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Investigations into the regulatory mechanism of the multifunctional PutA flavoprotein
The regulation of proline utilization in Escherichia coli involves the proline-dependent translocation of the PutA flavoprotein from the cytoplasm to a peripheral position on the membrane resulting in PutA switching functions from a put gene repressor to a membrane bound bifunctional enzyme that oxidizes L-proline to glutamate. The mechanism by which PutA switches function involves a proline-induced conformational change that is characterized by the appearance of a 119-kDa fragment during limited proteolysis. The effects that substrate binding and FAD reduction have on PutA conformation were analyzed and the proline-dependent conformational change was shown to depend solely on the FAD redox state. Nonreducing analogs such as L-tetrahydro-2-furoic acid, which was identified to be a new competitive inhibitor of PRODH activity with a Ki of 0.2 mM, also elicit a conformational change but it is different than that observed with proline or FAD reduction. Mapping of the protease susceptibility sites in PutA revealed that the conformational changes caused by FAD reduction and L-THFA binding are transmitted outside the proline dehydrogenase active site (residues 263–610) and involves a flexible domain of unknown function (residues 141–262). The proline dependent conformational change in PutA was further investigated by intrinsic Trp fluorescence using a truncated PutA mutant containing residues 86–601 (PutA86–601) which has only four Trp residues. The addition of proline to wild-type PutA86–601 decreases Trp fluorescence by 36% indicating a substantial conformational change. Stopped-flow Trp fluorescence measurements demonstrated that FAD reduction precedes the conformational transition which occurs on a time-scale >10-fold slower than the turnover number of PutA. Analysis of PutA86–601 Trp mutants indicated that Trp211 in a flexible domain near the PROM active site is the primary molecular marker of the conformational change caused by proline. Last, preliminary work indicates that the 2 ′-hydroxyl group of the ribityl chain of the FAD coenzyme is important for transmitting the FAD redox signal to a nearby domain in PutA and that a PutA membrane binding domain is located at the C-terminal end of PutA. ^
Zhu, Weidong, "Investigations into the regulatory mechanism of the multifunctional PutA flavoprotein" (2005). ETD collection for University of Nebraska - Lincoln. AAI3186892.