PMID:22013066
| Citation |
Singh, RK, Larson, JD, Zhu, W, Rambo, RP, Hura, GL, Becker, DF and Tanner, JJ (2011) Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli. J. Biol. Chem. 286:43144-53 |
|---|---|
| Abstract |
The trifunctional flavoprotein proline utilization A (PutA) links metabolism and gene regulation in Gram-negative bacteria by catalyzing the two-step oxidation of proline to glutamate and repressing transcription of the proline utilization regulon. Small-angle x-ray scattering (SAXS) and domain deletion analysis were used to obtain solution structural information for the 1320-residue PutA from Escherichia coli. Shape reconstructions show that PutA is a symmetric V-shaped dimer having dimensions of 205 × 85 × 55 Å. The particle consists of two large lobes connected by a 30-Å diameter cylinder. Domain deletion analysis shows that the N-terminal DNA-binding domain mediates dimerization. Rigid body modeling was performed using the crystal structure of the DNA-binding domain and a hybrid x-ray/homology model of residues 87-1113. The calculations suggest that the DNA-binding domain is located in the connecting cylinder, whereas residues 87-1113, which contain the two catalytic active sites, reside in the large lobes. The SAXS data and amino acid sequence analysis suggest that the Δ(1)-pyrroline-5-carboxylate dehydrogenase domains lack the conventional oligomerization flap, which is unprecedented for the aldehyde dehydrogenase superfamily. The data also provide insight into the function of the 200-residue C-terminal domain. It is proposed that this domain serves as a lid that covers the internal substrate channeling cavity, thus preventing escape of the catalytic intermediate into the bulk medium. Finally, the SAXS model is consistent with a cloaking mechanism of gene regulation whereby interaction of PutA with the membrane hides the DNA-binding surface from the put regulon thereby activating transcription. |
| Links |
PubMed PMC3234867 Online version:10.1074/jbc.M111.292474 |
| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Escherichia coli/metabolism; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Protein Multimerization; Protein Structure, Tertiary; Scattering, Small Angle; X-Rays |
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