Heterologous Expression and Characterization of Mouse Spermine Oxidase

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 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), 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 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, (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar, 2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 3Den Munckhof R.J.M. Denyn M. Tigchelaar-Gutter W. Schipper R.G. Verhofstad A.A.J. Van Noorden C.J.F. Frederiks W.M. J. Histochem. Cytochem. 1995; 43: 1155-1162Crossref PubMed Scopus (28) Google Scholar). PAOs with similar characteristics occur in methylotrophic yeasts (4Cohen S.S. Cohen S.S. A Guide to the Polyamines. Oxford University Press, NY1998: 82Google Scholar, 5Nishikawa M. Hagishita T. Yurimoto H. Kato N. Sakai Y. Hatanaka T. FEBS Lett. 2000; 476: 150-154Crossref PubMed Scopus (23) Google Scholar) and amoebae (6Shukla O.P. Muller S. Walter R.D. Mol. Biochem. Parasitol. 1992; 51: 91-98Crossref PubMed Scopus (12) Google Scholar). On the contrary, plant (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar), bacterial (7Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar), and protozoan (8Kim B.G. Sobota A. Bitonti A.J. McCann P.P. Byers T.J. J. Protozool. 1987; 34: 278-284Crossref PubMed Scopus (27) Google Scholar) 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 (9Pegg A.E. Hu R.H. Cancer Lett. 1995; 95: 247-252Crossref PubMed Scopus (25) Google Scholar, 10Bergeron R.J. Feng Y. Weimar W.R. McManis J.S. Dimova H. Porter C. Raisler B. Phanstiel O.A. J. Med. Chem. 1997; 40: 1475-1494Crossref PubMed Scopus (124) Google Scholar, 11Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 12Casero E.A. Woster P.M. J. Med. Chem. 2001; 44: 1-26Crossref PubMed Scopus (218) Google Scholar).As compared with large and detailed investigations on plant PAOs (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar,13Smith T.A. Phytochemistry. 1976; 15: 633-636Crossref Scopus (35) Google Scholar, 14Smith T.A. Barker J.H. Zappia V. Pegg A.E. Progress in Polyamine Research: Novel Biochemical, Pharmacological and Clinical Aspects. Plenum Press, NY1988: 573-589Google Scholar, 15Federico R. Alisi C. Forlani F. Phytochemistry. 1989; 28: 45-46Crossref Scopus (36) Google Scholar, 16Angelini R. Federico R. Bonfante P. J. Plant Physiol. 1995; 145: 686-692Crossref Scopus (40) Google Scholar, 17Tavladoraki P. Schininà M.E. Cecconi F. Di Agostino S. Manera F. Rea G. Mariottini P. Federico R. Angelini R. FEBS Lett. 1998; 426: 62-66Crossref PubMed Scopus (89) Google Scholar, 18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 19Laurenzi M. Rea G. Federico R. Tavladoraki P. Angelini R. Planta. 1999; 208: 146-154Crossref Scopus (47) Google Scholar, 20Cervelli M. Tavladoraki P. Di Agostino S. Angelini R. Federico R. Mariottini P. Plant Physiol. Biochem. 2000; 38: 667-677Crossref Scopus (39) Google Scholar, 21Cervelli M. Cona A. Angelini R. Polticelli F. Federico R. Mariottini P. Eur. J. Biochem. 2001; 268: 3816-3838Crossref PubMed Scopus (55) Google Scholar, 22Šebela M. Radová A. Angelini R. Tavladoraki P. Frébort I. Pêc P. Plant Sci. 2001; 160: 197-207Crossref PubMed Scopus (113) Google Scholar), only little attention has been devoted to the animal counterpart (2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 23Hölttä E. Biochemistry. 1977; 16: 91-100Crossref PubMed Scopus (266) Google Scholar, 24Bolkenius F.N. Bey P. Seiler N. Biochim. Biophys. Acta. 1984; 838: 69-76Crossref Scopus (68) Google Scholar, 25Suzuki O. Matsumoto T. Katsumata Y. Experentia (Basel). 1984; 40: 838-839Crossref PubMed Scopus (45) Google Scholar, 26Libby P.R. Porter C.W. Biochem. Biophys. Res. Commun. 1985; 144: 528-535Crossref Scopus (21) Google Scholar, 27Tsukada T. Furusako S. Maekawa S. Hibasami H. Nakashima K. Int. J. Biochem. 1988; 20: 695-702Crossref PubMed Scopus (17) Google Scholar, 28Gasparyan V.K. Nalbandyan R.M. Biokhimiya. 