The structure of the proline utilization a proline dehydrogenase domain inactivated by N-propargylglycine provides insight into conformational changes induced by substrate binding and flavin reduction

The oxidative conversion of proline to glutamate requires the coupled activities of proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) (Scheme 1). In some bacteria, these activities are combined in a single polypeptide known as proline utilization A (PutA). The polypeptide chain lengths of PutAs exceed 1000 residues, with the PRODH and P5CDH domains appearing in the N-terminal and C-terminal halves of the protein, respectively (1, 2). Some PutAs, including Escherichia coli PutA, also serve as transcriptional repressors of the put regulon, which comprises the divergently transcribed genes encoding PutA and the high-affinity Na+/proline transporter, PutP, plus an intervening 419-bp control DNA region (1, 3). PutA represses transcription of putA and putP by recognizing, via an N-terminal ribbon-helix-helix domain, a consensus binding sequence that appears at five different locations in the put control DNA region. In the absence of proline, PutA occupies the five operator sites, thereby blocking transcription of the put genes (3). In the presence of proline, PutA associates with the membrane where it couples the oxidation of proline to P5C with reduction of the electron transport chain (Scheme 1). Following the nonenzymatic hydrolysis of P5C, the P5CDH domain catalyzes the NAD+-dependent oxidation of γ-glutamate semialdehyde to glutamate (Scheme 1) (4-6). Thus, the association of PutA with the inner membrane is not only essential for efficient catalytic turnover, but also increases expression of putA and putP by preventing PutA from engaging its operator sites in the put control DNA region (1). Scheme 1 The process by which PutA is transformed from a DNA-bound repressor to a membrane-associated enzyme, known as functional switching, involves conformational changes triggered by proline binding and reduction of the flavin cofactor (1, 7-9). Recently, conformational changes in the FAD were deduced from a structure of the dithionite-reduced E. coli PutA PRODH domain with hyposulfite bound in the proline binding site (7). Upon reduction, the FAD was observed to adopt 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 (7). The 2′-OH group of the FAD was subsequently demonstrated to act as a redox-sensitive toggle switch that helps control association of PutA with the membrane (7). A second key interaction involves the FAD N(5) atom and Arg431. Arg431 is strategically placed within hydrogen bonding distance to the flavin N(5) and is thus potentially able to sense changes in electron density across the N(1)-N(5) enediamine system attendant to FAD reduction. Indeed, mutation of Arg431 to Met, or replacement of FAD with 5-deaza-FAD, abrogated the ability of proline to activate binding of PutA to the membrane (7). Thus, Arg431 was proposed to have an important role in transmitting redox signals out of the FAD active site (7), although the dithionite-reduced enzyme structure did not indicate any movement of this residue. The conformational changes in the FAD deduced from the structure of the dithionite-reduced PRODH domain (bending of the isoalloxazine ring and rotation of the 2’-OH) presumably represent the initial step in transmission of the flavin redox status to a remote membrane-binding domain. Signal transmission likely involves Arg431, but other residues important for broadcasting the flavin redox state remain largely unknown. Here, we have used mechanism-based inactivation of E. coli PutA by N-propargylglycine (PPG, 1 in Scheme 2) to elicit conformational changes similar to those involved in functional switching. This work was motivated by our recent study of the inactivation of PRODH from Thermus thermophilus (TtPRODH) by PPG (10). TtPRODH is a 307-residue monofunctional PRODH that has 27 % sequence identity to residues 235 - 569 of the 1320-residue E. coli PutA protein. The crystal structure of PPG-inactivated TtPRODH revealed a 3-carbon link between the e–amine of Lys99 and the N(5) of FAD. In one scenario, the mechanism of inactivation is initiated by the oxidation of PPG to N-propargyliminoglycine (2 in Scheme 2) and concomitant reduction of the FAD. It is proposed that attack of 2 by the N(5) of the reduced flavin, rearrangement of a putative allenic species (3) to an imminium intermediate (4), and nucleophilic attack by an active site lysine residue (Lys99) on the imminium intermediate lead to the covalent FAD-Lys99 adduct (5). As a result, the enzyme is, effectively, locked into the reduced state. Sequence alignments show that Lys99 is highly conserved among monofunctional PRODHs and PutAs, with Lys329 being the corresponding residue in E. coli PutA. Since PPG locks TtPRODH in the reduced state, we investigated the possibility of using PPG to stabilize the reduced conformation of PutA in order to facilitate examination of this important conformation. Results of these studies are described herein. Scheme 2