Maltose Uptake Research Articles

Inter-domain Communication Mechanisms in an ABC Importer: A Molecular Dynamics Study of the MalFGK2E Complex

ATP-Binding Cassette transporters are ubiquitous membrane proteins that convert the energy from ATP-binding and hydrolysis into conformational changes of the transmembrane region to allow the translocation of substrates against their concentration gradient. Despite the large amount of structural and biochemical data available for this family, it is still not clear how the energy obtained from ATP hydrolysis in the ATPase domains is “transmitted” to the transmembrane domains. In this work, we focus our attention on the consequences of hydrolysis and inorganic phosphate exit in the maltose uptake system (MalFGK2E) from Escherichia coli. The prime goal is to identify and map the structural changes occurring during an ATP-hydrolytic cycle. For that, we use extensive molecular dynamics simulations to study three potential intermediate states (with 10 replicates each): an ATP-bound, an ADP plus inorganic phosphate-bound and an ADP-bound state. Our results show that the residues presenting major rearrangements are located in the A-loop, in the helical sub-domain, and in the “EAA motif” (especially in the “coupling helices” region). Additionally, in one of the simulations with ADP we were able to observe the opening of the NBD dimer accompanied by the dissociation of ADP from the ABC signature motif, but not from its corresponding P-loop motif. This work, together with several other MD studies, suggests a common communication mechanism both for importers and exporters, in which ATP-hydrolysis induces conformational changes in the helical sub-domain region, in turn transferred to the transmembrane domains via the “coupling helices”.

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.

Binding Protein-Dependent Uptake of Maltose into Cells via an ATP-Binding Cassette Transporter.

Maltose and maltodextrins are actively transported across the cytoplasmic membrane of Escherichia coli and Salmonella by a periplasmic binding protein (BP)- dependent transport system. Since 1996, there have been many advances in the understanding of the structure and mechanism of the maltose transporter, in the assembly of the membrane-associated transporter complex, and in the mechanism of regulation of transport both at the DNA and the protein level. The transporter has been studied in detergent and reconstituted in liposome vesicles, and while many features, including the ability of maltose-binding protein (MBP) to stimulate ATPase activity, are retained in detergent, it has been noted that the basal ATPase activity of the transporter is elevated in detergent compared with liposomes. This review focuses on these recent developments, which have culminated in a high resolution structure of MBP in a complex with the MalFGK2 transporter. While this review focuses on the maltose system, complementary work has been carried out on many different ATP binding cassette (ABC) transporters, all of which has contributed in important ways to the understanding of the maltose transport system. The regulation of the maltose transport system, at the DNA level, is implemented by the synergistic action of MalT and cAMP/CAP complex and, at the protein level, by interactions of MalK with unphosphorylated EIIAglc, a signal-transducing component of the phosphoenolpyruvate-glucose phosphotransferase system.

Constitutive Expression of the Maltoporin LamB in the Absence of OmpR Damages the Cell Envelope

Cells experience multiple environmental stimuli simultaneously. To survive, they must respond accordingly. Unfortunately, the proper response to one stress easily could make the cell more susceptible to a second coexistent stress. To deal with such a problem, a cell must possess a mechanism that balances the need to respond simultaneously to both stresses. Our recent studies of ompR malT(Con) double mutants show that elevated expression of LamB, the outer membrane porin responsible for maltose uptake, causes cell death when the osmoregulator OmpR is disabled. To obtain insight into the nature of the death experienced by ompR malT(Con) mutants, we described the death process. On the basis of microscopic and biochemical approaches, we conclude that death results from a loss of membrane integrity. On the basis of an unbiased genome-wide search for suppressor mutations, we conclude that this loss of membrane integrity results from a LamB-induced envelope stress that the cells do not sufficiently perceive and thus do not adequately accommodate. Finally, we conclude that this envelope stress involves an imbalance in the lipopolysaccharide/porin composition of the outer membrane and an increased requirement for inorganic phosphate.

Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

Exploring the Potential of C. Pasteurianum to Produce Biofuels from Waste Products

Biofuels which can be produced by microorganisms from agricultural waste products will provide a renewable and non controversial source of energy for the future. The saccharolytic Clostridium sp. can ferment a wide range of sugars to produce organic acids and solvents. They are also anaerobic and in some cases thermophilic which is advantageous for large-scale industrial processes. C.pasteurianum shows potentially high solvent and hydrogen gas production capacity. The fermentation end products of C.pasteurianum, when grown in media of high sugar content, included significant quantities of acetone, butanol and ethanol, acetate, butyrate, CO2 and H2. The aim of this research was to investigate maltose uptake and metabolism in Clostridium pasteurianum NCIB 9486, to increase understanding of its starch utilisation. The organism was grown in different substrates and assayed for induction of glucosidase activities. Results confirmed that enzyme synthesis is induced by substrates.MaltaseandpNPGasewere found largely in soluble form (89.5% and 86.6% respectively) proving that maltase is produced intracellularly and hydrolysis of maltose takes place in the cytpoplasm. Fractionationof the soluble extract ofmaltose growncells yielded two maltase enzymes, maltase I (120-150 KDa) and maltase II (60 KDa). Further characterisation of maltase II showed high substrate specificity for maltose and its analogues, and sucrose. Maltase II exhibited similar rates of hydrolysis of maltose andmaltotriose: values for Km of 0.8 and 0.7 mmol l-1;Vmax of 3.3 and 4.2 nmol min-1, respectively. There is indication of specific activity of maltase II in cleaving the -1,4 (maltose) and -1,6-(isomaltose) linkages, but not -linkages (cellobiose). PEP-dependent phosphorylation of maltose was demonstrated suggesting the presence of a PEP-dependent PTS for maltose transport. This species has potential for use in biofuel production and a better understanding of its carbohydrate metabolism will facilitate its exploitation.

TrmB an archael transcriptional regulator

In Pyrococcus furiosus two binding protein–dependent, substrate–specific ABC transporters could be identified, one of it being a trehalose/maltose (TM)–transporter, which is responsible for the uptake of trehalose and maltose. By binding to the respective operator sites TrmB prevents the further binding of transcription factors and subsequent RNA polymerase recruitment, which leads to an inhibition of transcription. With the aid of in vitro transcription assays using purified components of P. furiosus it could be shown that TrmB–mediated inhibition of transcription on both promoters is released by specific carbohydrates with maltose and trehalose being inducers for the TM operon, and maltodextrins as well as sucrose releasing inhibition on the MD promoter.TrmBL1 is homologue to TrmB and is involved in the regulation of genes encoding glycolytic and gluconeogenetic enzymes, respectively, where it recognizes a conserved sequence motif called TGM. It inhibits transcription of glycolytic genes with maltose, maltotriose and fructose as inducers (3). On the other hand TrmBL1 activates the expression of gluconeogenetic genes as well. Since the TGM is not present in the promoter region of the TrmBL1 gene, the TGM is not essential for TrmBL1 target recognition (3).Both TrmB and TrmBL1 share the unique property of recognizing different DNA sequences on different promoters and showing distinctive, promoter–dependent sugar specificity.In vitro transcription assays, EMSA and DNaseI footprinting experiments proved that TrmB and TrmBL1 have at least two different HTH motifs and recognize their respective target promoters with different helices within their N–terminal DNA binding domains.

Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique

BackgroundSalmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches.ResultsIn this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX.ConclusionUsing a multi-step digest approach the LPI™ technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI™ technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for Salmonella Typhimurium.

Comparative in vivo gene expression of the closely related bacteria Photorhabdus temperata and Xenorhabdus koppenhoeferi upon infection of the same insect host, Rhizotrogus majalis

