In pursuit of mammalian polyamine oxidase crystal structure (774.4)

Abstract

Mouse Polyamine Oxidase (mPAO) is a mammalian flavoprotein that is necessary for the catabolism of polyamines. mPAO oxidizes the endo carbon nitrogen bonds of N1‐acetylspermine and N1‐acetylspermidine when oxidized to spermine and spermidine respectively. It is known that normal levels of polyamines are important for cell growth, yet their actual function is not well understood. The structure of mammalian PAO has yet to be determined; however structures for maize and yeast PAO (Fms1) exist. Although the identity between mPAO, Fms1, and maize PAO is only 20%, it has been shown, through sequence comparison and mutational analysis, that a conserved histidine residue at position 67 of Fms1 and 64 of mPAO helps to properly position the amine substrate for oxidation. Determining the crystal structure of mPAO will help identify other residues that play a role in substrate binding and catalysis.mPAO was cloned into pAG8H, a modified pET19d vector that introduces a cleavable his‐tag at the N‐terminus of the protein. mPAO was expressed and purified using a Nickel column followed by an anion exchange column. Following the purification process, analytical ultracentrifugation sedimentation velocity experiments were performed to determine the oligomerization state and homogeneity of the protein.The sedimentation velocity experiments revealed a homogeneous, monomeric mPAO in solution, in contrast to the dimeric form of Fms1 in solution. Crystallization trials are underway to determine the structure of mPAO.