Dataset Information

In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA.

ABSTRACT: Pseudomonas putida PtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons, kgu and gad. PtxS controls the expression of the kgu and gad operons as well as its own transcription. The PtxS operator is a perfect palindrome, 5'-TGAAACCGGTTTCA-3', which is present in all three promoters. Crystallization of native PtxS failed, and PtxS-DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10×10?µm. The crystal belonged to the trigonal space group P3, with unit-cell parameters a=b=213.71, c=71.57?Å. Only unhandled crystals grown in capillaries of 0.1?mm inner diameter diffracted X-rays to 1.92?Å resolution.

SUBMITTER: Pineda-Molina E

PROVIDER: S-EPMC3515369 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA.

Pineda-Molina E E Daddaoua A A Krell T T Ramos J L JL García-Ruiz J M JM Gavira J A JA

Acta crystallographica. Section F, Structural biology and crystallization communications 20121030 Pt 11

Pseudomonas putida PtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons, kgu and gad. PtxS controls the expression of the kgu and gad operons as well as its own transcription. The PtxS operator is a perfect palindrome, 5'-TGAAACCGGTTTCA-3', which is present in all three promoters. Crystallization of native PtxS failed, and PtxS-DNA crystals were finally produced by the counter-d ...[more]

PMID: 23143237

Similar Datasets

Project description:Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K(D) (equilibrium dissociation constant) of 15 muM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1 degrees C and 57.6 degrees C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 muM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5'-TGAAACCGGTTTCA-3'. This palindrome overlaps with the RNA polymerase binding site.

Dataset Information

In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA.

Publications

In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets