title: Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization.
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.
authors.affiliation: Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, 00146, Rome, Italy.
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Tavladoraki
authors.forename: Manuela
authors.initials: M
authors.lastname: Cervelli
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Antonangeli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Minervini
authors.forename: Pasquale
authors.initials: P
authors.lastname: Stano
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Federico
authors.forename: Paolo
authors.initials: P
authors.lastname: Mariottini
authors.forename: Fabio
authors.initials: F
authors.lastname: Polticelli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008322:Mammals; D051379:Mice; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013096:Spermine / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000097667:Polyamine Oxidase
chemical_list: D006639:Histidine; D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013096:Spermine; D000097667:Polyamine Oxidase; C108504:FMS1 protein, S cerevisiae