Purification and properties of proline reductase from Clostridium sticklandii.
Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D003013:Clostridium / Q000201:enzymology*; D004228:Dithionitrobenzoic Acid; D005486:Flavin Mononucleotide; D005182:Flavin-Adenine Dinucleotide; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D004228:Dithionitrobenzoic Acid; C022452:D-proline reductase (dithiol); D000594:Amino Acid Oxidoreductases