Substrate specificity of the Escherichia coli maltodextrin transport system and its component proteins

Ferenci, Thomas; Muir, Marianne E.; Lee, Kin-Sang; Maris, Doris

doi:10.1016/0005-2736(86)90496-7

Cited by 53 publications

(47 citation statements)

References 19 publications

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“…However, it is known that MBP can bind maltotriose, maltotetraose, and other maltooligosaccharides (MOS) in addition to maltose with K d s as low as 0.2 M in the case of maltotriose (19). Nonetheless, mutants may posses an altered specificity.…”

Section: Resultsmentioning

confidence: 99%

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Fehr

Frommer

Lalonde

2002

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

318

279

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Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing proteinbased nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications. Limited information is derived from methods requiring fixation or fractionation of tissue (1, 2). Static analysis of metabolite composition in organs, tissues, and cellular compartments involves cell disruption. Most techniques neither measure metabolite changes in real-time nor account for likely variations in local metabolite concentration at the cellular level. Current methods have low resolution and are prone to artifacts, e.g., contamination by other cell types or subcellular compartments. Thus, little is known about the dynamic changes in concentration of metabolites such as sugars and amino acids͞neurotransmit-ters at the site of transport, i.e., in the synaptic cleft relative to the cytosol of adjacent neurons and glia or at the loading site of the phloem in plants, but also in the distribution of different sugars within cellular compartments. To better understand metabolism and compartmentation, a noninvasive technique would be of significant advantage.To generate a set of multifunctional nanosensors with specificity to a large number of different compounds, suitable binding proteins fulfilling a number of criteria are required. First, the binding proteins must undergo a conformational change on substrate binding. Ideally, they should belong to a family covering a wide spectrum of substrates. Furthermore, high-affinity binding would be advantageous, because it would provide a comparatively fast way to generate mutants with lower affinities suited for an optimal physiological detection range. Finally, for measurements in eukaryotes, ...

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Section: Resultsmentioning

confidence: 99%

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Fehr

Frommer

Lalonde

2002

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

318

279

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“…High affinity requires a free reducing end of the maltodextrin molecule, while binding to extended polysaccharide chains is of lower affinity (29). Maltodextrins whose reducing end is blocked by residues larger than a methyl group (21), even though bound well by the binding protein, cannot be transported, indicating a polarity in the mechanism in which linear dextrins are channeled into the transporter (25,52,62). While maltose transport has been studied to some extent (21,23,38,60), the transport of maltodextrins is less well understood.…”

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confidence: 99%

The Maltodextrin System ofEscherichia coli: Metabolism and Transport

2005

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The maltose/maltodextrin regulon of Escherichia coli consists of 10 genes which encode a binding proteindependent ABC transporter and four enzymes acting on maltodextrins. All mal genes are controlled by MalT, a transcriptional activator that is exclusively activated by maltotriose. By the action of amylomaltase, we prepared uniformly labeled [ 14 C]maltodextrins from maltose up to maltoheptaose with identical specific radioactivities with respect to their glucosyl residues, which made it possible to quantitatively follow the rate of transport for each maltodextrin. Isogenic malQ mutants lacking maltodextrin phosphorylase (MalP) or maltodextrin glucosidase (MalZ) or both were constructed. The resulting in vivo pattern of maltodextrin metabolism was determined by analyzing accumulated [ 14 C]maltodextrins. MalP ؊ MalZ ؉ strains degraded all dextrins to maltose, whereas MalP ؉ MalZ ؊ strains degraded them to maltotriose. The labeled dextrins were used to measure the rate of transport in the absence of cytoplasmic metabolism. Irrespective of the length of the dextrin, the rates of transport at a submicromolar concentration were similar for the maltodextrins when the rate was calculated per glucosyl residue, suggesting a novel mode for substrate translocation. Strains lacking MalQ and maltose transacetylase were tested for their ability to accumulate maltose. At 1.8 nM external maltose, the ratio of internal to external maltose concentration under equilibrium conditions reached 10 6 to 1 but declined at higher external maltose concentrations. The maximal internal level of maltose at increasing external maltose concentrations was around 100 mM. A strain lacking malQ, malP, and malZ as well as glycogen synthesis and in which maltodextrins are not chemically altered could be induced by external maltose as well as by all other maltodextrins, demonstrating the role of transport per se for induction.

