Heterologous expression and biochemical characterization of a polyamine oxidase from Arabidopsis involved in polyamine back conversion.
Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H2O2, an aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H2O2, and an aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N1-acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.
Tavladoraki
Paraskevi
P
Department of Biology, University Roma Tre, 00146 Rome, Italy. tavlador@uniroma3.it
Rossi
Marianna Nicoletta
MN
Saccuti
Giuseppe
G
Perez-Amador
Miguel Angel
MA
Polticelli
Fabio
F
Angelini
Riccardo
R
Federico
Rodolfo
R
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives / Q000378:metabolism; D000097667:Polyamine Oxidase
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011993:Recombinant Fusion Proteins; D013095:Spermidine; D013096:Spermine; D000097667:Polyamine Oxidase; C017921:norspermine; C006760:norspermidine