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Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase

Amino Acids volume 48, pages 2283–2291 (2016)Cite this article

Abstract

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.

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Abbreviations

APAO:

N 1-acetylpolyamine oxidase

BSAO:

Bovine serum amine oxidase

CD:

Circular dichroism

DSC:

Differential scanning calorimetry

DTT:

Dithiothreitol

FAD:

Flavin adenine dinucleotide

PAO:

Polyamine oxidase

SMOX:

Spermine oxidase

SPD:

Spermidine

SPM:

Spermine

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Acknowledgments

The Authors M.C., A.L., P.S., R.F., F.P. and P.M. wish to thank Roma Tre University for financial support. E.A. thanks the Sapienza University of Rome and the Italian MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) for Grant support, as well as REGIONE LAZIO for the Grant FILAS-RU-2014-1020. Thanks are also due to Fondazione ‘Enrico ed Enrica Sovena’ for the scholarship given to Marla Xhani for supporting her Ph.D. (E.A.).

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Affiliations

  1. Department of Sciences, Roma Tre University, V.le Guglielmo Marconi 446, 00146, Rome, Italy

    Manuela Cervelli, Alessia Leonetti, Pasquale Stano, Rodolfo Federico, Fabio Polticelli & Paolo Mariottini

  2. Department of Biochemical Sciences “A. Rossi Fanelli”, SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy

    Laura Cervoni, Shinji Ohkubo, Marla Xhani & Enzo Agostinelli

  3. National Institute of Nuclear Physics, Roma Tre Section, Via della Vasca Navale 84, 00146, Rome, Italy

    Fabio Polticelli

Authors
  1. Manuela Cervelli
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  2. Alessia Leonetti
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  3. Laura Cervoni
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  4. Shinji Ohkubo
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  5. Marla Xhani
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  6. Pasquale Stano
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  7. Rodolfo Federico
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  8. Fabio Polticelli
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  9. Paolo Mariottini
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  10. Enzo Agostinelli
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Corresponding author

Correspondence to Enzo Agostinelli.

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Handling Editor: E. Agostinelli.

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Cervelli, M., Leonetti, A., Cervoni, L. et al. Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase. Amino Acids 48, 2283–2291 (2016). https://doi.org/10.1007/s00726-016-2273-5

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  • DOI: https://doi.org/10.1007/s00726-016-2273-5

Keywords

  • Spermine oxidase
  • Bovine serum amine oxidase
  • Enzyme stability
  • Quaternary structure