Identification, Expression And Functional Characterization Of PAO Gene In Amphioxus Branchiostoma Japonicum

Polyamine oxidase enzymes catalyze the oxidation of polyamines andacetylpolyamines. Since polyamines are basic regulators of cell growth andproliferation, their homeostasis is thus crucial for cell life. Polyamine oxidases (PAOs)from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine,however, in vertebrates two different enzymes, namely spermine oxidase (SMO) andacetylpolyamine oxidase (APAO), are responsible for catalyzing the oxidation ofspermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Members of thepolyamine oxidase gene family has been identified in a wide variety of animals,including vertebrates and invertebrates such as arthropodes, nematodes, placozoa, aswell as in plants and fungi，but no information is available about PAO to date in theprimitive chordate amphioxus, an evolutionarily important organisms. The aims ofthis study were thus to identify the PAO gene of the amphioxus Branchiostomajaponicum, designated as Bjpao, to examine its expression and bioactivity.Bjpao open reading frame (ORF) obtained was1428bp long, which encodes adeduced protein of475amino acids with a calculated molecular weight of about52.0kDa. The mature protein possesses a FAD binding domain and a polyamine oxidasemulti-domain. It also has the residues Trp62, His64, Lys301, Tyr412and Thr460thatare expected to form the active sites highly conserved in all SMOs, APAOs and PAOsfrom prokaryotic and eukaryotic organisms. All these data indicate that the cDNAcodes for a PAO in amphioxus. qRT-PCR assay was used to examine the expressionpattern of Bjpao in the different tissues of B. japonicum. Bjpao transcripts were mostabundant in the notochord and at a lower level present in the testis and ovary. Incontrast, only slight Bjpao transcripts were detected in the muscle and hind-gut. Theseshow that Bjpao is expressed in a tissue-specific fashion in B. japonicum. To investigate the function of BjPAO, a recombinant plasmid has beenconstructed, transferred to E. coli and induced to express the recombinant protein.After purification and refolding of rBjPAO, the substrate specificity of rBjPAO wasfirst determined. It was founs that rBjPAO was able to efficiently oxidize spermineand spermidine. However, BjPAO was less efficient in oxidizing N1-acetylspermine.We have also determined the apparent Kmand Vmaxvalues of rBjPAO. Increasingconcentrations of spermine and spermidine ranging from1to100μM were used tocalculate the initial velocities of substrate oxidation.We then explored the active site of rBjPAO with the method of site-directedmutagenesis. We obtained the following amino acid substitutions in BjPAO：H64Q,H64E, K301M, T460Y, and T460S (residue numbering is for BjPAO). We havedetermined the enzymatic activity and kinetic parameters of the mutants. Theenzymatic activity in the mutants was reduced by a different degree. These indicatethat both H64, K301and T460residues are indispensible for enzymatic activity ofrBjPAO.