Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides

Jacobs, Mariken H. J.; Driessen, Arnold J.M.; Konings, Wil N.

doi:10.1128/jb.177.7.1812-1816.1995

Cited by 26 publications

(29 citation statements)

References 29 publications

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“…The system does not seem to use ATP for transport and is not inhibited by vanadate, a phosphate analog that acts as a strong inhibitor of plasma membrane P-type ATPases and ABC transporters 9 . By contrast, glutamate uptake via the SBP-dependent ATPdriven transporter is inhibited by vanadate and is insensitive to uncouplers 10 . Second, the residual glutamate transport activity in these cells is osmoticshock sensitive and can be restored in spheroplasts by the addition of osmotic shock fluid or by purified glutamatebinding protein 9 .…”

Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

“…The latter function is normally not contained in secondary transporters. The SBP that belongs to these transporters functionally resemble the ones that interact with ABC transporters 12,13 , but they are not interchangeable 9,10 .…”

Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

“…In vivo studies using uncouplers and vanadate suggest that the C 4 -dicarboxylate transporters of Rb. capsulatus 10 and Rb. sphaeroides 9 are other examples of such a novel system.…”

Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

“…These conclusions are drawn from the following observations. First, cells that are defective in the SBP-dependent ATP-driven transporter for glutamate and glutamine 10 are still able to accumulate glutamate. Uncouplers and ionophores that collapse the pmf inhibit this residual glutamate transport activity.…”

Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

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Diversity of transport mechanisms: common structural principles

Driessen

Rosen

Konings

2000

Trends in Biochemical Sciences

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Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

“…In vivo studies using uncouplers and vanadate suggest that the C 4 -dicarboxylate transporters of Rb. capsulatus 10 and Rb. sphaeroides 9 are other examples of such a novel system.…”

Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

Section: Secondary Solute-binding-protein-dependent Transporters: Varmentioning

confidence: 99%

See 2 more Smart Citations

Diversity of transport mechanisms: common structural principles

Driessen

Rosen

Konings

2000

Trends in Biochemical Sciences

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“…Apparently, Rhodobacter spp. rely only on these energy-demanding high-affinity transport systems for the accumulation of solutes (20). The only genetically analyzed binding-protein-dependent transport system in Rhodobacter spp.…”

Section: Resultsmentioning

confidence: 99%

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

1997

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The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(؉) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.The purple, nonsulfur bacterium Rhodobacter sphaeroides Si4 is capable of growing on a variety of sugar alcohols (polyols), including D-mannitol and D-glucitol (sorbitol). In the presence of either substrate, R. sphaeroides Si4 concomitantly induces two NAD ϩ -dependent enzymes: mannitol dehydrogenase (MDH; EC 1.1.1.67 [38]) and sorbitol dehydrogenase (SDH; EC 1.1.1.14 [36]). SDH and MDH are involved in R. sphaeroides Si4 D-glucitol metabolism, but due to its low K m for D-glucitol, SDH is the predominant enzyme for the metabolization of this substrate at low concentrations (36). Both enzymes were purified and characterized. SDH is a dimeric enzyme with a subunit molecular mass of 29,000 Da and is active on sorbitol and galactitol, with sorbitol being the preferred substrate. MDH is a monomeric enzyme with a subunit molecular mass of 51,404 Da and is active on D-mannitol, sorbitol, and D-arabitol, with a preference for D-mannitol. Because of the potential of MDH for biotechnological applications (37,38,41), the corresponding gene (mtlK) has been cloned, sequenced (39), and overexpressed (35).In this communication, we demonstrate that the smoS gene, which encodes SDH, is located within a previously unrecognized polyol operon. The gene coding for an ATP-binding protein of an ATP-binding cassette (ABC) transport system (smoK) and the structural genes smoS and mtlK have been identified. On the basis of sequence homology data, SDH can be attributed to the short-chain dehydrogenase/reductase (SDR) protein family (21). Finally, overexpression of SDH in Escherichia coli provides sufficient amounts of the recombinant enzyme for crystallization and structure analysis studies. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains used are listed in Table 1. R. sphaeroides Si4 is an isolate of this laboratory (31). E. coli XL1-Blue MRF...

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Flagellate Motility, Behavioral Responses and Active Transport in Purple Non-Sulfur Bacteria

Armitage

Kelly

Sockett

Advances in Photosynthesis and Respiration

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Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides

Abstract: The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (K t of 1.2 M), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH.

Cited by 26 publications

References 29 publications

Diversity of transport mechanisms: common structural principles

Diversity of transport mechanisms: common structural principles

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

Flagellate Motility, Behavioral Responses and Active Transport in Purple Non-Sulfur Bacteria

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