Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.
Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.
Department of Sciences, Roma Tre University, V.le Guglielmo Marconi 446, 00146, Rome, Italy.
Manuela
M
Cervelli
Department of Sciences, Roma Tre University, V.le Guglielmo Marconi 446, 00146, Rome, Italy.
Alessia
A
Leonetti
Department of Biochemical Sciences "A. Rossi Fanelli", SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy.
Laura
L
Cervoni
Department of Biochemical Sciences "A. Rossi Fanelli", SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy.
Shinji
S
Ohkubo
Department of Biochemical Sciences "A. Rossi Fanelli", SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy.
Marla
M
Xhani
Department of Sciences, Roma Tre University, V.le Guglielmo Marconi 446, 00146, Rome, Italy.
Pasquale
P
Stano
Department of Sciences, Roma Tre University, V.le Guglielmo Marconi 446, 00146, Rome, Italy.
Rodolfo
R
Federico
Department of Sciences, Roma Tre University, V.le Guglielmo Marconi 446, 00146, Rome, Italy.|National Institute of Nuclear Physics, Roma Tre Section, Via della Vasca Navale 84, 00146, Rome, Italy.
Fabio
F
Polticelli
Department of Sciences, Roma Tre University, V.le Guglielmo Marconi 446, 00146, Rome, Italy.
Paolo
P
Mariottini
Department of Biochemical Sciences "A. Rossi Fanelli", SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy. enzo.agostinelli@uniroma1.it.
Enzo
E
Agostinelli
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D000097667:Polyamine Oxidase
D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors; D011994:Recombinant Proteins; D000097667:Polyamine Oxidase
Bovine serum amine oxidase; Enzyme stability; Quaternary structure; Spermine oxidase