The PutA Protein of Salmonella typhimurium Catalyzes the Two Steps of Proline Degradation via a Leaky Channel Mark W. Surber and Stanley Maloy Department of Microbiology, University of Illinois, Urbana, Illinois 61801 Received December 4, 1997, and in revised form February 23, 1998 Proline utilization in Salmonella typhimurium re- quires two proteins encoded by the put operon: PutP, the major proline permease, and PutA. PutA is a mul- tifunctional, peripheral membrane protein which acts both as a transcriptional repressor for the put operon and enzyme catalyzing the two-step conversion of pro- line to glutamate. In the first enzymatic reaction cat- alyzed by PutA, proline oxidation to pyrroline-5-car- boxylate (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 con- tribute to the regulation of proline utilization. © 1998 Academic Press Key Words: PutA; substrate channeling; proline de- hydrogenase; P5C dehydrogenase; Salmonella typhi- murium; leaky channel. In a sequence of metabolic reactions, the direct transfer of a reaction intermediate to the subsequent reaction without equilibration with the surrounding environment is referred to as substrate channeling (1). Substrate channeling facilitates certain otherwise un- feasible steps in intracellular metabolism. There are several reasons why channeling may be required in vivo. First, some reaction intermediates are used in multiple pathways, thus if allowed to equilibrate with the surrounding environment the intermediate might be purloined by another pathway. Second, some reac- tion intermediates are labile, thus if allowed to equili- brate with the surrounding environment the interme- diate might be rapidly degraded. Third, some enzy- matic reactions may require a high local concentration of reaction intermediates to function at optimal effi- ciency. Finally, some pathways utilize a membrane permeable intermediate, which if not retained would be lost from the cell. Proline utilization in Salmonella typhimurium is regulated by the put operon. The put operon encodes two proteins (2, 3): PutP, the major proline permease, and PutA, a multifunctional, peripheral-membrane protein which carries out the two enzymatic reactions in the catabolism of proline to glutamate (Fig. 1). The first enzymatic reaction, proline dehydrogenase (EC 1.5.99.8), couples the oxidation of proline to pyrroline- 5-carboxylate (P5C) 1 with reduction of a tightly asso- ciated FAD cofactor (2, 4). For this reaction to function catalytically, PutA must interact with the membrane- associated electron transport chain for reoxidation of the reduced FAD (5, 6). The second enzymatic reaction catalyzed by PutA is the oxidation of P5C to glutamate by P5C dehydrogenase (EC 1.5.1.12). P5C is first hy- drolyzed to glutamate semialdehyde (GSA) and the oxidation of GSA to glutamate is coupled with the reduction of a soluble NAD (3). 1 Abbreviations used: GSA, glutamate semialdehyde; P5C, pyrro- line-5-carboxylate; o-ab, o-aminobenzaldehyde; PMS, phenazine methosulfate; INT, p-iodonitrotetrazolium violet. 0003-9861/98 $25.00 281 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 354, No. 2, June 15, pp. 281–287, 1998 Article No. BB980697