Selectivity for maltose and maltodextrins of maltoporin, a pore‐forming protein of <i>E. coli</i> outer membrane

Dargent, Bénédicte; Rosenbusch, Jürg P.; Pattus, Franc

doi:10.1016/0014-5793(87)80891-8

Cited by 45 publications

(39 citation statements)

References 24 publications

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“…If binding blocked all three channels, then the inhibition constants for black lipid ion conductance would be roughly one-fourth of the binding dissociation constant and the inhibition assays would show a small deviation from hyberbolic (Michaelis-Menten) kinetics. These constants are roughly equal and no deviation has been observed (Table 1) (2,3,8).…”

Section: Discussionmentioning

confidence: 69%

“…In light of our results, it would appear that the "selectivity filter" in LamB (namely, the maltodextrin binding site) is located in the three channels. The single channel conductance of LamB in black lipid bilayers in 150 pS in 1 M KCI (2,8). While it is not possible at present to determine whether the 150 pS conductance steps are due to whole trimers or to single channels in a trimer, we can show that the binding of a single maltodextrin to a LamB trimer blocks only one-third of the total trimer conductance.…”

Section: Discussionmentioning

confidence: 93%

“…A variety of experiments have shown that LamB acts as a passive pore with a binding site for its substrates (2,3,7,8,13,18,19). Binding of maltodextrins inhibits pore activity and has allowed measurements of LamB's binding affinity for maltose and longer maltodextrins.…”

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confidence: 99%

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Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

et al. 1991

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We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 ,uM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.LamB (also called maltoporin) is a trimeric outer membrane protein that functions as a pore for the passage of maltose and maltodextrins [linear a(1-4)-linked D-glucose polymers] across the outer membrane of Escherichia coli (10,24,25,27). A variety of experiments have shown that LamB acts as a passive pore with a binding site for its substrates (2,3,7,8,13,18,19). Binding of maltodextrins inhibits pore activity and has allowed measurements of LamB's binding affinity for maltose and longer maltodextrins. In whole cells, LamB has been shown to act as a saturable pore for certain chromogenic substrate analogs with a measurable Km and Vmax (14).The number of channels (and maltodextrin-binding sites) per LamB trimer has widely been presumed to be either one or three. This is partially based on images of negatively stained two-dimensional crystals of LamB that show three stain-filled channels merging into one (17). Genetic studies of dominance between mutant LamB proteins have suggested that each LamB monomer forms a single independent pore (11). Here, we confirm the results of this genetic study and show that maltodextrin binding to LamB can be detected in solution and the stoichiometry of binding can be measured. MATERIALS AND METHODSFlow dialysis. Flow dialysis (rate of dialysis) was carried out as described by Colowick and Womack (6), using a homemade dialysis apparatus consisting of two chambers separated by a dialysis membrane. The upper chamber was loaded with 1 ml of concentrated, purified LamB (between 11 and 35 mg/ml) in a solution containing 50 mM Tris * Cl, 100 mM NaCl, 5 mM EDTA, 3 mM NaN3, 10 mM MgCl2, and 0.5% sodium dodecyl sulfate (SDS), pH 7.5. To start the experiment, -50 ,uCi of high-specific-activity [1-3H]maltodextrin (2.5 to 8 Ci/mmol) was added to the upper chamber.The bottom chamber (volume, -100 RI) was constantly flushed with buffer at 1.1 ml/min, and fractions were collected every minute. Both upper and lower chambers were constantly stirred. The radioactivity in the fractions was determined by liquid scintillation counting and used as a measure of the free [1-3H]maltodextrin concentration. Unlabeled maltodextrin was added at 5-min intervals to the * Corresponding author. The results of the flow dialysis experiments were analyzed by two methods. The first, Scatchard plot analysis, required estimating the rate of [1-3H]maltodextrin elution in the absence of LamB binding sites (the "100% free" rate). In general, at the highest plateau (lowest specific activity), most of the [1-3H]maltodextrin is blocked from the LamB binding site...

