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Rodolfo Federico - One of the best experts on this subject based on the ideXlab platform.

  • probing mammalian spermine Oxidase enzyme substrate complex through molecular modeling site directed mutagenesis and biochemical characterization
    Amino Acids, 2011
    Co-Authors: Paraskevi Tavladoraki, Manuela Cervelli, Fabrizio Antonangeli, Giovanni Minervini, Pasquale Stano, Paolo Mariottini, Rodolfo Federico, Fabio Polticelli
    Abstract:

    Spermine Oxidase (SMO) and acetylPolyamine Oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of Polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in Polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in Polyamine metabolism and related pathologies. However, none of the available Polyamine Oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure–function relationships, SMO enzyme–substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast Polyamine Oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme–substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

  • functional diversity inside the arabidopsis Polyamine Oxidase gene family
    Journal of Experimental Botany, 2011
    Co-Authors: Paola Fincato, Rodolfo Federico, Riccardo Angelini, Kalliopi A Roubelakisangelakis, Panagiotis N Moschou, Raffaela Tavazza, Valentina Spedaletti, Paraskevi Tavladoraki
    Abstract:

    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, H2O2, 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 H2O2 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 backconversion 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.

  • synthesis and biological evaluation of guanidino compounds endowed with subnanomolar affinity as competitive inhibitors of maize Polyamine Oxidase
    Journal of Medicinal Chemistry, 2009
    Co-Authors: Fabrizio Manetti, Alessandra Cona, Lucilla Angeli, Claudia Mugnaini, Francesco Raffi, Caterina Capone, Elena Dreassi, Alessandra Tania Zizzari, Alessandra Tisi, Rodolfo Federico
    Abstract:

    Previous studies on agmatine and its derivatives suggested that the presence of hydrophobic groups on the guanidine moiety was a crucial key for inhibitory activity of maize Polyamine Oxidase. Accordingly, new lipophilic agmatine and iminoctadine derivatives were synthesized and tested for their ability to inhibit this enzyme. Several compounds showed an affinity in the nanomolar range, while a cyclopropylmethyl derivative of iminoctadine was found to be the most potent inhibitor of maize Polyamine Oxidase reported so far (Ki = 0.08 nM).

  • inducible expression of maize Polyamine Oxidase in the nucleus of mcf 7 human breast cancer cells confers sensitivity to etoposide
    Amino Acids, 2008
    Co-Authors: Lucia Marcocci, Pasquale Stano, Rodolfo Federico, M Casadei, Carmen Faso, A Antoccia, S Leone, B Mondovi, Paraskevi Tavladoraki
    Abstract:

    In this study, Polyamine Oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H2O2, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with Polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest Polyamine Oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine.

  • involvement of Polyamine Oxidase in wound healing
    Plant Physiology, 2008
    Co-Authors: Riccardo Angelini, Rodolfo Federico, Maurizio Botta, Alessandra Tisi, Giuseppina Rea, Martha M Chen, Alessandra Cona
    Abstract:

    Hydrogen peroxide (H2O2) is involved in plant defense responses that follow mechanical damage, such as those that occur during herbivore or insect attacks, as well as pathogen attack. H2O2 accumulation is induced during wound healing processes as well as by treatment with the wound signal jasmonic acid. Plant Polyamine Oxidases (PAOs) are H2O2 producing enzymes supposedly involved in cell wall differentiation processes and defense responses. Maize (Zea mays) PAO (ZmPAO) is a developmentally regulated flavoprotein abundant in primary and secondary cell walls of several tissues. In this study, we investigated the effect of wounding on ZmPAO gene expression in the outer tissues of the maize mesocotyl and provide evidence that ZmPAO enzyme activity, protein, and mRNA levels increased in response to wounding as well as jasmonic acid treatment. Histochemically detected ZmPAO activity especially intensified in the epidermis and in the wound periderm, suggesting a tissue-specific involvement of ZmPAO in wound healing. The role played by ZmPAO-derived H2O2 production in perOxidase-mediated wall stiffening events was further investigated by exploiting the in vivo use of N-prenylagmatine (G3), a selective and powerful ZmPAO inhibitor, representing a reliable diagnostic tool in discriminating ZmPAO-mediated H2O2 production from that generated by perOxidase, oxalate Oxidase, or by NADPH Oxidase activity. Here, we demonstrate that G3 inhibits wound-induced H2O2 production and strongly reduces lignin and suberin polyphenolic domain deposition along the wound, while it is ineffective in inhibiting the deposition of suberin aliphatic domain. Moreover, ZmPAO ectopic expression in the cell wall of transgenic tobacco (Nicotiana tabacum) plants strongly enhanced lignosuberization along the wound periderm, providing evidence for a causal relationship between PAO and perOxidase-mediated events during wound healing.

