Post-translational modification of spermine oxidase (Smo): An in vitro approach

Cysteine (Cys) residues, especially those located on protein surface, are considered to be important for metal coordination, catalysis, protein structure by forming disulfide bonds and protein function regulation. Crucial Cys residues can be involved in the modulation of protein activity and signalling events via reactions of their thiol groups. These reactions can take several forms, such as redox events, chelation of transition metals or S-nitrosylation. In several cases, these disparate reactions can compete with each other for the same thiol group on a single Cys residue, forming a molecular switch composed of possible redox, NO or Zn(2+) modifications to control protein function. This work investigates the basis for these molecular Cys switches in spermine oxidase (SMO), an enzyme involved in the polyamine homeostasis of animal cells, which oxidizes spermine (Spm) to produce spermidine (Spd), 3-aminopropanal and H2O2. The murine SMO enzyme contains 14 Cys (correspond to 2.5% amino acid content), mainly localized on the protein surface. We carried out an in vitro chemical modifications of Cys residues to analyze their effect on the biochemical features of SMOWT (containing 14 Cys) and SMOC263-C429 mutant (containing only 2 Cys) recombinant proteins. Regarding the mutant protein, the rationale was that of mutating all the surface Cys residues into non reactive and isosteric Ser residues, while leaving the Cys263 and Cys429 residues which potentially play an important structural role. We used the NO-generating donor NOR-3 for S-nitrosylation, glutathione (GSH) for S-glutathionylation and S-nitrosoglutathione (GSNO) for both S-glutathionylation and S-nitrosylation. We could observed that treatment with NOR-3, but not with GSH and GSNO, decreased both SMOWT and SMOC263-C429 enzymatic activity.