Sites Important for Na+ and Substrate Binding in the Na+/Proline Transporter of Escherichia coli, a Member of the Na+/Solute Symporter Family

Pirch, Torsten; Quick, Matthias; Nietschke, Monika; Langkamp, Markus; Jung, Heinrich

doi:10.1074/jbc.m111008200

Cited by 30 publications

(43 citation statements)

References 33 publications

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“…As controls, uptake of [ 14 C]mannitol (PEP dependent) and [ 14 C]proline (sodium motive force dependent) was studied (13,14,16). With the ratio of the concentrations of arsenate and CCCP at 4,000 (i.e., 1 M CCCP and 4 mM arsenate or 5 M CCCP and 20 mM arsenate), proline uptake was always more strongly inhibited by CCCP, while mannitol uptake was always more strongly inhibited by arsenate.…”

Section: Resultsmentioning

confidence: 99%

The Ascorbate Transporter of Escherichia coli

et al. 2003

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The sgaTBA genes of Escherichia coli encode a putative 12-transmembrane ␣-helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively. We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of L-ascorbate in vivo and its phosphoenolpyruvatedependent phosphorylation in vitro. The transporter exhibits an apparent K m for L-ascorbate of 9 M and is highly specific. The sgaTBA genes are regulated at the transcriptional level by the yjfQ gene product, as well as by Crp and Fnr. The yjfR gene product is essential for L-ascorbate utilization and probably encodes a cytoplasmic L-ascorbate 6-phosphate lactonase. We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to L-ascorbate via the phosphoryl transfer pathway: PEP ¡ enzyme I-P ¡ HPr-P ¡ IIA-PIn 1996, we reported computational analyses of three operons present in the Escherichia coli genome that appeared to encode enzymes and a transporter concerned with sugar metabolism (18). These operons, sga, sgb, and sgc, encode enzymes homologous to pentose-phosphate 3-and 4-epimerases, and sga and sgc also encode homologues of constituents of the bacterial phosphotransferase system (PTS) (22). The sga operon includes two genes, sgaA and sgaB, that encode homologues of fructose/mannitol enzymes IIA and lactose/N,NЈ-diacetylchitobiose enzymes IIB, respectively ( Fig. 1). No IIC homologue was identified, but upstream of the sgaB and sgaA genes is a gene, sgaT, that encodes a membrane protein with 12 putative transmembrane helical segments, a characteristic of many secondary carriers (25). SgaT proved not to be homologous to any functionally characterized protein. Two possibilities were considered: (i) SgaT-SgaB-SgaA could be a novel type of enzyme II complex, or (ii) SgaT could be a secondary transporter, while SgaA and SgaB might regulate expression of the sga operon or the activity of one or more of its gene products (18,25).It has been known for over 60 years that various bacteria, including E. coli, can ferment L-ascorbate (L-xyloascorbate [vitamin C]) under anaerobic but not aerobic conditions (7,19,32). The unstable hydrolysis product of L-ascorbate, 3-keto-Lgulonate, has been implicated as an intermediate in its catabolism (28). The dissimilation of L-ascorbate, both during coculture with other carbon sources and as a sole carbon source, has been documented for E. coli (19,31). In animal tissues, the principal route of L-ascorbate metabolism involves enzymatic and nonenzymatic oxidation to dehydroascorbate, an unstable lactone that hydrolyzes spontaneously to 2,3-di-keto-L-gulonate (9).Recently, Yew and Gerlt (31) showed that the anaerobic utilization of L-ascorbate is dependent on three enzymes encoded downstream of sgaTBA within the sga operon (Fig. 1). These enzymes catalyze the conversion of the phosp...

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Section: Resultsmentioning

confidence: 99%

The Ascorbate Transporter of Escherichia coli

et al. 2003

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“…2 A, G, and H). The equivalent position in PutP is also important (64,65). In NIS, the Na + at the Na2 site interacts with the -CO .…”

Section: Discussionmentioning

confidence: 99%

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Ferrandino

Nicola

Sánchez

et al. 2016

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

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The sodium/iodide symporter (NIS) mediates active I− transport in the thyroid—the first step in thyroid hormone biosynthesis—with a 2 Na+: 1 I− stoichiometry. The two Na+ binding sites (Na1 and Na2) and the I− binding site interact allosterically: when Na+ binds to a Na+ site, the affinity of NIS for the other Na+ and for I− increases significantly. In all Na+-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na+ ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues—S66, D191, Q194, and Q263—are also involved in Na+ coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I−. These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance.

