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PMID:10085076
| Citation |
Kabisch, UC, Gräntzdörffer, A, Schierhorn, A, Rücknagel, KP, Andreesen, JR and Pich, A (1999) 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. J. Biol. Chem. 274:8445-54 |
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| 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. |
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| Keywords |
Amino Acid Oxidoreductases/chemistry; Amino Acid Oxidoreductases/genetics; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Base Sequence; Catalysis; Cloning, Molecular; Clostridium/enzymology; Fluoresceins; Molecular Sequence Data; Protein Precursors/genetics; Protein Processing, Post-Translational; RNA, Messenger/genetics; Selenocysteine/chemistry; Sequence Alignment; Sequence Analysis, DNA |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0050002: D-proline reductase (dithiol) activity |
ECO:0000314: |
F |
Figure 3b uses labeled D-proline (with C14) in the presence of Proline reductase and Proline racemase. The 23kDa subunit of the proline reductase became extremely radioactive when placed onto a gel. This indicates that Proline was bound to this peptide. |
complete | ||||
|
enables |
GO:0050002: D-proline reductase (dithiol) activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
See also
References
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