PMID:16156643
| Citation |
Zhu, W and Becker, DF (2005) Exploring the proline-dependent conformational change in the multifunctional PutA flavoprotein by tryptophan fluorescence spectroscopy. Biochemistry 44:12297-306 |
|---|---|
| Abstract |
The multifunctional PutA flavoprotein regulates proline utilization in Escherichia coli by switching from a cytosolic DNA-binding protein to a membrane-bound enzyme with proline dehydrogenase (PRODH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. The transformation of PutA from a transcriptional repressor of the proline utilization (put) regulon to a peripheral membrane associated enzyme is mediated by a proline-dependent conformational change. Previously, limited proteolysis of PutA indicated that the conformational change involves a flexible domain of unknown function (residues 141-262) which is nearby the FAD-binding and PRODH active sites (residues 263-610). Here, we extend our understanding of the proline-dependent conformational change in PutA by investigating the intrinsic Trp fluorescence spectroscopic properties of a truncated PutA protein which contains residues 86-601 (PutA86-601) and only four Trp residues. The addition of proline to wild-type PutA86-601 decreases Trp fluorescence by 36%, indicating a substantial conformational change. An apparent rate constant of 0.59 +/- 0.06 s(-)(1) was determined for the fluorescence change by stopped-flow fluorescence measurements. The limiting rate constant for proline reduction of the FAD cofactor in PutA is 133 +/- 6 s(-)(1), demonstrating that FAD reduction precedes the conformational transition observed by Trp fluorescence. The nonreducing ligand l-tetrahydro-2-furoic acid mimics the decrease in Trp fluorescence induced by proline, indicating that both FAD reduction and ligand binding contribute to the observed conformational change in PutA86-601. W194 and W211, which are located in the flexible domain, were replaced by Phe in the PutA86-601 mutants W194F, W211F, and W194F/W211F to determine which residue is involved in the observed fluorescence change. Analysis of the PutA86-601 mutants indicated that W211 is the primary molecular marker of the conformational change caused by proline. Altogether, this work shows that the switching of PutA from a transcriptional repressor to a membrane-bound protein involves W211 in a flexible domain near the PRODH active site and occurs on a time scale that is >10-fold slower than the turnover number of PutA. |
| Links |
PubMed Online version:10.1021/bi051026b |
| Keywords |
Amino Acid Substitution; Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Crystallography, X-Ray; Escherichia coli/chemistry; Escherichia coli/enzymology; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Models, Molecular; Mutagenesis, Site-Directed; Oxidoreductases Acting on CH-NH Group Donors/chemistry; Oxidoreductases Acting on CH-NH Group Donors/metabolism; Proline; Proline Oxidase/chemistry; Proline Oxidase/metabolism; Protein Conformation; Protein Structure, Secondary; Spectrometry, Fluorescence; Tryptophan |
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