Identification,Expression And Functional Characterization Of Bipao2 From Amphioxus Branchiostoma Japonicum

Polyamine oxidases (PAO) are flavin adenine dinucleotide-(FAD-) containing enzymes that catalyze the oxidation of polyamines. Recently 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) and spermidine (Spd) and their acetylated derivatives, N’-acetylspermine (N’-Aspm) and N’-acetylspermidine (N1-Aspd), while yeast PAOs oxidize Spm, N’-Aspm and N’-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’-Aspm/N’-Aspd, respectively. Bjpaol from amphioxus Branchiostoma japonicum an evolutionarily important organism can oxidase spermine and spermidine but not N’-Aspm. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic.In this study, Bjpao2 from amphioxus(Branchiostoma japonicum) is cloned and characterized. The full-length cDNA (GenBank accession number:KP693601) of Bjpao2 obtained was 2050 bp and contained an ORF of 1473 bp, coding for a protein of 491 amino acids with a molecular mass of 55.3 kDa. BjPAO2 includes a FAD binding domain at the N-terminus and a PAO domain at the residues 21 to 483, SignalP software analysis showed that BjPAO2 has no signal peptide. Sequence comparison showed that BjPAO2, which shared was 37.7% identity with BjPAO1, was 38.3% to 40.9% and 35.9% to 39.7% identical with SMOs and APAOs from mammal, birds, reptiles, amphibians and fishes, respectively, and 31.0% to 38.5% identical with PAOs from invertebrates. It had the residues Trp67, His69, Lys315, Tyr424 and Thr467 that are expected to form the active sites highly conserved in all SMOs, APAOs and PAOs from prokaryotic and eukaryotic organisms. All these data indicated that the cDNA coded for a PAO in amphioxus. Bjpao2 abundantly expressed in the hepatic caecum and hind-gut, indicating that Bjpao2 expressed in a tissue-specific fashion.For investigating the function of rBjPAO2, the recombinant plasmid has been constructed, transferred to E.coli and induced to express. After refold and purification of the rBjPAO2, we determined the substrate specificity of rBjPAO2. The result demonstrated that rBjPAO2 oxidizes both spermidine and N’-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAO2s generated by site-directed mutagenesis and expressed in E. coli are examined, which indicate that the residues H69, K315 and T467 in rBjPAO2 are all involved to some extent in substrate binding and enzyme catalytic activity. 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.