Riccardo Angelini - One of the best experts on this subject based on the ideXlab platform.

  • maize Polyamine Oxidase in the presence of spermine spermidine induces the apoptosis of lovo human colon adenocarcinoma cells
    International Journal of Oncology, 2019
    Co-Authors: Shinji Ohkubo, Alessandra Cona, Riccardo Angelini, Da Spandidos, Romina Mancinelli, Selenia Miglietta, Gianluca Canettieri, Eugenio Gaudio, Enzo Agostinelli
    Abstract:

    Amine Oxidases, which contribute to the regulation of Polyamine levels, catalyze the oxidative deamination of Polyamines to generate H2O2 and aldehyde(s). In this study, and at least to the best of our knowledge, maize Polyamine Oxidase (ZmPAO) was used for the first time with the aim of identifying a novel strategy for cancer therapy. The cytotoxicity and the mechanisms of cell death induced by the enzymatic oxidation products of Polyamine generated by ZmPAO were investigated. Exogenous spermine and ZmPAO treatment decreased cell viability in a spermine dose‑ and time‑dependent manner, particularly, the viability of the multidrug‑resistant (MDR) colon adenocarcinoma cells, LoVo DX, when compared with drug‑sensitive ones (LoVo WT). Further analyses revealed that H2O2 derived from spermine was mainly responsible for the cytotoxicity. Flow cytometric analysis revealed that treatment with ZmPAO and spermine increased the apoptotic population of LoVo WT and LoVo DX cells. In addition, we found that treatment with ZmPAO and spermine markedly reduced mitochondrial membrane potential in the LoVo DX cells, in agreement with the results of cell viability and apoptosis assays. Transmission electron microscopic observations supported the involvement of mitochondrial depolarization in the apoptotic process. Therefore, the dysregulation of Polyamine metabolism in tumor cells may be a potential therapeutic target. In addition, the development of MDR tumor cells is recognized as a major obstacle in cancer therapy. Therefore, the design of a novel therapeutic strategy based on the use of this combination may be taken into account, making this approach attractive mainly in treating MDR cancer patients.

  • functional diversity inside the arabidopsis Polyamine Oxidase gene family
    Journal of Experimental Botany, 2011
    Co-Authors: Paola Fincato, Rodolfo Federico, Riccardo Angelini, Kalliopi A Roubelakisangelakis, Panagiotis N Moschou, Raffaela Tavazza, Valentina Spedaletti, Paraskevi Tavladoraki
    Abstract:

    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, H2O2, 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 H2O2 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 backconversion 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.

  • involvement of Polyamine Oxidase in wound healing
    Plant Physiology, 2008
    Co-Authors: Riccardo Angelini, Rodolfo Federico, Maurizio Botta, Alessandra Tisi, Giuseppina Rea, Martha M Chen, Alessandra Cona
    Abstract:

    Hydrogen peroxide (H2O2) is involved in plant defense responses that follow mechanical damage, such as those that occur during herbivore or insect attacks, as well as pathogen attack. H2O2 accumulation is induced during wound healing processes as well as by treatment with the wound signal jasmonic acid. Plant Polyamine Oxidases (PAOs) are H2O2 producing enzymes supposedly involved in cell wall differentiation processes and defense responses. Maize (Zea mays) PAO (ZmPAO) is a developmentally regulated flavoprotein abundant in primary and secondary cell walls of several tissues. In this study, we investigated the effect of wounding on ZmPAO gene expression in the outer tissues of the maize mesocotyl and provide evidence that ZmPAO enzyme activity, protein, and mRNA levels increased in response to wounding as well as jasmonic acid treatment. Histochemically detected ZmPAO activity especially intensified in the epidermis and in the wound periderm, suggesting a tissue-specific involvement of ZmPAO in wound healing. The role played by ZmPAO-derived H2O2 production in perOxidase-mediated wall stiffening events was further investigated by exploiting the in vivo use of N-prenylagmatine (G3), a selective and powerful ZmPAO inhibitor, representing a reliable diagnostic tool in discriminating ZmPAO-mediated H2O2 production from that generated by perOxidase, oxalate Oxidase, or by NADPH Oxidase activity. Here, we demonstrate that G3 inhibits wound-induced H2O2 production and strongly reduces lignin and suberin polyphenolic domain deposition along the wound, while it is ineffective in inhibiting the deposition of suberin aliphatic domain. Moreover, ZmPAO ectopic expression in the cell wall of transgenic tobacco (Nicotiana tabacum) plants strongly enhanced lignosuberization along the wound periderm, providing evidence for a causal relationship between PAO and perOxidase-mediated events during wound healing.

  • heterologous expression and biochemical characterization of a Polyamine Oxidase from arabidopsis involved in Polyamine back conversion
    Plant Physiology, 2006
    Co-Authors: Paraskevi Tavladoraki, Fabio Polticelli, Riccardo Angelini, Marianna Nicoletta Rossi, Giuseppe Saccuti, Miguel A Perezamador, Rodolfo Federico
    Abstract:

    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 H2O2, 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, H2O2, 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 N1-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.

  • flavin containing Polyamine Oxidase is a hydrogen peroxide source in the oxidative response to the protein phosphatase inhibitor cantharidin in zea mays l
    Journal of Experimental Botany, 2006
    Co-Authors: Alessandra Cona, Rodolfo Federico, Federico Corelli, Maurizio Botta, Giuseppina Rea, Riccardo Angelini
    Abstract:

    In this study, the specific contribution of Polyamine Oxidase (PAO), a hydrogen peroxide (H2O2)-producing enzyme, to the oxidative burst induced in maize mesocotyl by the phosphatase inhibitor cantharidin was examined. For this purpose, a pharmacological approach was applied using, either in vitro or in vivo, two strong inhibitors of maize PAO (MPAO), N-prenylagmatine (G3) and its structural analogue Ro5, as well as diphenyleneiodonium (DPI), an inhibitor of the phagocyte NAD(P)H Oxidase. DPI was shown to be a good MPAO inhibitor in vitro. G3, Ro5, and DPI were very effective in inhibiting in vivo the extracellular accumulation of H2O2 that is released by mesocotyl segments upon spermidine supply. G3 and Ro5 did not show any inhibition in vitro of either horseradish perOxidase or barley oxalate Oxidase. Moreover, G3 and Ro5 did not inhibit the extracellular accumulation of superoxide radical that is released in vivo upon NADH supply. G3, Ro5, and DPI strongly affected H2O2 production induced in maize mesocotyl by cantharidin. Histochemical localization of H2O2 in cantharidin-treated mesocotyl cross-sections revealed an increase of H2O2-specific staining in the epidermal and subepidermal tissues. The effect was also inhibited by G3 and DPI. Moreover, an increase in MPAO activity was observed in the same tissues upon cantharidin treatment. All these data suggest that G3 and Ro5 behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo and that MPAO activity contributes to a major part of the cantharidin-induced H2O2 synthesis in the apoplastic milieu of maize mesocotyl.

Tomonobu Kusano - One of the best experts on this subject based on the ideXlab platform.