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“…Although speculative, such inferences may be appropriate since receptor B proteins show distant homology to these transporters, leading to their classification as a subgroup of the amino acid-polyamine-organocation superfamily (17). If the molecular basis of transport in prokaryotic amino acid transporters does represent a functional precedent for germinant-receptor B proteins, then it seems likely that residues that participate directly in germinant binding are also to be found in TM domains, since this is where ligand binding sites in a number of amino acid transporters are often located (24,25,36). The observation that residues spanning TM9 and TM10 in GerVB confer proline recognition to the GerUV fusion protein while OL5 residues alone do not would appear to substantiate this hypothesis.…”

Section: Discussionmentioning

confidence: 99%

Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the Bacillus megaterium Spore Germinant Receptor

2008

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The extreme metabolic dormancy and resistance properties of spores formed by members of the Bacillus and Clostridium genera are lost upon exposure to a variety of small-molecule germinants. Germinants are known to interact in an as yet undefined manner with cognate receptor complexes that reside in the inner membrane that surrounds the spore protoplast. The receptor itself is a complex of at least three proteins, and in this study we identify amino acid residues, predicted to lie in loop regions of GerVB on the exterior aspect of the membrane, that influence the Bacillus megaterium spore germination response. Three consecutive residues adjacent to putative transmembrane domain 10 (TM10) were demonstrated to mediate to various degrees the proline germinative response while also influencing germination in response to leucine, glucose, and inorganic salts, suggesting that this region may be part of a ligand binding pocket. Alternatively, substitutions in this region may affect the conformation of associated functionally important TM regions. Leucine-and KBrmediated germination was also influenced by substitutions in other outer loop regions. These observations, when considered with accompanying kinetic analyses that demonstrate cooperativity between germinants, suggest that binding sites for the respective germinants are in close spatial proximity in the receptor but do not overlap. Additionally, proline recognition was conferred to a chimeric receptor when TM regions associated with the putative binding loop were present, indicating that residues in TM9 and/or TM10 of GerVB are also of functional importance in the proline-induced germinative response.Endospores formed in response to nutrient starvation by members of the genera Bacillus and Clostridium display remarkable properties of resistance and dormancy that permit their survival in the environment for extended periods. Despite this extreme dormancy, spores retain the ability to initiate vegetative metabolism rapidly upon exposure to appropriate nutrients via the process of germination (20, 30). Significant advances have been made in recent years through the application of mainly genetic techniques to characterize the molecular apparatus involved in spore germination, identifying structural genes that encode germinant receptors (4,8,14,23,37), ion channels (31, 35), and hydrolytic enzymes involved in the degradation of the cortical peptidoglycan that surrounds the spore protoplast (6,11,16,21). Cytological observations on spore coat degradation during germination (29) have also recently been extended by the application of atomic force microscopy to provide insights to the structural basis for the disassembly of the outer layers that provide the primary barrier to environmental insult (26).Despite these advances, however, little is known of the molecular mechanism of the primary event of spore germination-the interaction of the germinant, typically an amino acid, sugar, or riboside, with its cognate receptor-and how this interaction triggers the subsequent casc...

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Sites Important for Na+ and Substrate Binding in the Na+/Proline Transporter of Escherichia coli, a Member of the Na+/Solute Symporter Family

Cited by 30 publications

References 33 publications

The Ascorbate Transporter of Escherichia coli

The Ascorbate Transporter of Escherichia coli

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the Bacillus megaterium Spore Germinant Receptor

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Sites Important for Na+ and Substrate Binding in the Na+/Proline Transporter of Escherichia coli, a Member of the Na+/Solute Symporter Family

Cited by 30 publications

References 33 publications

The Ascorbate Transporter of Escherichia coli

The Ascorbate Transporter of Escherichia coli

Na + coordination at the Na2 site of the Na + /I − symporter

Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the Bacillus megaterium Spore Germinant Receptor

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Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter