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 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) approximately 68,000) has K(m) and k(cat) values of 90 microm 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.
Cervelli
Manuela
M
Dipartimento di Biologia, Università Roma Tre, I-00146 Roma, Italy.
Polticelli
Fabio
F
Federico
Rodolfo
R
Mariottini
Paolo
P
D000595:Amino Acid Sequence; D000818:Animals; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000097667:Polyamine Oxidase
D000587:Oxidoreductases Acting on CH-NH Group Donors; D011994:Recombinant Proteins; D000097667:Polyamine Oxidase