The sugar‐specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate‐specific porins

Schülein, K.; Schmid, Kurt Werner; Benzl, R.

doi:10.1111/j.1365-2958.1991.tb02153.x

Cited by 68 publications

(60 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Both RafY and ScrY seem to be rather nonspecific, facilitating diffusion of all oligosaccharides tested. While in vitro studies on ScrY revealed a specific substrate binding (26), such experiments remain to be done for RafY. Thus, the designation "raffinose porin" reflects the genetic location of rafY as part of the raf operon rather than its molecular characteristics.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli

1997

View full text Add to dashboard Cite

The conjugative plasmid pRSD2 carries a raf operon that encodes a peripheral raffinose metabolic pathway in enterobacteria. In addition to the previously known raf genes, we identified another gene, rafY, which in Escherichia coli codes for an outer membrane protein (molecular mass, 53 kDa) similar in function to the known glycoporins LamB (maltoporin) and ScrY (sucrose porin). Sequence comparisons with LamB and ScrY revealed no significant similarities; however, both lamB and scrY mutants are functionally complemented by RafY. Expressed from the tac promoter, RafY significantly increases the uptake rates for maltose, sucrose, and raffinose at low substrate concentrations; in particular it shifts the apparent K m for raffinose transport from 2 mM to 130 M. Moreover, RafY permits diffusion of the tetrasaccharide stachyose and of maltodextrins up to maltoheptaose through the outer membrane of E. coli. A comparison of all three glycoporins in regard to their substrate selectivity revealed that both ScrY and RafY have a broad substrate range which includes ␣-galactosides while LamB seems to be restricted to malto-oligosaccharides. It supports growth only on maltodextrins but not, like the others, on raffinose and stachyose.The first step in carbohydrate metabolism of gram-negative bacteria is the permeation of the substrate through the outer membrane. Molecules up to a mass of about 600 Da can enter the periplasm through the general porins OmpF and OmpC in Escherichia coli; however, tetrasaccharides and larger molecules need specific glycoporins, such as LamB or ScrY (for recent reviews, see references 16 and 31).The maltoporin LamB of E. coli, which also functions as a phage receptor (19), is required for maltodextrin catabolism. In contrast to the general porins, it contains specific sugarbinding sites that facilitate diffusion of oligosaccharides through the pore (4, 14, 15). Moreover, the maltoporin also enhances the uptake of mono-and disaccharides (e.g., glucose and trehalose) at low substrate concentrations. The gene lamB, which is part of the chromosomal mal regulon, is normally induced by maltose, maltodextrins, and trehalose. It seems, however, that the endogenously induced level of the mal regulon is high enough for LamB to play an important general role in the carbohydrate transport of E. coli (8,12).The second glycoporin, ScrY (11,22,23), was found to be involved in the sucrose catabolic pathway encoded by the enterobacterial conjugative plasmid pUR400 (25). Although the sequence similarity between LamB and ScrY is very low, the latter can substitute for the maltoporin in lamB mutants, except for the function as a receptor (23). ScrY also contains sugar-binding sites (26).Here we describe a third glycoporin in enterobacteria, which is also encoded by a conjugative plasmid first isolated from an E. coli strain found in a chicken egg (7). This plasmid, pRSD2, is known to enable E. coli K-12 cells to grow on raffinose by providing a raffinose permease (encoded by rafB), an invertase (encoded by rafD), and ...

show abstract

Section: Discussionmentioning

confidence: 99%

“…Although the sequence similarity between LamB and ScrY is very low, the latter can substitute for the maltoporin in lamB mutants, except for the function as a receptor (23). ScrY also contains sugar-binding sites (26).…”

mentioning

confidence: 99%

Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli

1997

View full text Add to dashboard Cite

show abstract

“…This means that it is possible that RafY contains a binding site for carbohydrates comparable a trimer) had a smaller conductance than that of the other two monomeric channels. This result indicated that the vestibule of to those of LamB of E. coli [11,15], of maltoporin of S. typhimurium [25] and of ScrY [21]. To test this possibility we per-the trimeric channel facing the outside of the cell, similarly to OmpF and PhoE of E. coli [33], limited the current through the formed similar titration experiments as described earlier for specific porins of E. coli [11,21].…”

Section: Selectivity Of the Rafy Channel Further Information About Thementioning

confidence: 99%

“…porin [11] and sucroseporin [21]. These specific porins contain RafY does not contain a binding site for carbohydrates in-binding sites for carbohydrates.…”

Section: Selectivity Of the Rafy Channel Further Information About Thementioning

confidence: 99%

The porin RafY encoded by the raffinose plasmid pRSD2 of Escherichia coli forms a general diffusion pore and not a carbohydrate‐specific porin

