Purification of the putA gene product. A bifunctional membrane-bound protein from Salmonella typhimurium responsible for the two-step oxidation of proline to glutamate.
In this paper we report the purification of a protein which is able to catalyze both the proline oxidase and the pyrroline-5-carboxylic acid dehydrogenase activities necessary for the oxidation of proline to glutamic acid. The purification involves the preparation of a crude membrane pellet, detergent solubilization, ammonium sulfate fractionation, and DEAE-chromatography. We are able to obtain an essentially pure preparation (greater than 95% pure) after only a 52-fold purification, demonstrating that the protein is a major protein in cells fully induced for proline utilization. Both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-purity throughout our purification. Velocity sedimentation of the purified protein demonstrates that both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-sediment. Early in the purification procedure we are able to detect two species of protein which have both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities. Our procedure purifies only the larger molecular weight species. The purified protein is a dimer composed of identical 132,000-dalton subunits. Analysis of mutants defective for proline utilization demonstrate that the bifunctional enzyme is the putA gene product.
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D002462:Cell Membrane / Q000201:enzymology; D005796:Genes; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000302:isolation & purification; D011394:Proline Oxidase / Q000235:genetics* / Q000302:isolation & purification; D011762:Pyrroline Carboxylate Reductases / Q000235:genetics* / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011762:Pyrroline Carboxylate Reductases; D011394:Proline Oxidase