Substrate-specific selenoprotein B of glycine reductase from Eubacterium acidaminophilum. Biochemical and molecular analysis.
The substrate-specific selenoprotein B of glycine reductase (PBglycine) from Eubacterium acidaminophilum was purified and characterized. The enzyme consisted of three different subunits with molecular masses of about 22 (alpha), 25 (beta) and 47 kDa (gamma), probably in an alpha 2 beta 2 gamma 2 composition. PBglycine purified from cells grown in the presence of [75Se]selenite was labeled in the 47-kDa subunit. The 22-kDa and 47-kDa subunits both reacted with fluorescein thiosemicarbazide, indicating the presence of a carbonyl compound. This carbonyl residue prevented N-terminal sequencing of the 22-kDa (alpha) subunit, but it could be removed for Edman degradation by incubation with o-phenylenediamine. A DNA fragment was isolated and sequenced which encoded beta and alpha subunits of PBglycine (grdE), followed by a gene encoding selenoprotein A (grdA2) and the gamma subunit of PBglycine (grdB2). The cloned DNA fragment represented a second GrdB-encoding gene slightly different from a previously identified partial grdBl-containing fragment. Both grdB genes contained an in-frame UGA codon which confirmed the observed selenium content of the 47-kDa (gamma) subunit. Peptide sequence analyses suggest that grdE encodes a proprotein which is cleaved into the previously sequenced N-terminal 25-kDa (beta) subunit and a 22-kDa (alpha) subunit of PBglycine. Cleavage most probably occurred at an -Asn-Cys- site concomitantly with the generation of the blocking carbonyl moiety from cysteine at the alpha subunit.
Wagner
M
M
Institut für Mikrobiologie, Martin-Luther-Universität Halle, Germany.
Sonntag
D
D
Grimm
R
R
Pich
A
A
Eckerskorn
C
C
Söhling
B
B
Andreesen
J R
JR
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D005051:Eubacterium / Q000201:enzymology*; D005798:Genes, Bacterial / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics*; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D012644:Selenium Radioisotopes / Q000378:metabolism; D051140:Selenoproteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011506:Proteins; D012644:Selenium Radioisotopes; D051140:Selenoproteins; C097253:selenoprotein B, Bacteria; D000594:Amino Acid Oxidoreductases; C020815:glycine reductase