Crystallization and preliminary crystallographic analysis of the proline dehydrogenase domain of the multifunctional PutA flavoprotein from Escherichia coli

The PutA flavoprotein from Escherichia coli is a multifunctional protein th at plays pivotal roles in proline catabolism by functioning as both a membr ane-associated bifunctional enzyme and a transcriptional repressor. Periphe rally membrane-bound PutA catalyzes the two-step oxidation of proline to gl utamate, while cytoplasmic PutA represses the transcription of its own gene and the gene for a proline-transporter protein. X-ray crystallographic stu dies on PutA have been initiated to determine how the PutA structural scaff old 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 func tions. To facilitate crystallization, a recombinant protein (PutA669) corre sponding to the N-terminal 669 amino-acid residues of the 1320 residues of PutA was engineered. Activity assays demonstrated that PutA669 catalyzes th e first step of chemistry performed by PutA, the conversion of proline to D elta1-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 Angstrom; a 2.1 5 Angstrom data set was collected using a rotating-anode source. Assuming o ne molecule per asymmetric unit, the Matthews coefficient V-M is 2.5 Angstr om (3) Da(-1), with a solvent content of 50%. The structure of PutA669 will be solved by multiple isomorphous replacement.