  • expression profile of seven Polyamine Oxidase genes in rice oryza sativa in response to abiotic stresses phytohormones and Polyamines
    Physiology and Molecular Biology of Plants, 2021
    Co-Authors: G H M Sagor, Tomonobu Kusano, Masataka Inoue, Thomas Berberich
    Abstract:

    Polyamine levels are controlled by biosynthesis, intra- and inter-cellular flux by the respective transporters, and catabolism. The catabolism is catalyzed by two groups of enzymes. One is copper-containing amine Oxidases and the other is Polyamine Oxidases (PAOs). In Oryza sativa, seven PAO genes exist and they are termed as OsPAO1 to OsPAO7. However, their physiological function has not been elucidated yet. Here, we examined the expressional changes of seven OsPAO genes upon abiotic and oxidative stress, phytohormone, and exogenous Polyamines application. The transcript of extracellular Polyamine Oxidase OsPAO2 and OsPAO6 are strongly induced upon wounding, drought, salinity, oxidative stress (H2O2), and exogenous application of jasmonic acid, spermidine, spermine, thermospermine and negatively regulated upon indole acetic acid, isopentenyl adenine (iPT), gibberellic acid (GA), abscisic acid; OsPAO7 is to iPT, GA and all Polyamines; OsPAO4 and OsPAO5 are mildly responsive to heat, cold, oxidative stress. These results suggest that Polyamine Oxidase encoding extracellular enzyme may play a pivotal role during exogenous stimulus to protect the plant cell.

  • Polyamine Oxidase 1 from rice oryza sativa is a functional ortholog of arabidopsis Polyamine Oxidase 5
    Plant Signaling & Behavior, 2014
    Co-Authors: Taibo Liu, Masaru Niitsu, Thomas Berberich, Dong Wook Kim, Tomonobu Kusano
    Abstract:

    Polyamine Oxidase 1 (OsPAO1), from rice (Oryza sativa), and Polyamine Oxidase 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as Polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5-2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5-2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized Polyamine Oxidase, into Atpao5-2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5.

  • Polyamine Oxidase 7 is a terminal catabolism type enzyme in oryza sativa and is specifically expressed in anthers
    Plant and Cell Physiology, 2014
    Co-Authors: Taibo Liu, Masaru Niitsu, Thomas Berberich, Dong Wook Kim, Shunsuke Maeda, Masao Watanabe, Yoshiyuki Kamio, Tomonobu Kusano
    Abstract:

    Polyamine Oxidase (PAO), which requires FAD as a cofactor, functions in Polyamine catabolism. Plant PAOs are classified into two groups based on their reaction modes. The terminal catabolism (TC) reaction always produces 1,3-diaminopropane (DAP), H2O2, and the respective aldehydes, while the back-conversion (BC) reaction produces spermidine (Spd) from tetraamines, spermine (Spm) and thermospermine (T-Spm) and/or putrescine from Spd, along with 3-aminopropanal and H2O2. The Oryza sativa genome contains seven PAO-encoded genes termed OsPAO1-OsPAO7. To date, we have characterized four OsPAO genes. The products of these genes, i.e. OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze BC-type reactions. Whereas OsPAO1 remains in the cytoplasm, the other three PAOs localize to peroxisomes. Here, we examined OsPAO7 and its gene product. OsPAO7 shows high identity to maize ZmPAO1, the best characterized plant PAO having TC-type activity. OsPAO7 seems to remain in a peripheral layer of the plant cell with the aid of its predicted signal peptide and transmembrane domain. Recombinant OsPAO7 prefers Spm and Spd as substrates, and it produces DAP from both substrates in a time-dependent manner, indicating that OsPAO7 is the first TC-type enzyme identified in O. sativa. The results clearly show that two types of PAOs co-exist in O. sativa. Furthermore, OsPAO7 is specifically expressed in anthers, with an expressional peak at the bicellular pollen stage. The physiological function of OsPAO7 in anthers is discussed.

  • oryza sativa Polyamine Oxidase 1 back converts tetraamines spermine and thermospermine to spermidine
    Plant Cell Reports, 2014
    Co-Authors: Masaru Niitsu, Thomas Berberich, Tomonobu Kusano
    Abstract:

    Key message Oryza sativa Polyamine Oxidase 1 back-converts spermine (or thermospermine) to spermidine. Considering the previous work, major path of Polyamine catabolism in rice plant is suggestive to be back-conversion but not terminal catabolism.

