The PutA flavoprotein from Escherichia coli plays multiple roles in proline catabolism by functioning as a membrane-associated bi-functional enzyme and a transcriptional repressor of proline utilization genes. The human homolog of the PutA proline dehydrogenase (PRODH) domain is critical in p53-mediated apoptosis and schizophrenia. Here we report the crystal structure of a 669-residue truncated form of PutA that shows both PRODH and DNA-binding activities, representing the first structure of a PutA protein and a PRODH enzyme from any organism. The structure is a domain-swapped dimer with each subunit comprising three domains: a helical dimerization arm, a 120-residue domain containing a three-helix bundle similar to that in the helix-turn-helix superfamily of DNA-binding proteins and a beta/alpha-barrel PRODH domain with a bound lactate inhibitor. Analysis of the structure provides insight into the mechanism of proline oxidation to pyrroline-5-carboxylate, and functional studies of a mutant protein suggest that the DNA-binding domain is located within the N-terminal 261 residues of E. coli PutA.

The PutA flavoprotein from Escherichia coli is a multifunctional protein that plays pivotal roles in proline catabolism by functioning as both a membrane‐associated bifunctional enzyme and a transcriptional repressor. Peripherally membrane‐bound PutA catalyzes the two‐step oxidation of proline to glutamate, while cytoplasmic PutA represses the transcription of its own gene and the gene for a proline‐transporter protein. X‐ray crystallographic studies on PutA have been initiated to determine how the PutA structural scaffold enables it to be both an enzyme and a repressor, and to understand the mechanism by which PutA switches between its enzymatic and DNA‐­binding functions. To facilitate crystallization, a recombinant protein (PutA669) corresponding to the N‐terminal 669 amino‐acid residues of the 1320 residues of PutA was engineered. Activity assays demonstrated that PutA669 catalyzes the first step of chemistry performed by PutA, the conversion of proline to Δ1‐pyrroline‐5‐­carboxylate. Crystals of PutA669 have been obtained from PEG 3000 buffered at pH 6–7. The crystals occupy an I‐centered orthorhombic lattice with unit‐cell parameters a = 72.5, b = 140.2, c = 146.8 Å; a 2.15 Å data set was collected using a rotating‐anode source. Assuming one molecule per asymmetric unit, the Matthews coefficient VM is 2.5 Å3 Da−1, with a solvent content of 50%. The structure of PutA669 will be solved by multiple isomorphous replacement.

Enzymes are the metabolic catalysts that affect a multitude of physiological processes and responses. Tight control of enzyme activity is therefore essential in maintaining the steady state of all organisms.