Amino Acids (2001) 21: 161–174 Substrate recognition by proline permease in Salmonella M.-K. Liao 1 and S. Maloy 2 1 Department of Biology, Furman University, Greenville, South Carolina, U.S.A. 2 Department of Microbiology, University of Illinois, Urbana, Illinois, U.S.A. Accepted December 1, 2000 Summary. Proline transport is required for catabolism of proline as a carbon, nitrogen, and energy source, and for accumulation of proline during adapta- tion to osmotic stress. These physiological processes are widespread in nature, and play essential roles in the virulence of both prokaryotic and eukaryotic pathogens. In enteric bacteria, the major proline permease is encoded by the putP gene. To identify the structural features required for substrate recogni- tion by PutP, we assayed the transport and toxicity of a variety of natural and synthetic derivatives of proline. The results indicate that the substrate binding site of proline permease consists of a hydrophobic pocket that accommodates C3, C4, and C5 of the pyrrolidine ring. Both 4- and 5-membered rings fit into the substrate binding pocket, but 6-membered rings are excluded. Analogs with substituents on the C4 position are also excluded. In addition, the binding site includes a hydrophilic region that recognizes the imino and carbonyl groups. A free carboxyl group is not required. Taken together, these results may be used to design new synthetic inhibitors of proline transport that can effectively block proline uptake by microbial pathogens. Keywords: Amino acids – Proline permease substrate specificity Introduction Solute transport is an essential and often rate-limiting step in a metabolic pathway. In Salmonella and Escherichia coli, approximately 40% of all trans- port is catalyzed by ion/solute co-transport systems (permeases) (Wilson, 1978). The binding of the substrate and the counter-ion to their respective binding sites in the permease presumably leads to a conformational change that results in the release of substrate into the cytoplasm. Hence, permeases must display a high degree of specificity in substrate recognition in order to discriminate between different molecules presented to the cell (Saier, 2000). Four general approaches have been taken to gain insight on the structure and function of the substrate binding sites in permeases: (i) labeling with inhibitors such as N-ethylmaleimide that bind tightly to a permease and