Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.

The oxidation of proline to glutamate has significant ramifications in energy and nitrogen metabolism in a variety of organisms.1 For example, proline oxidative metabolism is important for Helicobacter pylori colonization of the gut and in the closely related mouse pathogen Helicobacter hepaticus.2, 3 Patients infected with H. pylori were reported to have 10-fold higher proline levels than non-infected individuals in the gut, where L-proline is a preferred respiratory substrate of H. pylori.4 In humans, proline serves two roles as a microenvironment energy source and as a pro-apoptotic substrate.5 Upregulation of proline oxidative metabolism, which occurs in the mitochondrion, leads to increased production of reactive oxygen species and induction of cell death pathways.6 Two enzymes, proline dehydrogenase (PRODH)2 and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH), catalyze the 4-electron oxidation of proline to glutamate. In Gram-positive bacteria and eukaryotes, PRODH and P5CDH are separate enzymes, whereas in Gram-negative bacteria such as H. pylori and Escherichia coli, PRODH and P5CDH are combined into a single polypeptide known as the proline utilization A (PutA) protein.1, 7 Scheme 1 shows the coordinated activities of the PRODH and P5CDH domains in PutA. The PRODH domain of PutA contains a flavin adenine dinucleotide (FAD) cofactor which couples the oxidation of proline (reductive half-reaction) to the reduction of ubiquinone in the membrane (oxidative half-reaction).8–10 The product of the PRODH reaction, P5C, is next hydrolyzed to γ-glutamate semialdehyde (GSA), which is then oxidized to glutamate in a NAD+-dependent step catalyzed by the P5CDH domain of PutA.7, 9 Scheme 1 Oxidation of proline to glutamate catalyzed by the PRODH and P5CDH domains of PutA. In certain Gram-negative bacteria such as E. coli and Salmonella typhimurium, PutA also contains a N-terminal ribbon-helix-helix (RHH) DNA binding domain that enables PutA to act as a transcriptional regulatory protein.11, 12 PutA regulates the transcription of the putA and putP (Na+/proline transporter) genes according to intracellular proline levels with increases in proline leading to activation of the put genes.13 The mechanism by which PutA regulates put gene expression hinges on the redox state of the flavin cofactor and PutA membrane interactions.14–18 In the oxidized state, cytoplasmic PutA binds to the put promoter and represses transcription.13 When intracellular proline levels increase, the flavin cofactor becomes reduced causing a dramatic increase in PutA membrane binding affinity.17 Thus, proline mediated reduction of the flavin cofactor switches PutA from a transcriptional repressor to a membrane-bound enzyme, which relieves PutA repression of the put genes. PutA from E. coli contains 1320 residues with the RHH, PRODH and P5CDH domains localized at residues 1–52, 261–612, and 650–1130, respectively. X-ray crystal structures have been obtained for the separate RHH/DNA-binding and PRODH domains.1, 13, 19 Figure 1A shows the structure of the PutA/PRODH domain from E. coli. The PRODH domain has a core (βα)8 barrel structure that noncovalently binds the flavin adenine dinucleotide (FAD) cofactor (Figure 1A). The (βα)8 barrel core is also found in monfunctional PRODHs from Gram-positive bacteria and is predicted to be similar to the catalytic core of human PRODH.20 Insights into proline binding were provided by an X-ray crystal structure of the PutA/PRODH domain in complex with the proline analog, L-tetrahydro-2-furoic acid (L-THFA).19, 21 THFA was observed to bind to the active site with the carboxylate group coordinated by two active site arginine residues (Arg555 and 556) that help position the proline analog near the si-face of flavin (Figure 1B).21 Thus far, the only X-ray structure of a full-length PutA is that of the PutA homolog from Bradyrhizobium japonicum.22 PutA from B. japonicum is a smaller polypeptide of 999 residues which lacks the RHH/DNA binding domain. The recently solved structure of PutA revealed a 41 A long cavity linking the PRODH and P5CDH active sites, suggesting that P5C and/or GSA are channeled within PutA.22 Figure 1 Structure of the PRODH domain and FAD conformations of E. coli PutA in oxidized and reduced states. (A) The (βα)8 barrel core structure of the PRODH domain is shown, highlighting the locations of the FAD cofactor (yellow) and THFA (green) ... The transformation of PutA from a transcriptional repressor to a membrane-associated enzyme, known as functional switching, involves conformational changes that are concomitant with proline reduction of the flavin.16–18, 23, 24 A structure of the PutA/PRODH domain reduced with dithionite showed that the FAD adopted a new conformation characterized by a significant “butterfly” bend (22°) of the isoalloxazine ring and rotation of the 2′-OH group of the ribityl chain resulting in formation of a new hydrogen bond between the 2′-OH and the FAD N(1) atom.18 Figure 1B highlights the conformational differences of the FAD cofactor between the THFA-bound (i.e., oxidized state) and dithionite-reduced PRODH domain structures. The 2′-OH group of the FAD was subsequently demonstrated to act as a redox-sensitive switch that helps control association of PutA with the membrane.18 Thus, conformational changes in the FAD upon proline reduction may represent the first step in activating PutA-membrane binding. Another key feature observed in the structures of the PutA/PRODH domain is a hydrogen bond interaction between Arg431 and the FAD N(5) atom. Although no significant conformational changes were observed for Arg431 in the dithionite-reduced structure, Arg431 is proposed to have a critical role in activating PutA membrane binding.18 Significant progress has been made toward characterizing key features of PutA such as domain organization and structure, DNA and membrane binding properties, and redox dependent functional switching.13, 18, 23, 25 A thorough understanding of the mechanisms of PRODH and P5CDH in PutA, however, is still lacking. In particular, rapid reaction kinetics of PutA/PRODH or any related monofunctional PRODH has not yet been performed. Here, we address this gap and provide a comprehensive stopped-flow kinetic analysis of PutA/PRODH. Conventional and global fitting of single turnover data to simulated mechanisms were performed to obtain microscopic rate constants for individual steps in the reductive and oxidative half-reactions of PutA/PRODH from E. coli. Steady-state kinetic parameters calculated from the microscopic rate constants showed excellent agreement with previously determined steady-state parameters for the PutA/PRODH reaction.10 The kinetic data and mechanistic insights reported herein will be of benefit in understanding the mechanisms of PRODH action, intermediate-substrate channeling in PutA, as well as systems-wide approaches for modeling proline homeostasis in biological systems.26, 27