  • oryza sativa Polyamine Oxidase 1 back converts tetraamines spermine and thermospermine to spermidine
    Plant Cell Reports, 2014
    Co-Authors: Taibo Liu, Masaru Niitsu, Thomas Berberich, Dong Wook Kim, Tomonobu Kusano
    Abstract:

    Oryza sativa Polyamine Oxidase 1 back-converts spermine (or thermospermine) to spermidine. Considering the previous work, major path of Polyamine catabolism in rice plant is suggestive to be back-conversion but not terminal catabolism. Rice (Oryza sativa) contains seven genes encoding Polyamine Oxidases (PAOs), termed OsPAO1 to OsPAO7, based on their chromosomal number and gene ID number. We previously showed that three of these members, OsPAO3, OsPAO4 and OsPAO5, are abundantly expressed, that their products localize to peroxisomes and that they catalyze the Polyamine back-conversion reaction. Here, we have focused on OsPAO1. The OsPAO1 gene product shares a high level of identity with those of Arabidopsis PAO5 and Brassica juncea PAO. Expression of OsPAO1 appears to be quite low under physiological conditions, but is markedly induced in rice roots by spermine (Spm) or T-Spm treatment. Consistent with the above finding, the recombinant OsPAO1 prefers T-Spm as a substrate at pH 6.0 and Spm at pH 8.5 and, in both cases, back-converts these tetraamines to spermidine, but not to putrescine. OsPAO1 localizes to the cytoplasm of onion epidermal cells. Differing in subcellular localization, four out of seven rice PAOs, OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze back-conversion reactions of PAs. Based on the results, we discuss the catabolic path(s) of PAs in rice plant.

Paraskevi Tavladoraki - One of the best experts on this subject based on the ideXlab platform.

  • probing mammalian spermine Oxidase enzyme substrate complex through molecular modeling site directed mutagenesis and biochemical characterization
    Amino Acids, 2011
    Co-Authors: Paraskevi Tavladoraki, Manuela Cervelli, Fabrizio Antonangeli, Giovanni Minervini, Pasquale Stano, Paolo Mariottini, Rodolfo Federico, Fabio Polticelli
    Abstract:

    Spermine Oxidase (SMO) and acetylPolyamine Oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of Polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in Polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in Polyamine metabolism and related pathologies. However, none of the available Polyamine Oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure–function relationships, SMO enzyme–substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast Polyamine Oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme–substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

  • functional diversity inside the arabidopsis Polyamine Oxidase gene family
    Journal of Experimental Botany, 2011
    Co-Authors: Paola Fincato, Rodolfo Federico, Riccardo Angelini, Kalliopi A Roubelakisangelakis, Panagiotis N Moschou, Raffaela Tavazza, Valentina Spedaletti, Paraskevi Tavladoraki
    Abstract:

    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, H2O2, 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 H2O2 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 backconversion 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.

  • inducible expression of maize Polyamine Oxidase in the nucleus of mcf 7 human breast cancer cells confers sensitivity to etoposide
    Amino Acids, 2008
    Co-Authors: Lucia Marcocci, Pasquale Stano, Rodolfo Federico, M Casadei, Carmen Faso, A Antoccia, S Leone, B Mondovi, Paraskevi Tavladoraki
    Abstract:

    In this study, Polyamine Oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H2O2, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with Polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest Polyamine Oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine.

  • heterologous expression and biochemical characterization of a Polyamine Oxidase from arabidopsis involved in Polyamine back conversion
    Plant Physiology, 2006
    Co-Authors: Paraskevi Tavladoraki, Fabio Polticelli, Riccardo Angelini, Marianna Nicoletta Rossi, Giuseppe Saccuti, Miguel A Perezamador, Rodolfo Federico
    Abstract:

    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 H2O2, 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, H2O2, 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 N1-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.

  • lys300 plays a major role in the catalytic mechanism of maize Polyamine Oxidase
    Biochemistry, 2005
    Co-Authors: Fabio Polticelli, Giovanni Minervini, Rodolfo Federico, Alessandra Cona, Riccardo Angelini, Carmen Faso, Jaswir Basran, Nigel S Scrutton, Paraskevi Tavladoraki
    Abstract:

    Maize Polyamine Oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N 5 atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady- state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pKa of 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met- spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE ) 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE ) 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.

