Identification of D-proline reductase from Clostridium sticklandii as a selenoenzyme and indications for a catalytically active pyruvoyl group derived from a cysteine residue by cleavage of a proprotein

Highly active D-proline reductase was obtained from Clostridium sticklandii by a modified purification scheme. The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular mas ses of 23, 26, and 45 kDa. The 23-kDa subunit contained a carbonyl group at its N terminus, which could either be labeled with fluorescein thiosemicar bazide or removed by o-phenylenediamine; thus, N-terminal sequencing became feasible for this subunit, L-[C-14]proline was covalently bound to the 23- kDa subunit if proline racemase and NaBH4 were added. Selenocysteine was de tected in the 26-kDa subunit, which correlated with an observed selenium co ntent of 10.6 g-atoms in D-proline reductase. No other non-proteinaceous co factor was identified in the enzyme, A 4.8-kilobase pair (kb) EcoRI fragmen t was isolated and sequenced containing the two genes prdA and prdB. prdA c oding for a 68-kDa protein was most likely translated as a proprotein that was posttranslationally cleaved at a threonine-cysteine site to give the 45 -kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit. The ge ne prdB encoded the 26-kDa subunit and contained an in frame UGA codon for selenocysteine insertion. prdA and prdB were transcribed together on a tran script of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-k b mRNA species.