Abstract
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 masses 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 thiosemicarbazide or removed by o-phenylenediamine; thus, N-terminal sequencing became feasible for this subunit. L-[14C]proline was covalently bound to the 23-kDa subunit if proline racemase and NaBH4 were added. Selenocysteine was detected in the 26-kDa subunit, which correlated with an observed selenium content of 10.6 g-atoms in D-proline reductase. No other non-proteinaceous cofactor was identified in the enzyme. A 4.8-kilobase pair (kb) EcoRI fragment was isolated and sequenced containing the two genes prdA and prdB. prdA coding 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 gene prdB encoded the 26-kDa subunit and contained an in frame UGA codon for selenocysteine insertion. prdA and prdB were transcribed together on a transcript of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-kb mRNA species.
Highlights
D-Proline reductase (EC 1.4.1.6) is involved in amino acid metabolism of several clostridia and catalyzes the reductive ring cleavage of D-proline to 5-aminovalerate [1,2,3].1 In a typical Stickland reaction, this reduction is coupled to the oxidation of other amino acids, but an utilization of electron donors like formate is possible [3, 5, 6]
The acetyl group is released as acetyl phosphate from protein C and its energy can be conserved by acetate kinase [13]
We developed a modified purification procedure for D-proline reductase and characterized the enzyme as a cytoplasmic protein with three subunits and a molecular mass of about 870,000 Da containing selenium in form of selenocysteine and a carbonyl moiety, most probably a pyruvoyl group
Summary
Materials—All chemicals were obtained from commercial sources unless otherwise specified. To get internal amino acid sequence information of proline reductase subunits, two approaches were used to isolate internal peptides: (a) the subunits were separated by SDS-PAGE and digested in the gel overnight at 37 °C with trypsin in 120 mM NH4HCO3 buffer, pH 8.5; (b) purified D-proline reductase (1 nmol) was dissolved in 50 l of 6 M guanidine hydrochloride containing 250 mM Tris-HCl buffer, pH 8.5, reduced with 2.5 l of 2-mercaptoethanol for 2 h in the dark at room temperature, and alkylated with 2 l of 4-vinylpyridine by incubation for 2 h in the dark at room temperature.
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