1990; 55: 1632-1637PubMed Google Scholar). Recently, Wang et al. (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and Vujcic et al. (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar) have reported the cloning and characterization of novel mammalian PAO enzymes capable of oxidizing preferentially Spm and for this reason named spermine oxidase (SMO) (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). In particular, this enzyme was expressed in an in vitro transcription/translation system (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and into transiently transfected human kidney cells (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). 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 (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar).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 (18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) suggests that the general features of MPAO active site are conserved in mSMO.DISCUSSIONPlant 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.(30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar).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 the typical spectrum for the addition of the Spm substrate in in a of the enzyme the the of a in the analysis of the molecular of mSMO as compared with MPAO three-dimensional structure suggests that the of the enzyme is is to that the for Spm Spd as a substrate for mSMO is to the different of the in which the Spd substrate be in a the enzyme which has been to on an of the of by a His in mSMO an for the pH of the In fact, it be that is at pH the of the polyamine be and enzyme activity be low as pH substrate and to an in enzyme which is at pH of in mammalian polyamine catabolism to be by the activity of PAO and the novel SMO, described in this (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). The precise of both polyamine oxidase in the metabolism of polyamines to be the role of enzyme in polyamine in mammalian in with and Polyamine oxidase (PAO), 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 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, (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar, 2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 3Den Munckhof R.J.M. Denyn M. Tigchelaar-Gutter W. Schipper R.G. Verhofstad A.A.J. Van Noorden C.J.F. Frederiks W.M. J. Histochem. Cytochem. 1995; 43: 1155-1162Crossref PubMed Scopus (28) Google Scholar). PAOs with similar characteristics occur in methylotrophic yeasts (4Cohen S.S. Cohen S.S. A Guide to the Polyamines. Oxford University Press, NY1998: 82Google Scholar, 5Nishikawa M. Hagishita T. Yurimoto H. Kato N. Sakai Y. Hatanaka T. FEBS Lett. 2000; 476: 150-154Crossref PubMed Scopus (23) Google Scholar) and amoebae (6Shukla O.P. Muller S. Walter R.D. Mol. Biochem. Parasitol. 1992; 51: 91-98Crossref PubMed Scopus (12) Google Scholar). On the contrary, plant (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar), bacterial (7Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar), and protozoan (8Kim B.G. Sobota A. Bitonti A.J. McCann P.P. Byers T.J. J. Protozool. 1987; 34: 278-284Crossref PubMed Scopus (27) Google Scholar) 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 (9Pegg A.E. Hu R.H. Cancer Lett. 1995; 95: 247-252Crossref PubMed Scopus (25) Google Scholar, 10Bergeron R.J. Feng Y. Weimar W.R. McManis J.S. Dimova H. Porter C. Raisler B. Phanstiel O.A. J. Med. Chem. 1997; 40: 1475-1494Crossref PubMed Scopus (124) Google Scholar, 11Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 12Casero E.A. Woster P.M. J. Med. Chem. 2001; 44: 1-26Crossref PubMed Scopus (218) Google Scholar). As compared with large and detailed investigations on plant PAOs (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar,13Smith T.A. Phytochemistry. 1976; 15: 633-636Crossref Scopus (35) Google Scholar, 14Smith T.A. Barker J.H. Zappia V. Pegg A.E. Progress in Polyamine Research: Novel Biochemical, Pharmacological and Clinical Aspects. Plenum Press, NY1988: 573-589Google Scholar, 15Federico R. Alisi C. Forlani F. Phytochemistry. 1989; 28: 45-46Crossref Scopus (36) Google Scholar, 16Angelini R. Federico R. Bonfante P. J. Plant Physiol. 1995; 145: 686-692Crossref Scopus (40) Google Scholar, 17Tavladoraki P. Schininà M.E. Cecconi F. Di Agostino S. Manera F. Rea G. Mariottini P. Federico R. Angelini R. FEBS Lett. 1998; 426: 62-66Crossref PubMed Scopus (89) Google Scholar, 18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 19Laurenzi M. Rea G. Federico R. Tavladoraki P. Angelini R. Planta. 