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Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 93%

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Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

et al. 1991

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“…A well studied example is that of LamB (64,65), which facilitates the diffusion of maltose and maltodextrins. Although in this case, a well resolved block of the channel, at the single-channel level, can be observed (66), in the case of KdgM the short lived blocked states could not be resolved.…”

Section: Discussionmentioning

confidence: 99%

The Oligogalacturonate-specific Porin KdgM of Erwinia chrysanthemi Belongs to a New Porin Family

Blot¹,

Berrier²,

Hugouvieux‐Cotte‐Pattat³

et al. 2002

Journal of Biological Chemistry

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The phytopathogenic Gram-negative bacteria Erwinia chrysanthemi secretes pectinases, which are able to degrade the pectic polymers of plant cell walls, and uses the degradation products as a carbon source for growth. We characterized a major outer membrane protein, KdgM, whose synthesis is strongly induced in the presence of pectic derivatives. The corresponding gene was characterized. Analysis of transcriptional fusions showed that the kdgM expression is controlled by the general repressor of pectinolytic genes, KdgR, by the repressor of hexuronate catabolism genes, ExuR, by the pectinase gene repressor, PecS, and by catabolite repression via the cyclic AMP receptor protein (CRP) transcriptional activator. A kdgM mutant is unable to grow on oligogalacturonides longer than trimers, and its virulence is affected. Electrophysiological experiments with planar lipid bilayers showed that KdgM behaves like a voltage-dependent porin that is slightly selective for anions and that exhibits fast block in the presence of trigalacturonate. In contrast to most porins, KdgM seems to be monomeric. KdgM has no homology with currently known porins, but proteins similar to KdgM are present in several bacteria. Therefore, these proteins might constitute a new family of porin channels.

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“…The most abundant outer membrane proteins made by serovar Typhi are the porins. These include OmpC and OmpF, which have more general substrate specificities (6,33,54), and PhoE, MalL (LamB), and Tsx, which facilitate the uptake of phosphates, maltodextrins, and nucleosides, respectively (16,45,71). Like OmpC and OmpF, the specialized porins are waterfilled channels and function without an energy requirement; however, they contain saturable substrate-binding sites that may limit transport at low substrate concentrations (8,43,53).…”

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confidence: 99%

TheSalmonella entericaSerovar TyphitsxGene, Encoding a Nucleoside-Specific Porin, Is Essential for Prototrophic Growth in the Absence of Nucleosides

et al. 2005

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The Salmonella enterica serovar Typhi tsx gene encodes a porin that facilitates the import of nucleosides. When serovar Typhi is grown under anaerobic conditions, Tsx is among the outer membrane proteins whose expression increases dramatically. This increase in expression is due, at least in part, to increased transcription and is dependent on Fnr but not on ArcA. A mutant derivative of serovar Typhi strain STH2370 with a deletion of the tsx gene is an auxotroph that requires either adenosine or thymidine for growth on minimal medium. In contrast, an otherwise isogenic nupG nupC double mutant, defective in the inner membrane nucleoside permeases, is a prototroph. Because anaerobic growth enhances the virulence of serovar Typhi in vitro, we assessed the role that the tsx gene plays in pathogenicity and found that the serovar Typhi STH2370 ⌬tsx mutant is defective in survival within human macrophage-like U937 cells. To understand why the ⌬tsx mutant is an auxotroph, we selected for insertions of minitransposon T-POP in the ⌬tsx genetic background that restored prototrophy. One T-POP insertion that suppressed the ⌬tsx mutation in the presence of the inducer tetracycline was located upstream of the pyrD gene. The results of reverse transcription-PCR analysis showed that addition of the inducer decreased the rate of pyrD transcription. These results suggest that the Tsx porin and the balance of products of the tsx and pyrD genes play critical roles in membrane assembly and integrity and thus in the virulence of serovar Typhi.

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Selectivity for maltose and maltodextrins of maltoporin, a pore‐forming protein of E. coli outer membrane

Cited by 45 publications

References 24 publications

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

The Oligogalacturonate-specific Porin KdgM of Erwinia chrysanthemi Belongs to a New Porin Family

TheSalmonella entericaSerovar TyphitsxGene, Encoding a Nucleoside-Specific Porin, Is Essential for Prototrophic Growth in the Absence of Nucleosides

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