Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Amendola R, Cervelli M, Fratini E, Polticelli F, Sallustio D, Mariottini P (2009) Spermine metabolism and anticancer therapy. Curr Cancer Drug Targets 9:118–130

    Article  PubMed  CAS  Google Scholar 

  • Babbar N, Murray-Stewart T, Casero RJ (2007) Inflammation and polyamine catabolism: the good, the bad and the ugly. Biochem Soc Trans 35:300–304

    Article  PubMed  CAS  Google Scholar 

  • Bas D, Rogers D, Jensen J (2008) Very fast prediction and rationalization of pK(a) values for protein-ligand complexes. Proteins Struct Funct Bioinform 73:765–783

    Article  CAS  Google Scholar 

  • 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 322:1–8

    Article  PubMed  CAS  Google Scholar 

  • 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 28:1–2

    Article  PubMed  CAS  Google Scholar 

  • 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 273:1115–1123

    Article  PubMed  CAS  Google Scholar 

  • 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 7:265–276

    Article  PubMed  CAS  Google Scholar 

  • Binda C, Angelini R, Federico R, Ascenzi P, Mattevi A (2001) Structural bases for inhibitor binding and catalysis in polyamine oxidase. Biochemistry 40:2766–2776

    Article  PubMed  CAS  Google Scholar 

  • 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 277:23973–23976

    Article  PubMed  CAS  Google Scholar 

  • 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 4:187–212

    Article  CAS  Google Scholar 

  • Casero RJ, Marton L (2007) Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug Discov 6:373–390

    Article  PubMed  CAS  Google Scholar 

  • Casero RA, Pegg AE (2009) Polyamine catabolism and disease. Biochem J 421:323–338

    Article  PubMed  CAS  Google Scholar 

  • 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 31:361–365

    Article  PubMed  CAS  Google Scholar 

  • Cervelli M, Polticelli F, Federico R, Mariottini P (2003) Heterologous expression and characterization of mouse spermine oxidase. J Biol Chem 278:5271–5276

    Article  PubMed  CAS  Google Scholar 

  • 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 271:760–770

    Article  PubMed  CAS  Google Scholar 

  • 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 279:40161–40173

    Article  PubMed  CAS  Google Scholar 

  • Cohen SS (1998) A guide to the polyamines. Oxford University Press, New York

    Google Scholar 

  • Deleage G, Geourjon C (1993) An interactive graphic program for calculating the secondary structure-content of proteins from circular-dichroism spectrum. Comput Appl Biosci 9:197–199

    PubMed  CAS  Google Scholar 

  • Gerner E, Meyskens FJ (2004) Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4:781–792

    Article  PubMed  CAS  Google Scholar 

  • 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 68:766–772

    Article  PubMed  CAS  Google Scholar 

  • 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 48:1508–1516

    Article  PubMed  CAS  Google Scholar 

  • Huang Q, Liu Q, Hao Q (2005) Crystal structures of Fms1 and its complex with spermine reveal substrate specificity. J Mol Biol 348:951–959

    Article  PubMed  CAS  Google Scholar 

  • Landry J, Sternglanz R (2003) Yeast Fms1 is a FAD-utilizing polyamine oxidase. Biochem Biophys Res Commun 303:771–776

    Article  PubMed  CAS  Google Scholar 

  • 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 23:2947–2948

    Article  PubMed  CAS  Google Scholar 

  • Laskowski R, MacArthur M, Moss D, Thornton J (1993) PROCHECK—a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291

    Article  CAS  Google Scholar 

  • 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 102:3586–3616

    CAS  Google Scholar 

  • Pegg AE (2009) Mammalian polyamine metabolism and function. IUBMB Life 61:880–894

    Article  PubMed  CAS  Google Scholar 

  • Persson L (2009) Polyamine homoeostasis. Essays Biochem 46:11–24

    Article  PubMed  CAS  Google Scholar 

  • 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):430–435

    Article  PubMed  CAS  Google Scholar 

  • 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 280:39843–39851

    Article  PubMed  CAS  Google Scholar 

  • 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 44:16108–16120

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • 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 29:2994–3005

    Article  PubMed  Google Scholar 

  • Schipper R, Penning L, Verhofstad A (2000) Involvement of polyamines in apoptosis. Facts and controversies: effectors or protectors? Semin Cancer Biol 10:55–68

    Article  PubMed  CAS  Google Scholar 

  • Stavropoulos P, Blobel G, Hoelz A (2006) Crystal structure and mechanism of human lysine-specific demethylase-1. Nat Struct Mol Biol 13:626–632

    Article  PubMed  CAS  Google Scholar 

  • Thomas T, Thomas T (2001) Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 58:244–258

    Article  PubMed  CAS  Google Scholar 

  • Thomas T, Thomas T (2003) Polyamine metabolism and cancer. J Cell Mol Med 7:113–126

    Article  PubMed  CAS  Google Scholar 

  • 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 367:665–675

    Article  PubMed  CAS  Google Scholar 

  • Wallace H, Fraser A, Hughes A (2003) A perspective of polyamine metabolism. Biochem J 376:1–14

    Article  PubMed  CAS  Google Scholar 

  • 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 61:5370–5373

    PubMed  CAS  Google Scholar 

  • 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 304:605–611

    Article  PubMed  CAS  Google Scholar 

  • Waterhouse A, Procter J, Martin D, Clamp M, Barton G (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191

    Article  PubMed  CAS  Google Scholar 

  • Wu T, Yankovskaya V, McIntire W (2003) Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase. J Biol Chem 278:20514–20525

    Article  PubMed  CAS  Google Scholar 

  • 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 326:483–490

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinform 9:40

    Article  Google Scholar 

Download references

Acknowledgments

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

Author information

Authors and Affiliations

  1. Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, 00146, Rome, Italy

    Paraskevi Tavladoraki, Manuela Cervelli, Fabrizio Antonangeli, Giovanni Minervini, Pasquale Stano, Rodolfo Federico, Paolo Mariottini & Fabio Polticelli

Authors
  1. Paraskevi Tavladoraki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Manuela Cervelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fabrizio Antonangeli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Giovanni Minervini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pasquale Stano
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rodolfo Federico
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Paolo Mariottini
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fabio Polticelli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fabio Polticelli.

Additional information

P. Tavladoraki and M. Cervelli contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 173 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tavladoraki, P., Cervelli, M., Antonangeli, F. et al. Probing mammalian spermine oxidase enzyme–substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization. Amino Acids 40, 1115–1126 (2011). https://doi.org/10.1007/s00726-010-0735-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-010-0735-8

Keywords

Search

Navigation