The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

Haardt, Martin; Kempf, Bettina; Faatz, Elke; Bremer, Erhard

doi:10.1007/bf00290728

Cited by 132 publications

(146 citation statements)

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“…Cell-culture and protein-preparation methods were described previously (61). Uptake of [ 14 C]-betaine was measured in E. coli MKH13 cells (62). E. coli DH5αmcr (63) was used for the heterologous expression of strep-betP.…”

Section: Methodsmentioning

confidence: 99%

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K m and K d for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22 Na + binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.secondary transport | symmetry | membrane protein | alkali metal ion | osmoregulation

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Section: Methodsmentioning

confidence: 99%

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…1), respectively. The E. coli K-12 strain MKH13 [⌬(betTIBA)U169 ⌬(putPA)101 ⌬(proP)2 ⌬(proU)608], the E. coli B strain BL21 ( DE3), and the plasmids pBR322, pRB373, pPD100, and pHSG575 have been described previously (5,7,11,17,49,51). The kanamycin resistance cassette used for gene disruption experiments was isolated from plasmid pAT21 (52), and the recombinant betTIBA phage [135]6E6(ϩ) was from the Kohara bacteriophage clone miniset collection (27).…”

Section: Methodsmentioning

confidence: 99%

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

et al. 1996

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Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of the gbsAB genes destroyed the cholineglycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high-osmolarity stress in choline-or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to A -dependent promoters and was stimulated by the presence of choline in the growth medium.

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“…Access of GB present in the environment to the periplasmic ProX protein is provided by passive diffusion across the outer membrane through the OmpC and OmpF general porins of E. coli (36). ProX binds GB avidly with a K D of ϳ1 M (32,37,38) delivering the ligand to the substrate translocation complex that is embedded in the cytoplasmic membrane. Driven by ATP hydrolysis, the translocation complex then transports the ligand into the cytoplasm (34,35,39).…”

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confidence: 99%

Cation-π Interactions as Determinants for Binding of the Compatible Solutes Glycine Betaine and Proline Betaine by the Periplasmic Ligand-binding Protein ProX from Escherichia coli

Schiefner

Breed

Bösser

et al. 2004

Journal of Biological Chemistry

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118

137

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Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.

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The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

Cited by 132 publications

References 25 publications

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

Cation-π Interactions as Determinants for Binding of the Compatible Solutes Glycine Betaine and Proline Betaine by the Periplasmic Ligand-binding Protein ProX from Escherichia coli

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