Heterologous Expression and Characterization of Mouse Spermine Oxidase

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 dinucleotidecontaining 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 calculated M 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 into Escherichia 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 ϳ68,000) has K m and k cat values of 90 M 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), 1 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 H 2 O 2 , (1-3). PAOs with similar characteristics occur in methylotrophic yeasts (4, 5) and amoebae (6). On the contrary, plant (1), bacterial (7), and protozoan (8) PAOs oxidize spermidine and spermine to 4-aminobutanol or N-(3-aminopropyl)-4-aminobutanol, respectively, plus 1,3-diaminopropane and H 2 O 2 . 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 (9 -12).As compared with large and detailed investigations on plant PAOs (1, 13-22), only little attention has been devoted to the animal counterpart (2,(23)(24)(25)(26)(27)(28). Recently, Wang et al. (29) and Vujcic et al. (30) have reported the cloning and characterization of novel mammalian PAO enzymes capable of oxidizing preferentially Spm and for this reason named spermine oxidase (SMO) (30). In ...

Arabidopsis mutant plants with diverse defects in polyamine metabolism show unequal sensitivity to exogenous cadaverine probably based on their spermine content

“…On the other hand, mammalian PAOs are involved in a PA back-conversion pathway (Vujcic et al 2003;Wu et al 2003). In addition, mammalian Spm oxidase converts Spm to Spd along with the production of 3-aminopropanal and H 2 O 2 without the acetylation process (Wang et al 2001;Vujcic et al 2002;Cervelli et al 2003;Seiler 2004). Even in plant, PA back-conversion activity was reported for AtPAO1, an isoform of five Arabidopsis PAOs ).…”

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine

“…The recombinant APAO and SMO proteins were expressed in E. coli BL21 DE3 cells and purified as previously reported by Bianchi et al 20 and Cervelli et al, 11 respectively. SDS/ PAGE electrophoretic analysis was performed on purified recombinant proteins to assess the grade of purity.…”

“…At present, only the three-dimensional structures of two members of the polyamine oxidase family are available, namely the maize polyamine oxidase (ZmPAO 17,18 ) and the yeast polyamine oxidase (FMS1 19 ). Molecular modeling of mouse SMO based on the three-dimensional structure of ZmPAO and sequence analysis of animal polyamine oxidases indicated that the general features of the ZmPAO threedimensional structure are conserved in the mammalian enzymes 11,20,21 . Furthermore, molecular modeling, docking and site-directed mutagenesis studies demonstrated that Spm binds to mammalian SMO in the same fashion as observed in the yeast FMS1, which shares with SMO and APAO the substrate oxidation mode 21 .…”

“…In the last decade, APAO and SMO catabolic enzymes have been extensively characterized and well documented to play an essential role in maintaining vertebrate PA homeostasis, the latter being mandatory for cellular life 1,2,[7][8][9][10] . The recombinant protein expression of the mammalian APAO and SMO in different heterologous systems provided the basis for a deeper understanding of the structure/function relationships of these enzymes [11][12][13][14][15][16] . At present, only the three-dimensional structures of two members of the polyamine oxidase family are available, namely the maize polyamine oxidase (ZmPAO 17,18 ) and the yeast polyamine oxidase (FMS1 19 ).…”

Spermine oxidase, a polyamine catabolic enzyme that linksHelicobacter pyloriCagA and gastric cancer risk

“…15 Active splice variants are found both in the cytoplasm and the nucleus. [16][17][18] The predominant human splice variant, SMO1 (SMO/PAOh1), codes for a 61 kD protein containing 555 amino acids.…”