Probing mammalian spermine oxidase enzyme–substrate complex through molecular modeling,site-directed mutagenesis and biochemical characterization |
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Authors: | Paraskevi Tavladoraki Manuela Cervelli Fabrizio Antonangeli Giovanni Minervini Pasquale Stano Rodolfo Federico Paolo Mariottini Fabio Polticelli |
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Institution: | (1) Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, 00146 Rome, Italy; |
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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. |
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