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PAO specifically oxidizes substrates that have both primary and secondary amino groups. The complex with MDL72527 shows that the primary amino groups are essential for the proper alignment of the substrate with respect to the flavin. Conservation of an N-terminal sequence motif indicates that PAO is member of a novel family of flavoenzymes. Among these, monoamine oxidase displays significant sequence homology with PAO, suggesting a similar overall folding topology.

Polyamines (PAs) are nitrogenous molecules which play a well-established role in most cellular processes during growth and development under physiological or biotic/abiotic stress conditions. The molecular mode(s) of PA action have only recently started to be unveiled, and comprehensive models for their molecular interactions have been proposed. Their multiple roles are exerted, at least partially, through signalling by hydrogen peroxide (H(2)O(2)), which is generated by the oxidation/back-conversion of PAs by copper amine oxidases and PA oxidases. Accumulating evidence suggests that in plants the cellular titres of PAs are affected by other nitrogenous compounds. Here, we discuss the state of the art on the possible nitrogen flow in PAs, their interconnection with nitrogen metabolism, as well as the signalling roles of PA-derived H(2)O(2) during some developmental processes and stress responses.

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H(2)O(2), and an aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H(2)O(2) and an aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1-AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine back-conversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.

Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H 2 O 2 , an aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H 2 O 2 , and an aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N 1 -acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.

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Wounding chickpea (Cicer arietinum) internodes or cotyledons resulted in an increase in the steady-state level of copper amine oxidase (CuAO) expression both locally and systemically. Dissection of the molecular mechanisms controlling CuAO expression indicated that jasmonic acid worked as a potent inducer of the basal and wound-inducible CuAO expression, whereas salicylic acid and abscisic acid caused a strong reduction of the wound-induced CuAO expression, without having any effect on the basal levels. Epicotyl treatment with the CuAO mechanism-based inhibitor 2-bromoethylamine decreased hydrogen peroxide (H 2 O 2 ) levels in all the internodes, as evidenced in vivo by 3,3Ј-diaminobenzidine oxidation. Moreover, inhibitor pretreatment of wounded epicotyls resulted in a lower accumulation of H 2 O 2 both at the wound site and in distal organs. In vivo CuAO inhibition by 2-bromoethylamine after inoculation of resistant chickpea cv Sultano with Ascochyta rabiei resulted in the development of extended necrotic lesions, with extensive cell damage occurring in sclerenchyma and cortical parenchyma tissues. These results, besides stressing the fine-tuning by key signaling molecules in wound-induced CuAO regulation, demonstrate that local and systemic CuAO induction is essential for H 2 O 2 production in response to wounding and indicate the relevance of these enzymes in protection against pathogens.Plant defense responses are accomplished by the deployment of a complex array of events that are differentially modulated depending on the incoming stress (Maleck and Dietrich, 1999). Wounding different plant organs or interaction with pathogens induce local and systemic accumulation of defenserelated proteins (Hammond-Kosack and Jones, 1996;Ryals et al., 1996;Ryan, 2000). The study of signaling events inducing local and systemic responses led to the discovery of systemin, jasmonates, ethylene, salicylic acid (SA), and abscisic acid (ABA) as signal molecules (Peñ a-Cortés et al., 1989; Farmer and Ryan, 1990; Pearce et al., 1991;Xu et al., 1994; O'Donnell et al., 1996;Schweizer et al., 1998;van Loon et al., 1998; Knoester et al., 1999).The existence of multiple defense strategies and complex signaling networks leads to an enhanced defense capacity of the plants. The signal transduction pathways of wounding and pathogen attack may be common, different, or exclusive, depending on the biological system, but likewise the establishment of defense mechanisms requires the presence or accumulation of hydrogen peroxide (H 2 O 2 ; Sutherland, 1991; Mehdy, 1994; Hammond-Kosack et al., 1996). In particular, H 2 O 2 behaves as a direct cytotoxic compound against pathogens and as a second messenger in the activation of defense genes (Lamb and Dixon, 1997). Moreover, this compound is involved in systemic acquired resistance and acts synergistically with NO in the induction of hypersensitive cell death (Delledonne et al., 1998). As a cosubstrate of the peroxidases, H 2 O 2 has been implicated in the oxidative cross-linking of apoplastic st...

Polyamine oxidase (PAO) carries out the FAD-dependent oxidation of the secondary amino groups of spermidine and spermine, a key reaction in the polyamine catabolism. The active site of PAO consists of a 30 A long U-shaped catalytic tunnel, whose innermost part is located in front of the flavin ring. To provide insight into the PAO substrate specificity and amine oxidation mechanism, we have investigated the crystal structure of maize PAO in the reduced state and in complex with three different inhibitors, guazatine, 1,8-diaminooctane, and N(1)-ethyl-N(11)-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm). In the reduced state, the conformation of the isoalloxazine ring and the surrounding residues is identical to that of the oxidized enzyme. Only Lys300 moves away from the flavin to compensate for the change in cofactor protonation occurring upon reduction. The structure of the PAO.inhibitor complexes reveals an exact match between the inhibitors and the PAO catalytic tunnel. Inhibitor binding does not involve any protein conformational change. Such lock-and-key binding occurs also in the complex with CHENSpm, which forms a covalent adduct with the flavin N5 atom. Comparison of the enzyme complexes hints at an "out-of-register" mechanism of inhibition, in which the inhibitor secondary amino groups are not properly aligned with respect to the flavin to allow oxidation. Except for the Glu62-Glu170 pair, no negatively charged residues are involved in the recognition of substrate and inhibitor amino groups, which is in contrast to other polyamine binding proteins. This feature may be exploited in the design of drugs specifically targeting PAO.