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Heterologous Expression and Characterization of Mouse Spermine Oxidase*

  • Manuela Cervelli
    Affiliations
    From the Dipartimento di Biologia, Università “Roma Tre,” I-00146 Roma, Italy
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  • Fabio Polticelli
    Affiliations
    From the Dipartimento di Biologia, Università “Roma Tre,” I-00146 Roma, Italy
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  • Rodolfo Federico
    Affiliations
    From the Dipartimento di Biologia, Università “Roma Tre,” I-00146 Roma, Italy
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  • Paolo Mariottini
    Correspondence
    To whom correspondence should be addressed: Dipartimento di Biologia, Università degli Studi “Roma Tre,” Viale Guglielmo Marconi 446, 00146 Roma, Italy. Tel.: 39-06-55176359; Fax: 39-06-5517-6321;
    Affiliations
    From the Dipartimento di Biologia, Università “Roma Tre,” I-00146 Roma, Italy
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  • Author Footnotes
    * This research was supported by grants from Ministero del l'Università e della Ricerca Scientifica e Tecnologica (MURST).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 27, 2002DOI:https://doi.org/10.1074/jbc.M207888200
      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.
      Polyamine oxidase (PAO),
      The abbreviations used are: PAO, polyamine oxidase; FAD, flavin adenine dinucleotide; MPAO, maize polyamine oxidase; SMO, spermine oxidase; mSMO, mouse SMO; HT, His tag; PAOh1, human polyamine oxidase; spermidine, Spd; spermine, Spm; N 1-acetylSpm, N 1-acetyl derivative of spermine; N 1-acetylSpd, N 1-acetyl derivative of spermidine
      1The abbreviations used are: PAO, polyamine oxidase; FAD, flavin adenine dinucleotide; MPAO, maize polyamine oxidase; SMO, spermine oxidase; mSMO, mouse SMO; HT, His tag; PAOh1, human polyamine oxidase; spermidine, Spd; spermine, Spm; N 1-acetylSpm, N 1-acetyl derivative of spermine; N 1-acetylSpd, N 1-acetyl derivative of spermidine
      a flavin adenine dinucleotide (FAD)-containing enzyme, catalyzes the oxidation of polyamines at the secondary amino group, giving different products according to the organism considered. In particular, vertebrate PAOs (EC 1.5.3.11) participate in the interconversion metabolism of polyamines, converting N 1-acetyl derivatives of spermine (N 1-acetylSpm) and spermidine (N 1-acetylSpd) into Spd and putrescine, respectively, plus 3-aminopropanal and H2O2, (
      • McIntire W.S.
      • Hartman C.
      ,
      • Seiler N.
      ,
      • Den Munckhof R.J.M.
      • Denyn M.
      • Tigchelaar-Gutter W.
      • Schipper R.G.
      • Verhofstad A.A.J.
      • Van Noorden C.J.F.
      • Frederiks W.M.
      ). PAOs with similar characteristics occur in methylotrophic yeasts (
      • Cohen S.S.
      ,
      • Nishikawa M.
      • Hagishita T.
      • Yurimoto H.
      • Kato N.
      • Sakai Y.
      • Hatanaka T.
      ) and amoebae (
      • Shukla O.P.
      • Muller S.
      • Walter R.D.
      ). On the contrary, plant (
      • McIntire W.S.
      • Hartman C.
      ), bacterial (
      • Tabor C.W.
      • Tabor H.
      ), and protozoan (
      • Kim B.G.
      • Sobota A.
      • Bitonti A.J.
      • McCann P.P.
      • Byers T.J.
      ) PAOs oxidize spermidine and spermine to 4-aminobutanol or N-(3-aminopropyl)-4-aminobutanol, respectively, plus 1,3-diaminopropane and H2O2. As these compounds cannot be converted directly to other polyamines, this class of PAOs generally is considered to be involved in the terminal catabolism of polyamines. Since PAOs play a crucial role in polyamine catabolism, these enzymes are important drug targets, and in fact, it has been shown that a number of polyamine analogues have an antitumor effect in different cell lines (
      • Pegg A.E.
