Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli.
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.
Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Ranjan K
RK
Singh
John D
JD
Larson
Weidong
W
Zhu
Robert P
RP
Rambo
Greg L
GL
Hura
Donald F
DF
Becker
John J
JJ
Tanner
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D014965:X-Rays
D001426:Bacterial Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria