title: FAD binding in glycine oxidase from Bacillus subtilis.
abstract: The apoprotein of the FAD-containing flavoenzyme glycine oxidase from Bacillus subtilis was obtained at pH 8.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M Tris-HCl and 20% glycerol. The apoprotein of glycine oxidase shows high protein fluorescence, high exposure of hydrophobic surfaces, and low temperature stability as compared to the holoenzyme. The isolated apoprotein species is present in solution as a monomer which rapidly recovers its tertiary structure and converts into the tetrameric holoenzyme following incubation with free FAD. The reconstitution process follows a particular two-stage process; the spectral properties of the reconstituted holoenzyme were virtually indistinguishable from those observed with native glycine oxidase, while the activity was only partially (50%) recovered. The urea-induced unfolding process of glycine oxidase can be considered as a two-step (three-state) process: the presence of intermediate(s) in the unfolding process of the holoenzyme at approximately 2 M urea is evident in the changes of the flavin fluorescence intensity and can be also inferred from the different urea sensitivities of the spectral probes used. On the other hand, only a single transition at approximately 4.5 M urea concentration is observed for the apoprotein form. The chemical denaturation of glycine oxidase holoenzyme is partially reversible (e.g., no activity is recovered when starting the refolding from 4 M urea-denatured holoprotein). Finally, the introduction by site-directed mutagenesis of residues corresponding to those involved in the covalent link with FAD in the related flavoenzyme monomeric sarcosine oxidase failed to convert glycine oxidase into a covalent flavoprotein. These investigations show that the consequences of FAD binding for the stability and folding process distinguish glycine oxidase from enzymes active on similar compounds.
authors.affiliation: Dipartimento di Biotecnologie e Scienze Molecolari, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100 Varese, and Centro Interuniversitario di Biotecnologie Proteiche The Protein Factory, Politecnico di Milano and Università degli studi dell'Insubria, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.forename: Mattia
authors.initials: M
authors.lastname: Pedotti
authors.forename: Laura
authors.initials: L
authors.lastname: Motteran
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002942:Circular Dichroism; D003605:D-Amino-Acid Oxidase / Q000494:pharmacology; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D050879:Sarcosine Oxidase / Q000378:metabolism; D013696:Temperature
chemical_list: D001059:Apoproteins; D005415:Flavins; D005420:Flavoproteins; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase; D003605:D-Amino-Acid Oxidase; D050879:Sarcosine Oxidase