      • Hu R.H.
      ,
      • Bergeron R.J.
      • Feng Y.
      • Weimar W.R.
      • McManis J.S.
      • Dimova H.
      • Porter C.
      • Raisler B.
      • Phanstiel O.A.
      ,
      • Ha H.C.
      • Woster P.M.
      • Yager J.D.
      • Casero Jr., R.A.
      ,
      • Casero E.A.
      • Woster P.M.
      ).
      As compared with large and detailed investigations on plant PAOs (
      • McIntire W.S.
      • Hartman C.
      ,
      • Smith T.A.
      ,
      • Smith T.A.
      • Barker J.H.
      ,
      • Federico R.
      • Alisi C.
      • Forlani F.
      ,
      • Angelini R.
      • Federico R.
      • Bonfante P.
      ,
      • Tavladoraki P.
      • Schininà M.E.
      • Cecconi F.
      • Di Agostino S.
      • Manera F.
      • Rea G.
      • Mariottini P.
      • Federico R.
      • Angelini R.
      ,
      • Binda C.
      • Coda A.
      • Angelini R.
      • Federico R.
      • Ascenzi P.
      • Mattevi A.
      ,
      • Laurenzi M.
      • Rea G.
      • Federico R.
      • Tavladoraki P.
      • Angelini R.
      ,
      • Cervelli M.
      • Tavladoraki P.
      • Di Agostino S.
      • Angelini R.
      • Federico R.
      • Mariottini P.
      ,
      • Cervelli M.
      • Cona A.
      • Angelini R.
      • Polticelli F.
      • Federico R.
      • Mariottini P.
      ,
      • Šebela M.
      • Radová A.
      • Angelini R.
      • Tavladoraki P.
      • Frébort I.
      • Pêc P.
      ), only little attention has been devoted to the animal counterpart (
      • Seiler N.
      ,
      • Hölttä E.
      ,
      • Bolkenius F.N.
      • Bey P.
      • Seiler N.
      ,
      • Suzuki O.
      • Matsumoto T.
      • Katsumata Y.
      ,
      • Libby P.R.
      • Porter C.W.
      ,
      • Tsukada T.
      • Furusako S.
      • Maekawa S.
      • Hibasami H.
      • Nakashima K.
      ,
      • Gasparyan V.K.
      • Nalbandyan R.M.
      ). Recently, Wang et al. (
      • Wang Y.
      • Devereux W.
      • Woster P.M.
      • Stewart T.M.
      • Hacker A.
      • Casero Jr, R.A.
      ) and Vujcic et al. (
      • Vujcic S.
      • Diegelman P.
      • Bacchi C.J.
      • Kramer D.L.
      • Porter C.W.
      ) have reported the cloning and characterization of novel mammalian PAO enzymes capable of oxidizing preferentially Spm and for this reason named spermine oxidase (SMO) (
      • Vujcic S.
      • Diegelman P.
      • Bacchi C.J.
      • Kramer D.L.
      • Porter C.W.
      ). In particular, this enzyme was expressed in an in vitro transcription/translation system (
      • Wang Y.
      • Devereux W.
      • Woster P.M.
      • Stewart T.M.
      • Hacker A.
      • Casero Jr, R.A.
      ) and into transiently transfected human kidney cells (
      • Vujcic S.
      • Diegelman P.
      • Bacchi C.J.
      • Kramer D.L.
      • Porter C.W.
      ). Based on these studies, it was postulated that in addition to the traditional interconversion pathway in which Spm is first acetylated by spermidine/spermineN 1-acetyltransferase and then oxidized by PAO, mammalian cells contain an enzyme capable of directly oxidizing Spm to Spd (
      • Vujcic S.
      • Diegelman P.
      • Bacchi C.J.
      • Kramer D.L.
