Amino Acids

, Volume 40, Issue 4, pp 1115–1126 | Cite as

Probing mammalian spermine oxidase enzyme–substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization

  • Paraskevi Tavladoraki
  • Manuela Cervelli
  • Fabrizio Antonangeli
  • Giovanni Minervini
  • Pasquale Stano
  • Rodolfo Federico
  • Paolo Mariottini
  • Fabio Polticelli
Original Article

Abstract

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure–function relationships, SMO enzyme–substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme–substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

Keywords

Polyamines Spermine oxidase Molecular modeling Site-directed mutagenesis Enzyme–substrate complex 

Notes

Acknowledgments

The authors wish to thank the University of Roma Tre for financial support.

Supplementary material

726_2010_735_MOESM1_ESM.doc (173 kb)
Supplementary material 1 (DOC 173 kb)

References

  1. Amendola R, Cervelli M, Fratini E, Polticelli F, Sallustio D, Mariottini P (2009) Spermine metabolism and anticancer therapy. Curr Cancer Drug Targets 9:118–130PubMedCrossRefGoogle Scholar
  2. Babbar N, Murray-Stewart T, Casero RJ (2007) Inflammation and polyamine catabolism: the good, the bad and the ugly. Biochem Soc Trans 35:300–304PubMedCrossRefGoogle Scholar
  3. Bas D, Rogers D, Jensen J (2008) Very fast prediction and rationalization of pK(a) values for protein-ligand complexes. Proteins Struct Funct Bioinform 73:765–783CrossRefGoogle Scholar
  4. Bellelli A, Cavallo S, Nicolini L, Cervelli M, Bianchi M, Mariottini P, Zelli M, Federico R (2004) Mouse spermine oxidase: a model of the catalytic cycle and its inhibition by N, N1-bis(2, 3-butadienyl)-1, 4-butanediamine. Biochem Biophys Res Commun 322:1–8PubMedCrossRefGoogle Scholar
  5. Bey P, Bolkenius F, Seiler N, Casara P (1985) N-2, 3-Butadienyl-1, 4-butanediamine derivatives: potent irreversible inactivators of mammalian polyamine oxidase. J Med Chem 28:1–2PubMedCrossRefGoogle Scholar
  6. Bianchi M, Polticelli F, Ascenzi P, Botta M, Federico R, Mariottini P, Cona A (2006) Inhibition of polyamine and spermine oxidases by polyamine analogues. FEBS J 273:1115–1123PubMedCrossRefGoogle Scholar
  7. Binda C, Coda A, Angelini R, Federico R, Ascenzi P, Mattevi A (1999) A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase. Structure 7:265–276PubMedCrossRefGoogle Scholar
  8. Binda C, Angelini R, Federico R, Ascenzi P, Mattevi A (2001) Structural bases for inhibitor binding and catalysis in polyamine oxidase. Biochemistry 40:2766–2776PubMedCrossRefGoogle Scholar
  9. Binda C, Mattevi A, Edmondson D (2002) Structure-function relationships in flavoenzyme-dependent amine oxidations: a comparison of polyamine oxidase and monoamine oxidase. J Biol Chem 277:23973–23976PubMedCrossRefGoogle Scholar
  10. Brooks B, Bruccoleri R, Olafson B, States D, Swaminathan S, Karplus M (1983) CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comp Chem 4:187–212CrossRefGoogle Scholar
  11. Casero RJ, Marton L (2007) Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug Discov 6:373–390PubMedCrossRefGoogle Scholar
  12. Casero RA, Pegg AE (2009) Polyamine catabolism and disease. Biochem J 421:323–338PubMedCrossRefGoogle Scholar
  13. Casero RJ, Wang Y, Stewart T, Devereux W, Hacker A, Smith R, Woster P (2003) The role of polyamine catabolism in anti-tumour drug response. Biochem Soc Trans 31:361–365PubMedCrossRefGoogle Scholar
  14. Cervelli M, Polticelli F, Federico R, Mariottini P (2003) Heterologous expression and characterization of mouse spermine oxidase. J Biol Chem 278:5271–5276PubMedCrossRefGoogle Scholar
  15. Cervelli M, Bellini A, Bianchi M, Marcocci L, Nocera S, Polticelli F, Federico R, Amendola R, Mariottini P (2004) Mouse spermine oxidase gene splice variants—nuclear subcellular localization of a novel active isoform. Eur J Biochem 271:760–770PubMedCrossRefGoogle Scholar
  16. Chaturvedi R, Cheng Y, Asim M, Bussière F, Xu H, Gobert A, Hacker A, Casero RJ, Wilson K (2004) Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization. J Biol Chem 279:40161–40173PubMedCrossRefGoogle Scholar
  17. Cohen SS (1998) A guide to the polyamines. Oxford University Press, New YorkGoogle Scholar
  18. Deleage G, Geourjon C (1993) An interactive graphic program for calculating the secondary structure-content of proteins from circular-dichroism spectrum. Comput Appl Biosci 9:197–199PubMedGoogle Scholar
  19. Gerner E, Meyskens FJ (2004) Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4:781–792PubMedCrossRefGoogle Scholar
  20. Goodwin A, Jadallah S, Toubaji A, Lecksell K, Hicks J, Kowalski J, Bova G, De Marzo A, Netto G, Casero RJ (2008) Increased spermine oxidase expression in human prostate cancer and prostatic intraepithelial neoplasia tissues. Prostate 68:766–772PubMedCrossRefGoogle Scholar
