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

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, N-1-acetylspermine (N-1-Aspm) and N-1-acetylspermidine (N-1-Aspd), while yeast PAOs oxidize Spm, N-1-Aspm and N-1-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N-1-Aspm/N-1-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 N-1-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N-1-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. (C) 2015 Elsevier B.V. All rights reserved.