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Characterization of Human Polyamine Oxidase
Author(s) -
Zamora Diana,
Tormos Melendez Jose,
Galaleldeen Ahmad
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.15
Subject(s) - polyamine oxidase , polyamine , chemistry , biochemistry , spermidine , spermine , flavoprotein , histidine , protein crystallization , stereochemistry , amino acid , crystallization , enzyme , organic chemistry
Polyamine Oxidase (PAO) is a flavoprotein that is necessary for the catabolism of polyamines. PAO oxidizes the endo carbon nitrogen bonds of N1‐acetylspermine and N1‐acetylspermidine when oxidized to spermine and spermidine, respectively. Normal levels of polyamines are needed for cell growth, yet their actual function is not well understood. The crystal structures of maize and yeast PAO (Fms1) are available but no mammalian PAO structure has been determined to date. The identity between human PAO (hPAO), Fms1, and maize PAO is only 20%. Sequence homology and mutational analysis showed that a conserved histidine residue in mammalian PAOs that corresponds to position 67 of Fms1 helps to properly position the amine substrate for oxidation. Determining the crystal structure of hPAO will help identify the other residues that play a role in substrate binding and catalysis. hPAO was cloned into pAG8H, a modified pET19d vector that introduces a cleavable his‐tag at the N‐terminus of the protein. hPAO was expressed and purified using affinity and ion exchange columns. The his‐tag was removed and the protein is being assayed for activity. Analytical ultracentrifugation sedimentation velocity experiments are also underway to determine the oligomerization state of the protein in solution in addition to crystallization trials to determine the structure of hPAO.