BackgroundPhotorhabdus and Xenorhabdus are Gram-negative, phylogenetically related, enterobacteria, forming mutualism with the entomopathogenic nematodes Heterorhabditis and Steinernema, respectively. The mutualistic bacteria living in the intestines of the nematode infective juveniles are pathogenic to the insect upon release by the nematodes into the insect hemolymph. Such a switch needs activation of genes that promote bacterial virulence. We studied in vivo gene expression in Photorhabdus temperata and Xenorhabdus koppenhoeferi upon infection of the white grub Rhizotrogus majalis using selective capture of transcribed sequences technique.ResultsA total of 40 genes in P. temperata and 39 in X. koppenhoeferi were found to be upregulated in R. majalis hemolymph at 24 h post infection. Genomic presence or upregulation of these genes specific in either one of the bacterium was confirmed by the assay of comparative hybridization, and the changes of randomly selected genes were further validated by quantitative real-time PCR. The identified genes could be broadly divided into seven functional groups including cell surface structure, regulation, virulence and secretion, stress response, intracellular metabolism, nutrient scavenging, and unknown. The two bacteria shared more genes in stress response category than any other functional group. More than 60% of the identified genes were uniquely induced in either bacterium suggesting vastly different molecular mechanisms of pathogenicity to the same insect host. In P. temperata lysR gene encoding transcriptional activator was induced, while genes yijC and rseA encoding transcriptional repressors were induced in X. koppenhoeferi. Lipopolysaccharide synthesis gene lpsE was induced in X. koppenhoeferi but not in P. temperata. Except tcaC and hemolysin related genes, other virulence genes were different between the two bacteria. Genes involved in TCA cycle were induced in P. temperata whereas those involved in glyoxylate pathway were induced in X. koppenhoeferi, suggesting differences in metabolism between the two bacteria in the same insect host. Upregulation of genes encoding different types of nutrient uptake systems further emphasized the differences in nutritional requirements of the two bacteria in the same insect host. Photorhabdus temperata displayed upregulation of genes encoding siderophore-dependent iron uptake system, but X. koppenhoeferi upregulated genes encoding siderophore-independent ion uptake system. Photorhabdus temperata induced genes for amino acid acquisition but X. koppenhoeferi upregulated malF gene, encoding a maltose uptake system. Further analyses identified possible mechanistic associations between the identified gene products in metabolic pathways, providing an interactive model of pathogenesis for each bacterium species.ConclusionThis study identifies set of genes induced in P. temperata and X. koppenhoeferi upon infection of R. majalis, and highlights differences in molecular features used by these two closely related bacteria to promote their pathogenicity in the same insect host.

Sugar uptake byMycoplasma mycoides strain PT994 andMycoplasma canis strain 85 B97

A pathogenic strain of each ofMycoplasma mycoides subsp.mycoides (Large Colony type) (strain PT994) andMycoplasma canis (strain 85 B97) showed different patterns of sugar uptake when provided with glucose and a disaccharide. Maltose had no effect on the uptake of glucose or of α-methylglucoside byM. mycoides, whereas glucose inhibited the uptake of maltose. Maltose uptake by growing cultures ofM. mycoides recommenced immediately after glucose exhaustion. There was thus an interaction between the two sugars for uptake, with glucose inhibiting the uptake of maltose either by competing for a common transporter or by suppression of a specific maltose-binding system. In contrast, uptake of glucose and sucrose byM. canis showed no interaction, with both sugars being used concurrently, and were thus transported by independent systems. Sugar uptake by both species was inhibited by fluoride, consistent with the operation of glycolysis, and possibly phosphoenolpyruvate-dependent sugar uptake systems.

Molecular strategies for protein stabilization: The case of a trehalose/maltose‐binding protein from Thermus thermophilus

The trehalose/maltose-binding protein (MalE1) is one component of trehalose and maltose uptake system in the thermophilic organism Thermus thermophilus. MalE1 is a monomeric 48 kDa protein predominantly organized in alpha-helix conformation with a minor content of beta-structure. In this work, we used Fourier-infrared spectroscopy and in silico methodologies for investigating the structural stability properties of MalE1. The protein was studied in the absence and in the presence of maltose as well as in the absence and in the presence of SDS at different p(2)H values (neutral p(2)H and at p(2)H 9.8). In the absence of SDS, the results pointed out a high thermostability of the MalE1 alpha-helices, maintained also at basic p(2)H values. However, the obtained data also showed that at high temperatures the MalE1 beta-sheets underwent to structural rearrangements that were totally reversible when the temperature was lowered. At room temperature, the addition of SDS to the protein solution slightly modified the MalE1 secondary structure content by decreasing the protein thermostability. The infrared data, corroborated by molecular dynamics simulation experiments performed on the structure of MalE1, indicated that the protein hydrophobic interactions have an important role in the MalE1 high thermostability. Finally, the results obtained on MalE1 are also discussed in comparison with the data on similar thermostable proteins already studied in our laboratories.

