title: Peroxidase activity of selenoprotein GrdB of glycine reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum.
abstract: The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific "protein B"-complex of glycine reductase. The "protein B"-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)(-1) with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)(-1) in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.
authors.affiliation: Institute of Chemistry/Biochemistry, FU Berlin, Thielallee 63, 14195 Berlin, Germany.
authors.forename: Tina
authors.initials: T
authors.lastname: Gröbe
authors.forename: Michael
authors.initials: M
authors.lastname: Reuter
authors.forename: Torsten
authors.initials: T
authors.lastname: Gursinsky
authors.forename: Brigitte
authors.initials: B
authors.lastname: Söhling
authors.forename: Jan R
authors.initials: JR
authors.lastname: Andreesen
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005051:Eubacterium / Q000201:enzymology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism*; D051140:Selenoproteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D051140:Selenoproteins; C097253:selenoprotein B, Bacteria; D010544:Peroxidases; D000594:Amino Acid Oxidoreductases; C020815:glycine reductase