1999; 208: 146-154Crossref Scopus (47) Google Scholar, 20Cervelli M. Tavladoraki P. Di Agostino S. Angelini R. Federico R. Mariottini P. Plant Physiol. Biochem. 2000; 38: 667-677Crossref Scopus (39) Google Scholar, 21Cervelli M. Cona A. Angelini R. Polticelli F. Federico R. Mariottini P. Eur. J. Biochem. 2001; 268: 3816-3838Crossref PubMed Scopus (55) Google Scholar, 22Šebela M. Radová A. Angelini R. Tavladoraki P. Frébort I. Pêc P. Plant Sci. 2001; 160: 197-207Crossref PubMed Scopus (113) Google Scholar), only little attention has been devoted to the animal counterpart (2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 23Hölttä E. Biochemistry. 1977; 16: 91-100Crossref PubMed Scopus (266) Google Scholar, 24Bolkenius F.N. Bey P. Seiler N. Biochim. Biophys. Acta. 1984; 838: 69-76Crossref Scopus (68) Google Scholar, 25Suzuki O. Matsumoto T. Katsumata Y. Experentia (Basel). 1984; 40: 838-839Crossref PubMed Scopus (45) Google Scholar, 26Libby P.R. Porter C.W. Biochem. Biophys. Res. Commun. 1985; 144: 528-535Crossref Scopus (21) Google Scholar, 27Tsukada T. Furusako S. Maekawa S. Hibasami H. Nakashima K. Int. J. Biochem. 1988; 20: 695-702Crossref PubMed Scopus (17) Google Scholar, 28Gasparyan V.K. Nalbandyan R.M. Biokhimiya. 1990; 55: 1632-1637PubMed Google Scholar). Recently, Wang et al. (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and Vujcic et al. (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar) have reported the cloning and characterization of novel mammalian PAO enzymes capable of oxidizing preferentially Spm and for this reason named spermine oxidase (SMO) (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). In particular, this enzyme was expressed in an in vitro transcription/translation system (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and into transiently transfected human kidney cells (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). 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 (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). 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 (18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) suggests that the general features of MPAO active site are conserved in mSMO. 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.(30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar).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 the typical spectrum for the addition of the Spm substrate in in a of the enzyme the the of a in the analysis of the molecular of mSMO as compared with MPAO three-dimensional structure suggests that the of the enzyme is is to that the for Spm Spd as a substrate for mSMO is to the different of the in which the Spd substrate be in a the enzyme which has been to on an of the of by a His in mSMO an for the pH of the In fact, it be that is at pH the of the polyamine be and enzyme activity be low as pH substrate and to an in enzyme which is at pH of in mammalian polyamine catabolism to be by the activity of PAO and the novel SMO, described in this (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). The precise of both polyamine oxidase in the metabolism of polyamines to be the role of enzyme in polyamine in mammalian in with and 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 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.(30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). 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 The absorption spectrum of the native enzyme the typical spectrum for the addition of the Spm substrate in in a of the enzyme the the of a in the analysis of the molecular of mSMO as compared with MPAO three-dimensional structure suggests that the of the enzyme is is to that the for Spm Spd as a substrate for mSMO is to the different of the in which the Spd substrate be in a the enzyme which has been to on an of the The of by a His in mSMO an for the pH of the In fact, it be that is at pH the of the polyamine be and enzyme activity be low as pH substrate and to an in enzyme which is at pH of In in mammalian polyamine catabolism to be by the activity of PAO and the novel SMO, described in this (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). The precise of both polyamine oxidase in the metabolism of polyamines to be the role of enzyme in polyamine in mammalian in with and