PMID:11717519
| Citation |
Nadaraia, S, Lee, YH, Becker, DF and Tanner, JJ (2001) Crystallization and preliminary crystallographic analysis of the proline dehydrogenase domain of the multifunctional PutA flavoprotein from Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 57:1925-7 |
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| Abstract |
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 Delta1-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 A; a 2.15 A data set was collected using a rotating-anode source. Assuming one molecule per asymmetric unit, the Matthews coefficient V(M) is 2.5 A(3) Da(-1), with a solvent content of 50%. The structure of PutA669 will be solved by multiple isomorphous replacement. |
| Links | |
| Keywords |
Bacterial Proteins/chemistry; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Escherichia coli/enzymology; Membrane Proteins/chemistry; Proline Oxidase/chemistry; Protein Conformation; Protein Structure, Tertiary |
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