title: The PutA protein of Salmonella typhimurium catalyzes the two steps of proline degradation via a leaky channel.
abstract: Proline utilization in Salmonella typhimurium requires two proteins encoded by the put operon: PutP, the major proline permease, and PutA. PutA is a multifunctional, peripheral membrane protein which acts both as a transcriptional repressor for the put operon and enzyme catalyzing the two-step conversion of proline to glutamate. In the first enzymatic reaction catalyzed by PutA, proline oxidation to pyrroline-5-carboxylate (P5C) is coupled with the reduction of a tightly associated FAD. In the second reaction, P5C oxidation to glutamate is coupled with reduction of soluble NAD. Although PutA can use exogenous P5C, the concentration of exogenous P5C required for the P5C dehydrogenase reaction is much greater than the steady-state P5C concentration accumulated during proline degradation. Furthermore, exogenous P5C does not efficiently compete against endogenous P5C for the production of glutamate, and the endogenous P5C produced directly from proline is preferentially used by PutA for the production of glutamate. Kinetic assays indicate that in the presence of NAD the two enzymatic reactions of PutA function synchronously to increase the overall reaction rate over that of the two independent reactions, and the second reaction proceeds in the absence of a lag phase. These results indicate that PutA directly transfers the intermediate P5C between the two enzymatic functions via a "leaky channel" mechanism. Because both the reduction of FAD and the intermediate P5C stimulate membrane association of PutA, channeling of P5C may also contribute to the regulation of proline utilization.
authors.affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, 61801, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Surber
authors.forename: S
authors.initials: S
authors.lastname: Maloy
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D002384:Catalysis; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D011394:Proline Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011758:Pyrroles / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D011758:Pyrroles; D009243:NAD; C015485:delta-1-pyrroline-5-carboxylate; D011392:Proline; D011394:Proline Oxidase