Identification and biochemical characterization of polyamine oxidases in amphioxus: Implications for emergence of vertebrate-specific spermine and acetylpolyamine oxidases

• Two polyamine oxidase genes Bjpao1 and Bjpao2 are identified in amphioxus.
• Bjpao1 and Bjpao2 both show distinct tissue-specific expression pattern.
• rBjPAO1 and rBjPAO2 oxidize Spm and N1-Aspm respectively but both oxidase Spd.
• Key residues important for enzymatic activity are identified in BjPAO1 and BjPAO2.
• A model depicting evolution and specialization of SMO and APAO is proposed.

Polyamine oxidases (PAOs) have been identified in a wide variety of animals, as well as in fungi and plant. Generally, plant PAOs oxidize spermine (Spm), spermidine (Spd) and their acetylated derivatives, N1-acetylspermine (N1-Aspm) and N1-acetylspermidine (N1-Aspd), while yeast PAOs oxidize Spm, N1-Aspm and N1-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N1-Aspm/N1-Aspd, respectively. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic. In this study, two amphioxus (Branchiostoma japonicum) PAO genes, named Bjpao1 and Bjpao2, were cloned and characterized. Both Bjpao1 and Bjpao2 displayed distinct tissue-specific expression patterns. Notably, rBjPAO1 oxidized both spermine and spermidine, but not N1-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N1-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAOs that were generated by site-directed mutagenesis and expressed in E. coli were examined, The results indicate that the residues H64, K301 and T460 in rBjPAO1, and H69, K315 and T467 in rBjPAO2 were all involved in substrate binding and enzyme catalytic activity to some extent. Based on our results and those of others, a model depicting the divergent evolution and functional specialization of vertebrate SMO and APAO genes is proposed.