Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase

The aminoacyl sequences of three regions of pure bovine N-1-acetylated polyamine oxidase (PAO) were obtained and used to search GenBank(TM). This led to the cloning and sequencing of a complete coding cDNA for murine PAO (mPAO) and the 5'-truncated coding region of the bovine pao (bpao) gene. A search of GenBank(TM) indicated that mpao maps to murine chromosome 7 as seven exons. The translated amino acid sequences of mpao and bpao have a - Pro-Arg-Leu peroxisomal targeting signal at the extreme C termini. A beta-alpha-beta FAD-binding motif is present in the N-terminal portion of mPAO. This and several other regions of mPAO and bPAO are highly similar to corresponding sections of other flavoprotein amine oxidases, although the overall identity of aligned sequences indicates that PAO represents a new subfamily of flavoproteins. A fragment of mpao was used as a probe to establish the relative transcription levels of this gene in various mature murine tissues and murine embryonic and breast tissues at different developmental stages. An Escherichia coli expression system has been developed for manufacturing mPAO at a reasonable level. The mPAO so produced was purified to homogeneity and characterized. It was demonstrated definitively that PAO oxidizes N-1-acetylspermine to spermidine and 3-acetamidopropanal and that it also oxidizes N-1-acetylspermidine to putrescine and 3-acetamidopropanal. Thus, this is the classical polyamine oxidase (EC 1.5.3.11) that is defined as the enzyme that oxidizes these N-1-acetylated polyamines on the exo-side of their N-4-amino groups. This enzyme is distinguishable from the plant polyamine oxidase that oxidizes spermine on the endo-side of the N-4-nitrogen. It differs also from mammalian spermine oxidase that oxidizes spermine ( but not N-1-acetylspermine or N-1-acetylspermidine) at the exo-carbon of its N-4-amino group. This report provides details of the biochemical, spectral, oxidation-reduction, and steady-state kinetic properties of pure mPAO.