Substrate‐specific selenoprotein B of glycine reductase from 
Eubacterium acidaminophilum

Abstract

The substrate-specific selenoprotein B of glycine reductase (PB glycine ) from Eubacterium acidaminophilum was purified and characterized. The enzyme consisted of three different subunits with molecular masses of about 22 (a), 25 (b) and 47 kDa (g), probably in an a 2 b 2 g 2 composition. PB glycine purified from cells grown in the presence of [ 75 Se]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 (a) subunit, but it could be removed for Edman degradation by incubation with o-phenylenediamine.A DNA fragment was isolated and sequenced which encoded b and a subunits of PB glycine (grdE), followed by a gene encoding selenoprotein A (grdA2) and the g subunit of PB glycine (grdB2). The cloned DNA fragment represented a second GrdB-encoding gene slightly different from a previously identified partial grdB1-containing fragment. Both grdB genes contained an in-frame UGA codon which confirmed the observed selenium content of the 47-kDa (g) subunit. Peptide sequence analyses suggest that grdE encodes a proprotein which is cleaved into the previously sequenced N-terminal 25-kDa (b) subunit and a 22-kDa (a) subunit of PB glycine . Cleavage most probably occurred at an -Asn-Cys-site concomitantly with the generation of the blocking carbonyl moiety from cysteine at the a subunit.

Identification of d-Proline Reductase fromClostridium sticklandiias a Selenoenzyme and Indications for a Catalytically Active Pyruvoyl Group Derived from a Cysteine Residue by Cleavage of a Proprotein

“…5 and 7). PrdB exhibited similarities (23% identity) to GrdB (11), especially the region around the selenocysteine was conserved; however, grdB encodes an extension of about 200 amino acids at its N terminus that was not present in prdB. PrdA was similar to GrdE (16% identity), for both proteins are posttranslationally cleaved to give a 23-and a 22-kDa ␣-keto acid (pyruvoyl)-containing subunit, respectively (Fig.…”

“…Both sequences were preceded by a putative ribosome binding site, but no promotor-like structure was identified. A mRNA structure similar to the secondary mRNA structure necessary for selenocysteine incorporation in E. coli (38,39) was not identified in the prdB gene, similar to the situation in grdA and grdB (11) of glycine reductase.…”

“…Then, a nucleophilic attack at the ␣-carbon of proline was presumed to be carried out by an electron-donating dithiol (like DTT) 2 and the ring is cleaved to give 5-aminovalerate after hydrolytic release from the pyruvoyl adduct. Glycine reductase is composed of the proteins A, B, and C; protein B binds glycine as Schiff base via an enzyme-bound carbonyl group (11). A selenol anion attacks the ␣-carbon, and subsequently the carbon-nitrogen bond is cleaved to give ammonium and probably a protein B-bound carboxymethylselenoether that is transferred to protein A.…”

“…To start a new reaction cycle, protein A is reduced by the thioredoxin system that obtains its electrons from NADPH (14). The genes encoding protein A, protein B, protein C, thioredoxin, and thioredoxin reductase are organized in an operon-like structure (11,15). For D-proline reductase, the existence of an analogous high energy acyl-enzyme intermediate has been excluded, indicating that proline reduction is not coupled to energy conservation by substrate level phosphorylation (10).…”

Selenium in Biology: Facts and Medical Perspectives

“…Most prokaryotic selenoproteins, however, are unique and catalyze highly-varied processes that have not been discovered in eukarya. In the clostridial cluster XI (Kreimer and Andreesen, 1995;Wagner et al, 1999;Kabisch et al, 1999), selenoproteins are vital for energy production, particularly under stress, and appear to be important for additional metabolic performances and pathogenicity. A better understanding of such unique pathways in clinically relevant microorganisms might provide a rational basis for therapeutic intervention.…”