  21. Henderson Pozzi M, Gawandi V, Fitzpatrick P (2009) pH dependence of a mammalian polyamine oxidase: insights into substrate specificity and the role of lysine 315. Biochemistry 48:1508–1516PubMedCrossRefGoogle Scholar
  22. Huang Q, Liu Q, Hao Q (2005) Crystal structures of Fms1 and its complex with spermine reveal substrate specificity. J Mol Biol 348:951–959PubMedCrossRefGoogle Scholar
  23. Landry J, Sternglanz R (2003) Yeast Fms1 is a FAD-utilizing polyamine oxidase. Biochem Biophys Res Commun 303:771–776PubMedCrossRefGoogle Scholar
  24. Larkin M, Blackshields G, Brown N, Chenna R, McGettigan P, McWilliam H, Valentin F, Wallace I, Wilm A, Lopez R, Thompson J, Gibson T, Higgins D (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  25. Laskowski R, MacArthur M, Moss D, Thornton J (1993) PROCHECK—a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  26. MacKerell AJ, Bashford D, Bellott M, Dunbrack RJ, Evanseck J, Field M, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D, Kuchnir L, Kuczera K, Lau F, Mattos C, Michnick S, Ngo T, Nguyen D, Prodhom B, Reiher WI, Roux B, Schlenkrich M, Smith J, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J, Yin D, Karplus M (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem 102:3586–3616Google Scholar
  27. Pegg AE (2009) Mammalian polyamine metabolism and function. IUBMB Life 61:880–894PubMedCrossRefGoogle Scholar
  28. Persson L (2009) Polyamine homoeostasis. Essays Biochem 46:11–24PubMedCrossRefGoogle Scholar
  29. Petrey D, Xiang Z, Tang C, Xie L, Gimpelev M, Mitros T, Soto C, Goldsmith-Fischman S, Kernytsky A, Schlessinger A, Koh I, Alexov E, Honig B (2003) Using multiple structure alignments, fast model building, and energetic analysis in fold recognition and homology modeling. Proteins 53(Suppl 6):430–435PubMedCrossRefGoogle Scholar
  30. Pledgie A, Huang Y, Hacker A, Zhang Z, Woster P, Davidson N, Casero RJ (2005) Spermine oxidase SMO(PAOh1), not N1-acetylpolyamine oxidase PAO, is the primary source of cytotoxic H2O2 in polyamine analogue-treated human breast cancer cell lines. J Biol Chem 280:39843–39851PubMedCrossRefGoogle Scholar
  31. Polticelli F, Basran J, Faso C, Cona A, Minervini G, Angelini R, Federico R, Scrutton N, Tavladoraki P (2005) Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase. Biochemistry 44:16108–16120PubMedCrossRefGoogle Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  33. Schäffer A, Aravind L, Madden T, Shavirin S, Spouge J, Wolf Y, Koonin E, Altschul S (2001) Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res 29:2994–3005PubMedCrossRefGoogle Scholar
  34. Schipper R, Penning L, Verhofstad A (2000) Involvement of polyamines in apoptosis. Facts and controversies: effectors or protectors? Semin Cancer Biol 10:55–68PubMedCrossRefGoogle Scholar
  35. Stavropoulos P, Blobel G, Hoelz A (2006) Crystal structure and mechanism of human lysine-specific demethylase-1. Nat Struct Mol Biol 13:626–632PubMedCrossRefGoogle Scholar
  36. Thomas T, Thomas T (2001) Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 58:244–258PubMedCrossRefGoogle Scholar
  37. Thomas T, Thomas T (2003) Polyamine metabolism and cancer. J Cell Mol Med 7:113–126PubMedCrossRefGoogle Scholar
  38. Vujcic S, Diegelman P, Bacchi C, Kramer D, Porter C (2002) Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin. Biochem J 367:665–675PubMedCrossRefGoogle Scholar
  39. Wallace H, Fraser A, Hughes A (2003) A perspective of polyamine metabolism. Biochem J 376:1–14PubMedCrossRefGoogle Scholar
  40. Wang Y, Devereux W, Woster PM, Stewart TM, Hacker A, Casero RA Jr (2001) Cloning and characterization of a human polyamine oxidase that is inducible by polyamine analogue exposure. Cancer Res 61:5370–5373PubMedGoogle Scholar
  41. Wang Y, Murray-Stewart T, Devereux W, Hacker A, Frydman B, Woster P, Casero RJ (2003) Properties of purified recombinant human polyamine oxidase, PAOh1/SMO. Biochem Biophys Res Commun 304:605–611PubMedCrossRefGoogle Scholar
  42. Waterhouse A, Procter J, Martin D, Clamp M, Barton G (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191PubMedCrossRefGoogle Scholar
  43. Wu T, Yankovskaya V, McIntire W (2003) Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase. J Biol Chem 278:20514–20525PubMedCrossRefGoogle Scholar
  44. Wu T, Ling K, Sayre L, McIntire W (2005) Inhibition of murine N1-acetylated polyamine oxidase by an acetylenic amine and the allenic amine, MDL 72527. Biochem Biophys Res Commun 326:483–490PubMedCrossRefGoogle Scholar
  45. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinform 9:40CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Paraskevi Tavladoraki
    • 1
  • Manuela Cervelli
    • 1
  • Fabrizio Antonangeli
    • 1
  • Giovanni Minervini
    • 1
  • Pasquale Stano
    • 1
  • Rodolfo Federico
    • 1
  • Paolo Mariottini
    • 1
  • Fabio Polticelli
    • 1
  1. 1.Department of BiologyUniversity Roma TreRomeItaly

Personalised recommendations