor, as was shown by spectroscopy and by reaction with phenylhydrazine. Docking of MDL 72527 is difficult, owing to its size and two lipophilic moieties, and it has been shown that minor changes in reaction rate of substrates cause major changes in K m and k cat/ K m. We hypothesise that subtle conformational changes between 25 and 37 °C impair MDL 72527 from productive binding and prevent the nucleophilic group from reacting with the double bond system.

How important is the oxidative degradation of spermine?: minireview article.

Spermine is a constituent of most eucaryotic cells, however, it is not of vital importance for the vertebrate organism, as is demonstrated by the existence of transgenic (Gy) mice that lack spermine and spermine synthase. In contrast its degradation appears to be of vital importance, since mice die after chronic administration of N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72517). Under this condition spermine accumulates in red blood cells and blood plasma. Lethal toxicity can be avoided by intervals of MDL 72527-free periods. During these periods spermine appears to be directly degraded to spermidine without an intermediary acetylation step within the red blood cells. Since this reaction is of enormous physiological significance, it will be important to characterise the red blood cell spermine oxidase, and it will be particularly important to determine whether this oxidase is identical with the FAD-dependent polyamine oxidase that is considered to be involved in the polyamine interconversion sequence, or whether it is one of the recently characterised spermine oxidase isoenzymes.

Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2).

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N1-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded.

Polyamines and ripening of photoreceptor outer segments in chicken embryos

Polyamines and their related monoacetyl derivatives were studied in rod outer segment (ROS) and cone outer segment (COS) of photoreceptor cells from chick embryo retina during eye development (7th–18th days). Putrescine was found to be necessary, in the second phase of retinogenesis, to sustain both ROS and COS differentiation and, after acetylation, γ-aminobutyric acid synthesis. On the other hand, spermidine and even more spermine intervene in the third phase of development when photoreceptors mature. Moreover, the presence of N1-acetylspermidine already at the 7th day indicates that in the outer segment of photoreceptor cells too, as in the whole retina, putrescine synthesis comes about by two pathways. One pathway involves ornithine decarboxylase; the other, spermidine/spermine N1-acetyltransferase and FAD-dependent polyamine oxidase activities that convert spermidine to putrescine via N1-acetylspermidine. These different biosynthetic pathways are probably also decisive in permitting γ-aminobutyric acid synthesis, which is very important in the ripening process of neural retina.

Involvement of FAD-dependent polyamine oxidase in the metabolism of milacemide in the rat.

1. It has previously been established that monoamine oxidase (MAO)-B participates in the metabolism of milacemide [2-(pentylamino)acetamide]. Furthermore, in rats, inhibition of FAD-dependent polyamine oxidase (PAO) was found to decrease the urinary excretion of two milacemide metabolites, termed UK1 and UK2. 2. Using gas chromatography-mass spectrometry, UK1 was identified as oxamic acid and UK2 as 2-hydroxyacetamide, confirming that PAO is involved in the metabolism of milacemide. 3. Thus, two FAD-dependent amine-oxidizing enzymes, MAO and PAO, contribute to the metabolism of milacemide. Milacemide appears to be the first non-polyamine xenobiotic in the metabolism of which PAO participates.

Polyamine catabolism in rodent and human cells in culture

N1-Acetylspermidine (N1AcSpd) accumulates in late exponential phase, or after certain stresses such as heat shock, in both human tumour (A549) and rodent (HTC, CHO) cells, grown in medium containing an inhibitor of the FAD-dependent polyamine oxidase (PAO). Inhibition of PAO has little effect on cell growth or on the cellular content of the major polyamines, putrescine, spermidine or spermine, found in proliferating cells in culture, but decreases cellular putrescine content in heat shocked cells. Putrescine and spermidine are generated when N1AcSpd or N1-acetylspermine (N1AcSpm) respectively is added to either human or rodent cells depleted of the former amines by alpha-difluoromethylornithine. N1AcSpm is formed in polyamine-depleted human A549 cells when N1AcSpd is added to cultures treated with the PAO inhibitor. This reaction does not occur in either rodent line, suggesting that N1AcSpd can be converted directly into N1AcSpm in human, but not rodent, cells under specific conditions. The data presented demonstrate that a variety of human and rodent cells express PAO activity and catabolize polyamines by a mechanism which includes PAO. PAO activity is of little consequence to proliferating A549, HTC or CHO cells in culture, but does produce new putrescine in both late-exponential-phase and heat-shocked cells. These findings suggest that polyamine catabolism is part of a general response of both rodent and human cells to a variety of environmental and physiological stresses.

Gestational-age-dependent concentrations of polyamines, their conjugates and metabolites in urine and amniotic fluid.

The metabolism and fate of polyamines during cell proliferation, differentiation, and cell loss were investigated by measuring the concentration of polyamines, their conjugates and some of their metabolites in amniotic fluid of 24 subjects, and in urine of 85 women during pregnancy. The increase of putrescine and spermine in acid-hydrolysed urines during pregnancy appeared to be almost completely due to increases in monoacetylated putrescine and N1,N12 diacetylated spermine, respectively. The latter two were the quantitatively most important polyamines in amniotic fluid. In urine, monoacetylated putrescine showed the highest levels at the end of pregnancy, whereas N1,N12 diacetylated spermine reached the highest values at about 32 weeks gestation. It was impossible to establish whether extracellular monoacetylated putrescine is linked either to cell growth or cell loss. The appearance of N1,N12 diacetylated spermine is probably due to cell loss and dependent on the degree of differentiation during fetal development. The decline and eventual disappearance of urinary N1,N12 diacetylated spermine during the first 2 years after birth may be coherent with maturation of the FAD-dependent polyamine oxidase activity.