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 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.
Nadaraia
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Department of Chemistry, University of Missouri-Columbia, 65211, USA.
Lee
Y H
YH
Becker
D F
DF
Tanner
J J
JJ
D001426:Bacterial Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry*; D011394:Proline Oxidase / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D011394:Proline Oxidase