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“…LamB also functions as the receptor for phage (36). MBP is a soluble periplasmic protein that binds malto-oligosaccharides with a high affinity (K d ϭ 0.1 to 1 M) (15,28,40). MBP is thought to deliver the substrate to the inner membrane complex, and it is required for the transport of maltose (41, 50).…”

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confidence: 99%

“…MalK is a peripheral protein associated with the inner membrane probably through interactions with the MalF and MalG proteins (34,43). MalK binds and hydrolyzes ATP (34, 51) and has strong sequence homology with the ATP-binding domains of other ABC transporters (23).Although substrate binding by the periplasmic component (MBP) has been well-documented by several approaches (15,(19)(20)(21)(22)46), very little is known about how the inner membrane complex recognizes malto-oligosaccharides. MBP binds a substrate with a K d that reflects the K m of transport, and it is regarded as the main determinant of substrate specificity for the maltose system.…”

mentioning

confidence: 99%

“…Although substrate binding by the periplasmic component (MBP) has been well-documented by several approaches (15,(19)(20)(21)(22)46), very little is known about how the inner membrane complex recognizes malto-oligosaccharides. MBP binds a substrate with a K d that reflects the K m of transport, and it is regarded as the main determinant of substrate specificity for the maltose system.…”

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confidence: 99%

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Unliganded maltose-binding protein triggers lactose transport in an Escherichia coli mutant with an alteration in the maltose transport system

Merino

Shuman

1997

J Bacteriol

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Escherichia coli accumulates malto-oligosaccharides by the maltose transport system, which is a member of the ATP-binding-cassette (ABC) superfamily of transport systems. The proteins of this system are LamB in the outer membrane, maltose-binding protein (MBP) in the periplasm, and the proteins of the inner membrane complex (MalFGK 2 ), composed of one MalF, one MalG, and two MalK subunits. Substrate specificity is determined primarily by the periplasmic component, MBP. However, several studies of the maltose transport system as well as other members of the ABC transporter superfamily have suggested that the integral inner membrane components MalF and MalG may play an important role in determining the specificity of the system. We show here that residue L334 in the fifth transmembrane helix of MalF plays an important role in determining the substrate specificity of the system. A leucine-to-tryptophan alteration at this position (L334W) results in the ability to transport lactose in a saturable manner. This mutant requires functional MalK-ATPase activity and the presence of MBP, even though MBP is incapable of binding lactose. The requirement for MBP confirms that unliganded MBP interacts with the inner membrane MalFGK 2 complex and that MBP plays a crucial role in triggering the transport process.The maltose transport system of Escherichia coli mediates the unidirectional transport of ␣(1-4)-linked malto-oligosaccharides (up to maltoheptaose) from the extracellular medium into the cytoplasm of the cell (14). It is a member of the ATP-binding-cassette (ABC) superfamily of transport systems (2) by virtue of its strong sequence homology and similar domain structure. Members of this family are found in a variety of organisms and include the cystic fibrosis transmembrane conductance regulator in humans as well as the STE6 protein of Saccharomyces cerevisiae (24). The maltose transport system is composed of five proteins: LamB (maltoporin), maltosebinding protein (MBP), MalF, MalG, and MalK (44). The genes that encode these proteins are located in divergent operons in the malB region of the E. coli chromosome.The LamB protein is an outer membrane porin that facilitates the diffusion of malto-oligosaccharides into the periplasm of the cell (39, 47). LamB also functions as the receptor for phage (36). MBP is a soluble periplasmic protein that binds malto-oligosaccharides with a high affinity (K d ϭ 0.1 to 1 M) (15,28,40). MBP is thought to deliver the substrate to the inner membrane complex, and it is required for the transport of maltose (41, 50). The three-dimensional structure of MBP has been determined to the atomic level by X-ray crystallography (46). The subunit stoichiometry of the inner membrane MalF-MalG-MalK complex is 1:1:2 (MalFGK 2 ) (9). This complex transports the substrate across the inner membrane in an ATP-dependent manner. The MalF and MalG proteins are integral membrane proteins that are predicted to traverse the membrane eight and six times, respectively (3,8,16). MalK is a peripheral protein associ...

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Substrate specificity of the Escherichia coli maltodextrin transport system and its component proteins

Cited by 53 publications

References 19 publications

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

The Maltodextrin System ofEscherichia coli: Metabolism and Transport

Unliganded maltose-binding protein triggers lactose transport in an Escherichia coli mutant with an alteration in the maltose transport system

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