      • Porter C.W.
      ).
      Data base searching analysis using PAOh1 cDNA sequence recovered a mouse cDNA clone (Image Clone 264769) corresponding to the murine counterpart, which was supplied by the United Kingdom Medical Research Council Human Genome Mapping Resource Centre (Cambridge, United Kingdom) consortium and herein defined as mSMO. To enhance the knowledge of enzymology of mammalian SMO and shed light on the structure/function relationship of this enzyme, the mSMO cDNA was further subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. This paper describes the expression and the main biochemical features of mouse spermine oxidase. Notwithstanding the low amino acid sequence homology shown by the animal and plant PAOs (45% sequence homology between MPAO and mSMO), molecular modeling of mSMO based on MPAO three-dimensional structure (
      • Binda C.
      • Coda A.
      • Angelini R.
      • Federico R.
      • Ascenzi P.
      • Mattevi A.
      ) suggests that the general features of MPAO active site are conserved in mSMO.

      DISCUSSION

      Plant or animal recombinant PAOs have been never reported to be expressed in E. coli cells to our knowledge. This is the first report of a vertebrate polyamine oxidase overexpressed in secreted and secreted-tagged forms in such a heterologous system. SMO recombinant enzymes are targeted to the periplasmic membrane compartment, and the catalytically active proteins are expressed at a level of ∼6 units/liter of culture broth. The data obtained for the secreted recombinant mSMO perfectly match the ones obtained for the secreted-tagged enzyme form.
      The analysis of the expressed mSMO protein gave an improved understanding of the biochemical features of this enzyme, since the purified recombinant enzyme was able to oxidize only Spm and failed to act upon Spd and the N 1-acetyl derivatives. The enzyme specificity reported herein is in agreement with the one described in transfected human cells by Vujcic et al.(
      • Vujcic S.
      • Diegelman P.
      • Bacchi C.J.
      • Kramer D.L.
      • Porter C.W.
      ).
      The precise nature of the reaction products and the cleavage position on the Spm substrate are those typical of an animal PAO enzyme. In line with this finding, the specific inhibition of mSMO enzyme activity was obtained with MDL72527 but not with pargyline.
      The absorption spectrum of the native enzyme showed the typical three-banded spectrum for flavoproteins. Furthermore, the addition of the Spm substrate in anaerobic conditions resulted in a dramatic absorbance decrease, while reoxygenation of the enzyme restored the initial spectrum, indicating the involvement of a FAD group in the catalytic cycle.
      An analysis of the molecular model of mSMO as compared with MPAO three-dimensional structure suggests that the catalytic tunnel of the former enzyme is wider. It is tempting to speculate that the preference for Spm over Spd as a substrate observed for mSMO is linked to the different shape of the catalytic tunnel in which the short Spd substrate would be bound in a “floppy” fashion, thus rendering less efficient the enzyme catalysis, which has been hypothesized to rely on an “in register” binding of the substrates.
      The substitution of Glu-62 by a His residue in mSMO can provide an explanation for the peculiar pH dependence of the activity. In fact, it can be reasonably assumed that His-62 is partially protonated at pH values lower than 7.0. Thus, the binding of the cationic polyamine substrates would be unfavorable and enzyme activity would be very low as observed experimentally. At pH values higher than 7.0, His-62 would deprotonate, thus facilitating substrate binding and leading to an increase in enzyme activity, which is maximal at pH values of ∼8.0.
      In conclusion, in mammalian cells, polyamine catabolism seems to be mediated by the activity of two enzymes, PAO and the novel SMO, described in this work (
      • Vujcic S.
      • Diegelman P.
      • Bacchi C.J.
      • Kramer D.L.
      • Porter C.W.
      ). The precise significance of both polyamine oxidase activities in the metabolism of polyamines remains to be established, particularly regarding the role of each enzyme in regulating polyamine concentrations in mammalian tissues in relation with growth processes and malignant transformation.

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