Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli

Ranjan K. Singh, John D. Larson, Weidong Zhu, Robert P. Rambo, Greg L. Hura, Donald F Becker, John J. Tanner

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 x 85 x 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.

Original languageEnglish (US)
Pages (from-to)43144-43153
Number of pages10
JournalJournal of Biological Chemistry
Volume286
Issue number50
DOIs
StatePublished - Dec 16 2011

Fingerprint

Flavoproteins
X ray scattering
Proline
Escherichia coli
X-Rays
Regulon
X rays
DNA
Scattering
Transcription
Gene expression
1-Pyrroline-5-Carboxylate Dehydrogenase
Catalytic Domain
Oligomerization
Aldehyde Dehydrogenase
Dimerization
Protein Sequence Analysis
Gram-Negative Bacteria
Metabolism
Dimers

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli. / Singh, Ranjan K.; Larson, John D.; Zhu, Weidong; Rambo, Robert P.; Hura, Greg L.; Becker, Donald F; Tanner, John J.

In: Journal of Biological Chemistry, Vol. 286, No. 50, 16.12.2011, p. 43144-43153.

Research output: Contribution to journalArticle

Singh, Ranjan K. ; Larson, John D. ; Zhu, Weidong ; Rambo, Robert P. ; Hura, Greg L. ; Becker, Donald F ; Tanner, John J. / Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 50. pp. 43144-43153.
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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 x 85 x 55 {\AA}. The particle consists of two large lobes connected by a 30-{\AA} 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.",
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