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  • pacific oyster Polyamine Oxidase a protein missing link in invertebrate evolution
    Amino Acids, 2015
    Co-Authors: Manuela Cervelli, Fabio Polticelli, Pasquale Stano, Emanuela Angelucci, Elena Di Muzio, Paolo Mariottini
    Abstract:

    Polyamine Oxidases catalyse the oxidation of Polyamines and acetylPolyamines and are responsible for the Polyamine interconversion metabolism in animal cells. Polyamine Oxidases from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, while in vertebrates two different enzymes, namely spermine Oxidase and acetylPolyamine Oxidase, specifically catalyse the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylPolyamine Oxidases have arisen from an ancestor invertebrate Polyamine Oxidase with lower specificity for Polyamine substrates, as demonstrated by the enzymatic activity of the mollusc Polyamine Oxidase characterized here. This is the first report of an invertebrate Polyamine Oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to Oxidase both N1-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

  • molecular evolution of the Polyamine Oxidase gene family in metazoa
    BMC Evolutionary Biology, 2012
    Co-Authors: Fabio Polticelli, Paolo Mariottini, Daniele Salvi, Roberto Amendola, Manuela Cervelli
    Abstract:

    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. 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. 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.

  • probing mammalian spermine Oxidase enzyme substrate complex through molecular modeling site directed mutagenesis and biochemical characterization
    Amino Acids, 2011
    Co-Authors: Paraskevi Tavladoraki, Manuela Cervelli, Fabrizio Antonangeli, Giovanni Minervini, Pasquale Stano, Paolo Mariottini, Rodolfo Federico, Fabio Polticelli
    Abstract:

    Spermine Oxidase (SMO) and acetylPolyamine Oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of Polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in Polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in Polyamine metabolism and related pathologies. However, none of the available Polyamine Oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure–function relationships, SMO enzyme–substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast Polyamine Oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme–substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

  • a novel c terminal sequence from barley Polyamine Oxidase is a vacuolar sorting signal
    Plant Journal, 2004
    Co-Authors: Manuela Cervelli, Rodolfo Federico, Riccardo Angelini, Oriana Di Caro, Alessandra Di Penta, Alessandro Vitale, Paolo Mariottini
    Abstract:

    Barley contains two different isoforms of flavin-containing Polyamine Oxidase (BPAO1 and BPAO2). We have previously demonstrated that BPAO2 is a symplastic protein in barley leaves. On the contrary, maize Polyamine Oxidase (MPAO), the best characterized member of this enzyme class, is apoplastic. Comparison of the derived amino-acid sequences of BPAO2 and MPAO has revealed that both precursor proteins include a cleavable N-terminal signal peptide of 25 amino acid residues, but the barley enzyme shows an extra C-terminal extension of eight amino acids. By means of MPAO engineering with BPAO2 C-terminal tail (MPAO-T) and exploiting transient expression in Nicotiana tabacum protoplasts, we demonstrate that this oligopeptide is a signal for protein sorting to the plant vacuole. The vacuolar sorting of MPAO-T was saturable. Specific mutations of the C-terminal tail were constructed to determine which amino acid residues of this novel propeptide affect proper protein sorting. No consensus sequence or common structural determinant is required for the intracellular retention of the MPAO-T protein, but a gradual lowering of the efficiency was observed as a result of progressive deletion of the C-terminus.

  • heterologous expression and characterization of mouse spermine Oxidase
    Journal of Biological Chemistry, 2003
    Co-Authors: Manuela Cervelli, Fabio Polticelli, Rodolfo Federico, Paolo Mariottini
    Abstract:

    Abstract Polyamine Oxidases are key enzymes responsible of the Polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine Oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine Oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of Polyamine Oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculatedM r of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms intoEscherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N 1-acetylPolyamines. The purified recombinant-tagged form enzyme (M r∼68,000) has K m and k catvalues of 90 μm and 4.5 s−1, respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize Polyamine Oxidase three-dimensional structure suggests that the general features of maize Polyamine Oxidase active site are conserved in mSMO.