Redox-induced changes in flavin structure and roles of flavin N(5) and the ribityl 2'-OH group in regulating PutA--membrane binding.

Proline utilization A (PutA) from Escherichia coli and other Gram negative bacteria is a large multifunctional protein that uniquely combines enzymatic and transcriptional regulatory activities within a single 1320 amino acid polypeptide (1-4). As an enzyme, PutA peripherally associates with the inner cytoplasmic membrane to catalyze the four-electron oxidation of proline to glutamate via the coordinated actions of separate flavin-dependent proline dehydrogenase (PRODH) and NAD+-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains involving residues 263-612 and 650-1130, respectively (5, 6). An N-terminal ribbon-helix-helix motif (residues 2-43) endows PutA with DNA-binding activity enabling PutA to also function as a cytosolic autogenous transcriptional repressor of the proline utilization (put) genes putA and putP (encodes a high affinity proline transporter) (7-9). To fulfill its mutually exclusive roles as a transcriptional repressor and membrane-bound proline catabolic enzyme, PutA undergoes proline-dependent functional switching. Proline reduction of the FAD cofactor induces PutA-membrane binding which consequently disrupts the PutA-DNA complex equilibrium and activates put gene expression (10-14). To reveal the mechanism by which FAD reduction drives tight PutA-membrane associations and therefore regulates PutA function, we have begun to explore molecular interactions between the FAD cofactor and active site residues in the PRODH domain of PutA. Previously, the structure of a truncated form of oxidized PutA containing residues 86-669 (PutA86-669) was solved by X-ray crystallography (2.0-2.1 A) in complex with different active site inhibitors such as L-lactate (Ki = 1.4 mM) and L-tetrahydro-2-furoic acid (L-THFA, Ki = 0.2 mM) (15). Important ion pair interactions are evident between the substrate carboxylate group and the guanidinium groups of Arg555 and Arg556. Also, Arg556 is within hydrogen bonding distance of the 2′-OH ribityl group of the FAD cofactor. Another notable interaction is between Arg431 and the N(5) atom of the isoalloxazine ring. Because electron density changes dramatically across the N(1)-N(5) enediamine system upon FAD reduction, we hypothesized that Arg431 may have a role in enabling PutA to sense redox changes in the FAD cofactor. In this study, redox-dependent structural changes in the FAD cofactor were identified by comparing crystal structures of oxidized and dithionite reduced PutA86-669. Substantial movement of the 2′-OH group of the ribityl moiety was observed in the dithionite treated crystals relative to oxidized PutA86- 669 resulting in disruption of the Arg556-N(2)H-O(2′)-FAD interaction and the formation of a new hydrogen bond network between the ribityl 2′-OH group, the N(1) atom of the isoalloxazine ring, the ribityl 4′-OH, and Gly435. We subsequently analyzed the impact of these interactions on PutA function by measuring the kinetic membrane binding properties of PutA reconstituted with FAD analogues and matching PutA site directed mutants by surface plasmon resonance (SPR). Results are presented that provide an example of a unique mechanism by which the FAD cofactor controls membrane binding of a large multifunctional enzyme.