Andersen

Krones

Ulmke

et al. 1998

European Journal of Biochemistry

View full text Add to dashboard Cite

The gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin-deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-permeable channel with a single-channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high-molecular-mass carbohydrates through the outer membrane.Keywords : raffinose transport; carbohydrate specificity ; RafY channel ; enteric bacteria; lipid bilayer membrane.The cell envelope of gram-negative bacteria consists of dif-also contains the gene for a carbohydrate-specific outer-membrane porin [19Ϫ21]. ferent layers. The inner or cytoplasmic membrane contains the proteins for the transport of nutrients and proteins involved in E. coli is normally unable to grow on raffinose as sole carbon source. In some E. coli strains a plasmid has been recogthe synthesis of phospholipids, peptidoglycans and lipopolysaccharides [1]. The periplasmic space between the membranes is nized that allows them to grow on raffinose [22]. The structure of the plasmid has been investigated and several genes have an aqueous compartment isoosmolar to the cytoplasm [2]. The outer membrane is an asymmetric membrane composed on the been found to form an operon on the plasmid [23]. Its genes encode an inner membrane permease (rafB) and two cytoplasmic outside of lipopolysaccharides and on the inside of lipids [3]. It contains only a few major proteins. At least one of them is a enzymes (rafA and rafD), which split raffinose into galactose and sucrose and sucrose into fructose and glucose, respectively. constitutive transmembrane protein, called porin, which contains a general diffusion pore with a defined exclusion limit for hydro-Recently, another gene has been cloned and sequenced, which codes for an outer membrane protein and allows E. coli to grow philic solutes [4, 5]. In addition to the constitutive porins the outer membrane may contain porins that are induced under spe-on raffinose, sucrose and maltose [24]. RafY allows the diffusion of a variety of high-molecular-mass carbohydrates, such as cial growth conditions [6Ϫ9]. They often form solute-specific stachyose and maltoheptaose, through the outer membrane, pores and contain binding sites for neutral substrates, such as which means that it has a simil...

show abstract

“…We found that only NAD and NMN caused a decrease in conductivity. This indicates that not only is OmpP2 a general diffusion pore, but it also has a similar function as the carbohydrate-specific porins of the OMs of enteric bacteria such as LamB and ScrY (23)(24)(25).…”

mentioning

confidence: 99%

Porin OmpP2 of Haemophilus influenzae Shows Specificity for Nicotinamide-derived Nucleotide Substrates

Andersen

Maier

Kemmer

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with K S values of ϳ8 and 4 mM, respectively, in 0.1 M KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.The only known natural habitat of Haemophilus influenzae is the human nasopharynx. H. influenzae is Gram-negative and is able to grow under facultative anaerobic conditions. It is also a human pathogen and is responsible for significant morbidity and mortality in young children (1, 2). To cultivate H. influenzae, a complex medium is required; and if not blood-based, it must contain two growth factors: NAD and hemin (3). In the nasopharynx, H. influenzae finds a relatively constant environment, and the organism has retained a certain degree of flexibility in its ability to utilize nutrients (4) by possessing some limiting metabolic pathway redundancy (5).Early biochemical investigations have established that NMN 1 and nicotinamide riboside (NR) can substitute for NAD, whereas nicotinamide, niacin, or other nicotine-based intermediates of the Preiss-Handler pathway cannot (6 -8). The NAD dependence of H. influenzae was confirmed by the absence of genes encoding the enzymes necessary for the de novo biosynthesis of NAD (9). Accumulation of nicotinamide nucleotides derived from NAD or NR has been demonstrated in H. influenzae and Haemophilus parainfluenzae (10, 11). For H. parainfluenzae, the K m for transport is ϳ0.55 M for NAD and 0.14 M for NR, and the V max for NR is about four times that of NAD (11). This implies that NR is the substrate for an as yet unidentified inner membrane transporter. Recently, we presented data showing that two gene products appear to be involved in the NAD utilization pathway of H. influenzae (12)(13)(14). The gene products were identified as the hel-encoded outer membrane lipoprotein e(P4) and a periplasmic protein termed NadN. Knockout mutations of both genes resulted in growth-deficient phenotypes depending on the...

show abstract

The sugar‐specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate‐specific porins

Cited by 68 publications

References 47 publications

Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli

Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli

The porin RafY encoded by the raffinose plasmid pRSD2 of Escherichia coli forms a general diffusion pore and not a carbohydrate‐specific porin

Porin OmpP2 of Haemophilus influenzae Shows Specificity for Nicotinamide-derived Nucleotide Substrates

Contact Info

Product

Resources

About