Escherichia coli tat mutant strains are able to transport maltose in the absence of an active malE gene

The twin-arginine transport (Tat) system is a prokaryotic protein transport system. Escherichia coli mutants in this pathway show a defect in cell separation during cell division, resulting in destabilization and permeability of the outer membrane. Maltose uptake is catalysed by a membrane-bound transporter of the ATP binding cassette (ABC) superfamily, where MalE is the essential periplasmic binding protein component. Here, we report that tat mutants are unexpectedly able to transport maltose in the absence of malE. This observation is specific to the MalE component since co-inactivation of malF, which encodes one of the channel components of the transporter, completely abolishes maltose transport even when the Tat system is inactivated. Genetic repair of the outer membrane leaky phenotype of the tat mutant strain re-established the absolute requirement for MalE in maltose uptake. In addition, we demonstrate that phenotypic repair of the outer membrane defect of the tat strain can also be achieved chemically by the inclusion of high concentrations of calcium or magnesium in the growth medium.

Stimulation of Zero- trans Rates of Lactose and Maltose Uptake into Yeasts by Preincubation with Hexose To Increase the Adenylate Energy Charge

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three- to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular beta-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [(14)C]lactose uptake reactions were started. A short (1- to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5- to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 micromol x g dry yeast(-1)). Stimulation by glucose affected the transport V(max) values, with smaller increases in K(m) values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H(+) symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H(+) symports. A short exposure to glucose allows a closer approach to the sugar/H(+) symport capacity of actively metabolizing cells.

Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known alpha-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K(m), 36 +/- 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

Crystal structure of a catalytic intermediate of the maltose transporter

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-A crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP.

Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in LivingSaccharomyces cerevisiaeCells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).

ATP-driven MalK Dimer Closure and Reopening and Conformational Changes of the “EAA” Motifs Are Crucial for Function of the Maltose ATP-binding Cassette Transporter (MalFGK2)

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK(2)) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.

Molecular adaptation strategies to high temperature and thermal denaturation mechanism of the D‐trehalose/D‐maltose‐binding protein from the hyperthermophilic archaeon Thermococcus litoralis

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose uptake system. In the hyperthermophilic archaeon T. litoralis this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds both trehalose and maltose with high affinity. In this work we used differential scanning calorimetry studies together with a detailed analysis, at the molecular level, of the three-dimensional protein structure to shed light on the basis of the high thermostability exhibited by the recombinant TMBP from the archaeon T. litoralis. The obtained data suggest that the presence of trehalose does not change the overall mechanism of the denaturation of this protein but it selectively modifies the stability of the TMBP structural domains.

Differences amongAGT1-encoded α-glucoside transporters and their ability to transport maltotriose inSaccharomyces yeasts

Improved fermentation of starch and its dextrin products would benefit the brewing and whiskey industries. Most strains ofSaccharomyces ferment glucose and maltose and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The maltotriose transporting efficiency varies between differentSaccharomyces strains. In this study, severalSaccharomyces strains, including whiskey strains, were screened for growth on maltotriose. TheAGT1 genes, which encode a maltose transporter that show affinity for maltotriose uptake, were isolated from the strains that grew strongest in media with maltotriose as sole carbon source. The isolatedAGT1 alleles were sequenced and their chromosomal locations determined in the strains from which they were cloned. Nucleotide and deduced amino acid sequences of the isolated genes shared 95% and 98% identity, respectively. The efficiency of maltotriose transport was determined by expressing theAGT1 variants in an identical genetic background. TheK m values obtained for all the permeases were very similar (≈3), but the permease with improved performance for maltotriose transport showed an approximately 30% higherV max value than for the others. The data obtained suggest that the genetic variation among theAGT1-encoded transporters is reason for the variation in maltotriose transport efficiency among differentSaccharomyces strains. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whiskey industries.

Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis for sugars monitoring

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection.